In vitro method for diagnosing, screening and / or monitoring chronic inflammatory diseases

Abstract

The present invention refers to an in vitro method for diagnosing or screening a chronic inflammatory disease selected from the group comprising: Inflammatory bowel disease (IBD), arthritis or psoriasis.The present invention also refers to an in vitro method for monitoring patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; and / or for differentiating active patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis from those patients who are in remission.

Description

[0001]IN VITRO METHOD FOR DIAGNOSING, SCREENING AND / OR MONITORINGCHRONIC INFLAMMATORY DISEASES FIELD OF THE INVENTION The present invention refers to the medical field. Particularly, the present invention refers toan in vitro method for diagnosing or screening a chronic inflammatory disease selected fromthe group comprising: Inflammatory bowel disease (IBD), arthritis or psoriasis. The presentinvention also refers to an in vitro method for monitoring patients suffering from a chronicinflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; and / or for differentiating active patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis from those patients who are in remission. STATE OF THE ART Inflammation is a physiological response of the immune system to harmful stimuli, such as that the host enacts to defend itself against viruses, bacteria, tissue damage, metabolic stress by recruiting immune and non-immune cells. In some situations, this process becomes chronic, and may result in systemic effects beyond their primary target organs, underscoring the complex nature of chronic inflammatory diseases (CID). This term encompass a diverse group of medical conditions such as: asthma, chronic obstructive pulmonary disease, cancer, cardiovascular disease, neurological diseases like Parkinson's disease, systemic lupus erythematosus, rheumatoid arthritis, psoriasis and IBD. IBD is a relapsing chronic disorder of the gastrointestinal tract associated with alteration in the epithelial barrier function and mucosal inflammation. The main idiopathic forms of IBD are Crohn's disease (CD) and ulcerative colitis (UC); both share some clinical and demographic characteristics but differ in the tissue damage they produce and their prognosis. CD can involve any part of the digestive tract and is characterized by transmural inflammation, which means it can affect multiple layers of the intestinal wall. UC, is characterized by inflammation and ulcerations that primarily affects the colon and rectum, causing inflammation limited to the mucosal layer. Rheumatoid Arthritis-Psoriasis (RAP) are two distinct autoimmune diseases. Rheumatoid arthritis (RA) specifically targets the joints, inducing chronic inflammation, joint pain, and potential deformities if not tackled. Additionally, RA can affect other organs, such as the heart and lungs. On the other hand, Psoriasis (P) represents a skin disorder that develops red, scaly patches on the skin, as a result of an overactive immune response. These conditions affect diverse bodily systems, yet they are linked by immune system dysfunction and persistent inflammation. These pathologies share the pharmacological treatment used by clinicians to reduce inflammation, alleviate symptoms, and prevent or manage complications. One of the overarching goals in CID remains to accelerate its diagnosis to adapt treatment and improve its symptoms. In general, the diagnosis of chronic inflammatory diseases takes long time because it involves an evaluation that integrates a medical history, physical examinations, and various diagnostic tests which include specialized imaging techniques like X-rays and magnetic resonance imaging (MRI). Moreover, in the case of IBD, invasive procedure such as endoscopyand endoscopy and colonoscopy, often couple with tissue biopsies, may be required for adefinitive diagnosis. Consequently, there is an unmet medical need of finding reliable and non-invasive tools for the diagnosis or screening chronic inflammatory diseases like IBD, arthritis or psoriasis. The present invention is focused on solving this problem by the determination of inflammatory markers in saliva thus providing clinicians with a simple and rapid test which, combined with a thorough anamnesis allow them an early diagnosis of CID and a prompt treatment to control the symptoms. DESCRIPTION OF THE INVENTION Brief description of the invention As indicated above, the present invention is directed to a reliable and non-invasive method for the diagnosis or screening chronic inflammatory diseases like IBD, arthritis or psoriasis byassessing specific biomarker in saliva. The present invention also refers to an in vitro methodfor monitoring patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; and / or for differentiating active patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis from those patients who are in remission.So, the special technical feature conferring unity of invention to the present invention is the useof saliva samples for the diagnosis or screening chronic inflammatory diseases like IBD,arthritis or psoriasis by assessing specific biomarkers.Such as it can be seen in the Examples dealing with the “discovery phase”, protein levels andRNA expression of several specific biomarkers in saliva are associated with the presence of CID, such as IBD, Rheumatoid arthritis-psoriasis (RPA). Particularly, such as it is shown in the Examples dealing with the “validation phase”, the expression of the protein combination IL1B and calprotectin, or the genes encoding thoseproteins, are associated with chronic inflammatory diseases selected from the groupcomprising: IBD, arthritis or psoriasis; so the combination of IL1B and calprotectin can beused for identifying biomarker signatures for the diagnosis or screening of a chronicinflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis, for monitoring patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; and / or for differentiating active patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis from those patients who are in remission. So, according to the discovery phase, the first embodiment of the present invention refers to anin vitro method for identifying biomarker signatures for the diagnosis or screening of a chronicinflammatory disease selected from the group comprising or consisting of: IBD, arthritis(preferably rheumatoid arthritis) or psoriasis in a subject, which comprises: a) Assessing theexpression level of at least one protein selected from the group consisting of: IL1B and / or SIRT1, or of the genes encoding those proteins, in a saliva sample obtained from the subject, and b) wherein a deviation of the expression level of the proteins of step a) or of the genes encoding those proteins, as compared to a pre-established reference value, is an indication that the biomarker signature may be used for the diagnosis or screening of a chronic inflammatorydisease selected from the group comprising or consisting of: IBD, arthritis or psoriasis.In a preferred embodiment, the first embodiment of invention further comprises assessing the expression level of the protein calprotectin, or of the gene encoding this protein, in a saliva sample obtained from the subject.The second embodiment of the present invention refers to an in vitro method for diagnosing orscreening a chronic inflammatory disease selected from the group comprising or consisting of:IBD, arthritis (preferably rheumatoid arthritis) or psoriasis in a subject, which comprises: a)Assessing the expression level of at least one protein selected from the group consisting of: IL1B and / or SIRT1, or of the genes encoding these proteins, in a saliva sample obtained from the subject, and b) wherein a higher expression level of the protein IL1B and / or a lower expression level of the protein SIRT1, or of the genes encoding those proteins, as compared to a pre-established reference value, is indicative that the subject suffers from IBD, arthritis and / or psoriasis. In a preferred embodiment, the second embodiment invention further comprises assessing: a) Assessing the expression level of the protein calprotectin or of the gene encoding this protein, b) wherein a higher expression level of the protein calprotectin, or of the gene encoding that protein, as compared to a pre-established reference value, is indicative that the subject suffersfrom IBD, or b) wherein a higher expression level of the protein calprotectin, as compared toa pre-established reference value, is indicative that the subject suffers from arthritis or psoriasis,or c) wherein a lower abundance of mRNA transcripts encoding calprotectin, as compared to apre-established reference value, is indicative that the subject suffers from arthritis or psoriasis.In a preferred embodiment, the present invention comprises: a) Assessing the expression levelof the proteins IL1B and calprotectin or of the gene encoding this protein, and b) wherein a higher expression level of the proteins IL1B and calprotectin, or of the gene encoding these proteins, as compared to a pre-established reference value, is indicative that the subject suffers from IBD.In a preferred embodiment, the present invention further comprises: a) Assessing theexpression level of the protein SIRT1 or of the gene encoding this protein, and b) wherein a decreased expression level of the protein SIRT1, or of the gene encoding that protein, as compared to a pre-established reference value, is indicative that the subject suffers from IBD. In a preferred embodiment, the IBD disease is selected from: Chron´s disease or ulcerative colitis.The third embodiment of the present invention refers to the in vitro use of the proteins IL1Band / or SIRT1, or of the transcripts encoding thereof, or of a kit comprising reagents for the identification of the proteins IL1B and / or SIRT1, or of the transcripts encoding thereof, for identifying biomarker signatures for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis, wherein the IBD is preferably selected from Chron’s disease or ulcerative colitis.In a preferred embodiment, the present invention refers to the in vitro use of the proteins IL1Band / or SIRT1, or of the transcripts encoding thereof, in combination with the proteincalprotectin, or of the transcripts encoding thereof, or of a kit comprising reagents for the identification of the proteins IL1B and / or SIRT1, or of the transcripts encoding thereof, incombination with calprotectin, or of the transcripts encoding thereof.In a preferred embodiment, the present invention refers to the in vitro use of the proteins IL1Band / or SIRT1, or of the transcripts encoding thereof, or of a kit comprising reagents for the identification of the proteins IL1B and / or SIRT1, or of the transcripts encoding thereof, for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising:IBD, arthritis or psoriasis, wherein the IBD is preferably selected from Chron’s disease orulcerative colitis.In a preferred embodiment, the present invention refers to the in vitro use of the proteins IL1Band / or SIRT1, or of the transcripts encoding thereof, in combination with calprotectin, or of akit comprising reagents for the identification of the proteins IL1B and / or SIRT1, or of thetranscripts encoding thereof, in combination with calprotectin.In a preferred embodiment, the present invention refers to the in vitro use of proteins IL1B andcalprotectin, or of the transcripts encoding thereof, or of a kit comprising reagents for the identification of the proteins IL1B and calprotectin, or of the transcripts encoding thereof, for the diagnosis or screening IBD, preferably selected from Chron’s disease or ulcerative colitis.In a preferred embodiment, the present invention refers to the in vitro use of the proteins IL1B,calprotectin and SIRT1, or of the transcripts encoding thereof, or of a kit comprising reagentsfor the identification of the proteins IL1B, calprotectin and SIRT1, or of the transcripts encoding thereof.In a preferred embodiment, the present invention refers to the in vitro use of biomarkersignatures identified by following the method of the invention, for the diagnosis or screening of a chronic inflammatory disease selected from the group comprises: IBD, arthritis or psoriasis, wherein the IBD is preferably selected from Chron’s disease or ulcerative colitis.In a preferred embodiment, the present invention refers to anti-inflammatory drugs (see Table1), preferably selected from the group comprising: a) salicylates; b) immunosuppressantsselected from azathioprine, methotrexate and mercaptopurine; c) biological treatments selectedfrom adalimumab, golimumab, infliximab, risankisumab, upadicitinib, ustekinumab andvedolizumab; or d) corticosteroids selected from: prednisone, cortisone, betamethasone,budesonide and beclomethasone; for use in the treatment of a chronic inflammatory diseaseselected from the group comprising: IBD, preferably selected from Chron’s disease or ulcerative colitis, arthritis or psoriasis, wherein the method comprises diagnosing patients that suffer from the chronic inflammatory disease by using the signatures identified by following the method of the invention. Table 1. Pharmacological treatment used for chronic inflammatory diseases.Salicylates MesalazineI The present invention also refers to an in vitro method for identifying biomarker signatures forthe diagnosis or screening of a chronic inflammatory disease selected from the groupcomprising: IBD, arthritis or psoriasis in a subject, which comprises: a) Assessing theexpression level of at least one protein selected from the group consisting of: IL1B, SIRT1 and / or calprotectin, or of the genes encoding those proteins, in a saliva sample obtained fromthe subject, and b) wherein a deviation of the expression level of the proteins of step a) or ofthe genes encoding those proteins, as compared to a pre-established reference value, is an indication that the subject suffers from: IBD, arthritis or psoriasis.The present invention refers to an in vitro method for the differential diagnosis between activeand remissive disease in a subject suffering from IBD, which comprises: a) Assessing theexpression level of at least one protein selected from the group consisting of: IL1B and / or calprotectin, or of the genes encoding those proteins, in a saliva sample obtained from thesubject, b) wherein a higher expression level of the protein IL1B, or of the gene encodingthereof, as compared to a pre-established reference value, is indicative that the subject suffersfrom active IBD, or c) wherein a higher abundance of mRNA transcripts encoding calprotectin,as compared to a pre-established reference value, is indicative that the subject suffers from IBD, or d) wherein a lower expression level of the protein IL1B, or of the gene encoding thereof, as compared to a pre-established reference value, is indicative that the subject suffersfrom remissive IBD, or e) wherein a lower abundance of mRNA transcripts encodingcalprotectin, as compared to a pre-established reference value, is indicative that the subject suffers from remissive IBD.The present invention also refers to an in vitro method for the differential diagnosis betweenChron’s disease and ulcerative colitis in a subject suffering from IBD, which comprises: a)Assessing the expression level of the protein SIRT1, or of the genes encoding thereof, in asaliva sample obtained from the subject, b) wherein a higher expression level of the proteinSIRT1, or of the gene encoding thereof, as compared to a pre-established reference value, isindicative that the subject suffers from Chron’s disease, or c) wherein a lower expression levelof the protein SIRT1, or of the gene encoding thereof, as compared to a pre-established reference value, is indicative that the subject suffers from ulcerative colitis. In a preferred embodiment, the chronic inflammatory disease, IBD, psoriasis or arthritis is active or remissive. In a preferred embodiment, the subject is a subject suffering from IBD that has received a treatment selected from: corticosteroids, biological or immunosuppressants. In a preferred embodiment, the IBD is selected from: Chron´s disease or ulcerative colitis.On the other hand, the present invention also refers to:A method for detecting a biomarker in a saliva sample from a human subject at risk ofdeveloping a chronic inflammatory disease selected from the group comprising: IBD, arthritisor psoriasis, the method comprising performing an assay for determining the concentration of the protein or level of expression of the genes of the above cited biomarkers, such as for instance, an immunoassay or PCR. In a preferred embodiment, the present invention is a computer-implemented invention, wherein a processing unit (hardware) and a software are configured to: a) Receive theconcentration level or level of expression values of any of the above cited biomarkers orsignatures, b) process the concentration level values received for finding substantial variations or deviations, and c) provide an output through a terminal display of the variation or deviation of the concentration or expression level, wherein the variation or deviation of the concentration or expression level indicates that the subject may be suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis. The present invention also refers to a computer program or a computer-readable media containing means for carrying out the present invention. Moreover, according to the “validation phase”, the present invention refers to an in vitro method for identifying biomarker signatures for the diagnosis and / or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis (preferablyrheumatoid arthritis) or psoriasis in a subject, which comprises assessing the expression levelof the protein combination IL1B and calprotectin, or of the genes encoding those proteins, in a saliva sample obtained from the subject.The present invention also refers to an in vitro method for the diagnosis or screening of achronic inflammatory disease selected from the group comprising: IBD, arthritis (preferablyrheumatoid arthritis) or psoriasis in a subject, which comprises assessing the expression levelof the protein combination IL1B and calprotectin, or of the genes encoding those proteins, in a saliva sample obtained from the subject.The present invention also refers to an in vitro method for monitoring patients suffering froma chronic inflammatory disease selected from the group comprising: IBD, arthritis (preferablyrheumatoid arthritis) or psoriasis; and / or for differentiating active patients suffering from achronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis from those patients who are in remission, which comprises assessing the expression level of the protein combination IL1B and calprotectin, or of the genes encoding those proteins, in a saliva sample obtained from the subject.The present invention also refers to the in vitro use of the protein combination IL1B andcalprotectin, or of the genes encoding those proteins, or of a kit for assessing the level expression of the proteins or the genes, in a saliva sample obtained from the subject, for identifying biomarker signatures for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis.The present invention also refers to the in vitro use of the protein combination IL1B andcalprotectin, or of the genes encoding those proteins, or of a kit for assessing the level expression of the proteins or the genes, in a saliva sample obtained from the subject, for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis.The present invention also refers to the in vitro use of the protein combination IL1B andcalprotectin, or of the genes encoding those proteins, or of a kit for assessing the level expression of the proteins or the genes, in a saliva sample obtained from the subject, for monitoring patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; and / or for differentiating active patients suffering froma chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasisfrom those patients who are in remission. In a preferred embodiment, a deviation of the expression level of the proteins or of the genes encoding those proteins, as compared to a pre-established reference value, is an indication that the biomarker signature may be used for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; or that the subject issuffering from a chronic inflammatory disease selected from the group comprising: IBD,arthritis or psoriasis. In a preferred embodiment the production of saliva is not stimulated (for instance through masticatory stimulation by chewing) and the saliva is not centrifugated. In a preferred embodiment, an increase of the protein level of IL1B and calprotectin as compared to a pre-established reference value, is an indication that the biomarker signature may be used for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; or that the subject is suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis. In a preferred embodiment, an increase of the gene expression level of IL1B and calprotectin as compared to a pre-established reference value, is an indication that the biomarker signature may be used for the diagnosis or screening of IBD; or that the subject is suffering from IBD. In a preferred embodiment, an increase of the gene expression level of IL1B and a decrease of the gene expression level of calprotectin as compared to a pre-established reference value, is an indication that the biomarker signature may be used for the diagnosis or screening of arthritis and / or psoriasis; or that the subject is suffering from arthritis and / or psoriasis. In a preferred embodiment, the IBD disease is selected from: Chron´s disease or ulcerative colitis. The present invention also refers to anti-inflammatory drugs selected from the group comprising: a) Salicylates;b) Immunosuppressants selected from azathioprine, methotrexate andmercaptopurine; c) Biological treatments selected from adalimumab, golimumab, infliximab,risankisumab, upadicitinib, ustekinumab and vedolizumab; d) Corticosteroids selected from: prednisone, cortisone, betamethasone, budesonideand beclomethasone; for use in the treatment of a chronic inflammatory disease selected from the group comprising: IBD, preferably selected from Chron’s disease or ulcerative colitis, arthritis or psoriasis, wherein the method comprises diagnosing patients that suffer from the chronic inflammatory disease by using the method of the invention. For the purpose of the present invention, the following terms are defined: ^The term "comprising" means including, but not limited to, whatever follows the word"comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. ^The term "consisting of” means including, and it is limited to, whatever follows thephrase “consisting of”. Thus, the phrase "consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.^ According to the present invention, a reference value can be a “pre-establishedthreshold value” or a “cut-off” value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. According to the present invention, the “pre-established threshold” value refers to a value previously determined in control subjects who are nonsuffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis. A “threshold value” can also be arbitrarily selected based upon the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The “threshold value” has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the “threshold value”) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Brief description of the figures Figure 1. Increased mRNA expression of inflammatory biomarkers in saliva of chronicinflammatory disease (CID) patients. A) mRNA expression of Il1β, B) mRNA expression ofs100s9 calprotectin and C) mRNA expression sirt1 in saliva of CID patients and controlsamples. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples Mann- Whitney U test was used and *P < 0.05 indicate the statistical significance vs controls. Figure 2. Increased mRNA expression of inflammatory biomarkers in saliva ofinflammatory bowel disease (IBD) patients. A) mRNA expression of Il1β, B) mRNAexpression of s100a9 calprotectin and C) mRNA expression sirt1 in saliva of IBD patients andcontrol samples. Data show median and the interquartile range of gene expression; each dotrepresents the value of an individual expressed as fold induction vs the mean of controlsamples. Mann-Whitney U test was used and *P < 0.05 indicate the statistical significance vs controls. Figure 3. Increased mRNA expression of inflammatory biomarkers in saliva of active vsremission IBD patients. A) mRNA expression of Il1β and B) mRNA expression of s100a9calprotectin in saliva from both remission and active IBD patients and healthy controls. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. The colors represent treatments received by of patients: pink (mesalazine), blue (Immunosuppressant), red (biological), sky blue (combined treatment), green (corticosteroids) and black (without drug). Mann-Whitney U test and one-way ANOVA were used and *P<0.05, **P<0.01 and ***P < 0.001 indicate the statistical significance vs controls while ##P<0.01 vs IBD patients in remission. Mann-Whitney U test. Figure 4. mRNA expression of inflammatory biomarkers in rheumatoid arthritis-psoriasis (ARP) patients. A) mRNA expression of Il1β, B) mRNA expression of s100a9calprotectin and C) mRNA expression of sirt1 in saliva of ARP patients and control samples. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test was used and ***P < 0.001 indicate the statistical significance vs controls. Figure 5. mRNA expression of inflammatory biomarkers in saliva of both, active andremission rheumatoid arthritis-psoriasis (ARP) patients. A) mRNA expression of Il1β andB) mRNA expression of s100a9 calprotectin in saliva from both remission and active ARPpatients and healthy controls. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and *P<0.05, indicate the statistical significance vs controls. Figure 6. Increased protein expression of IL1β, calprotectin and SIRT1 in chronicinflammatory disease (CID) patients. A, B, C) Area under the receiver operatingcharacteristic (ROC) curve of IL1β, calprotectin and SIRT1, respectively in saliva todiscriminate between individuals with CID patients and control subjects. D, E, F) Proteinexpression levels in saliva of CID patients and control individuals of IL1β, calprotectin and SIRT1, respectively. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and *P<0.05 and ***P<0.001 indicate the statistical significance vs controls. Figure 7. Increased protein expression of IL1β, calprotectin and SIRT1 in inflammatorybowel disease (IBD) patients. A, B, C) Area under the receiver operating characteristic (ROC)curve of IL1β, calprotectin and SIRT1, respectively in saliva to discriminate betweenindividuals with IBD patients and control subjects. D, E, F) Protein expression levels in salivaof IBD patients and control individuals of IL1β, calprotectin and SIRT1, respectively. Datashow median and the interquartile range of gene expression; each dot represents the value ofan individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and **P<0.01 and ***P<0.001 indicate the statistical significance vs controls. Figure 8. Comparison of salivary concentrations of biomarkers in saliva of inflammatory bowel disease (IBD) patients depending on the pharmacological treatment received.Graphs show protein levels of A) IL1β, B) calprotectin and C) SIRT1 levels in saliva of controlsubjects and IBD patients classified according to the pharmacological treatment received (CC: corticosteroids, biological and immunosuppressors). Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and *P<0.05, **P<0.01 and ***P<0.001 indicate the statistical significance vs controls. Figure 9. Analysis of IL1β, calprotectin and SIRT1 protein levels in Crohn disease (CD)and Ulcerative colitis (UC) patients. Graphs show protein levels of A) IL1β, B) calprotectinand C) SIRT1 levels in saliva of control subjects and patients with Crohn´s disease (CD) orUlcerative colitis (UC). Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and *P<0.05 and ***P<0.001 indicate the statistical significance vs controls and #P<0.05 vs CD. Figure 10. Protein expression of inflammatory biomarkers in rheumatoid arthritis-psoriasis (ARP) patients. Protein expression of A) IL1β, B) S100A9 calprotectin and C)SIRT-1 in saliva of ARP patients and control samples. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as foldinduction vs the mean of control samples. Mann-Whitney U test was used and **P < 0.01indicate the statistical significance vs controls. Figure 11. Correlation between the mRNA expression levels of IL1β and S100a9 calprotectin in saliva of IBD patients. A positive and significant correlation is detected between the mRNA expression of S100a9 calprotectin and IL1β (expressed as fold induction) in saliva of IBD patients. The Spearman’s correlation coefficient (R) and p value are also shown. Figure 12. Correlation between protein levels of IL1β and S100a9 calprotectin as well asIL1b and SIRT1 in saliva of IBD patients. A) A positive and significant correlation isdetected between the mRNA expression of S100a9 calprotectin and IL1β (expressed as foldinduction) in saliva of IBD patients. B) A positive and no significant correlation is detectedbetween the mRNA expression of SIRT1 and IL1β (expressed as fold induction) in saliva of IBD patients The Spearman’s correlation coefficient (R) and p value are also shown. Figure 13. Increased mRNA expression of inflammatory biomarkers in saliva of chronicinflammatory disease (CID) patients. A) mRNA expression of Il1β, B) mRNA expression ofs100a9 calprotectin. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples Mann-Whitney U test was used and *P <0.05 and ***P <0.001 indicate the statistical significance vs controls. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 14. Increased mRNA expression of inflammatory biomarkers in saliva of active vsremission CID patients. A) mRNA expression of Il1β and B) mRNA expression of s100a9calprotectin in saliva from both remission and active CID patients and healthy controls. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and *P<0.05, **P<0.01 and ***P <0.001 indicate the statistical significance vs controls while ##P<0.01 vs IBD patients in remission. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 15. Increased mRNA expression of inflammatory biomarkers in saliva ofinflammatory bowel disease (IBD) patients. A) mRNA expression of Il1β, B) mRNAexpression of s100a9 calprotectin in saliva of IBD patients and control samples. Data showmedian and the interquartile range of gene expression; each dot represents the value of anindividual expressed as fold induction vs the mean of control samples. Mann-Whitney U testwas used and **P<0.01 and ***P <0.001 indicate the statistical significance vs controls. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 16. Increased mRNA expression of inflammatory biomarkers in saliva of active vsremission IBD patients. A) mRNA expression of Il1β and B) mRNA expression of s100a9calprotectin in saliva from both remission and active IBD patients and healthy controls. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and *P<0.05, **P<0.01 and ***P <0.001 indicate the statistical significance vs controls while #P<0.05 vs IBD patients in remission. Table on the left express mean±SEM and table on the right express the 95% CI of the median. Figure 17. mRNA expression of inflammatory biomarkers in rheumatoid arthritis-psoriasis (RAP) patients. A) mRNA expression of Il1β, B) mRNA expression of s100a9calprotectin in saliva of RAP patients and control samples. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test was used and ***P <0.001 indicate the statistical significance vs controls. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 18. mRNA expression of inflammatory biomarkers in saliva of both, active andremission rheumatoid arthritis-psoriasis (RAP) patients. A) mRNA expression of Il1β andB) mRNA expression of s100a9 calprotectin in saliva from both remission and active RAPpatients and healthy controls. Data show median and the interquartile range of gene expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and ***P <0.001, indicate the statistical significance vs controls while ##P<0.01 vs IBD patients in remission. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 19. Increased protein expression of IL1β and calprotectin in chronic inflammatorydisease (CID) patients. A, B) Protein expression levels in saliva of CID patients and controlindividuals of IL1β and calprotectin, respectively. Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA wereused and ***P<0.001 indicate the statistical significance vs controls. C, D) Area under thereceiver operating characteristic (ROC) curve of IL1β and calprotectin, respectively in saliva to discriminate between individuals with CID patients and control subjects. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 20. Increased protein expression of inflammatory biomarkers in saliva of activevs remission CID patients. A) protein expression of IL1β and B) protein expressioncalprotectin in saliva from both remission and active CID patients and healthy controls. Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and ***P < 0.001 indicate the statistical significance vscontrols while #P<0.05, ##P<0.01 vs IBD patients in remission. C, D, E, F) Area under thereceiver operating characteristic (ROC) curve of IL1β and calprotectin, respectively in saliva to discriminate between individuals from both remission and active CID patients and control subjects. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 21. Increased protein expression of IL1β and calprotectin in inflammatory boweldisease (IBD) patients. A, B) Protein expression levels in saliva of IBD patients and controlindividuals of IL1β and calprotectin, respectively. Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA wereused and ***P<0.001 indicate the statistical significance vs controls. C, D) Area under thereceiver operating characteristic (ROC) curve of IL1β and calprotectin, respectively in saliva to discriminate between individuals with IBD patients and control subjects. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 22. Increased protein expression of inflammatory biomarkers in saliva of both,active and remission in inflammatory bowel disease (IBD) patients. A) Protein expressionof IL1β and B) protein expression of calprotectin in saliva from both remission and active IBDpatients and healthy controls. Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and ***P < 0.001, indicate the statistical significance vs controls while ##P<0.01 vs IBD patients in remission. C, D, E, F) Area under the receiver operating characteristic (ROC) curve of IL1β and calprotectin, respectively in saliva to discriminate between individuals from both remission and active IBD patients and control subjects. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 23. Comparison of salivary concentrations of biomarkers in saliva ofinflammatory bowel disease (IBD) patients depending on the pharmacological treatmentreceived. Graphs show protein levels of A) IL1β and B) calprotectin levels in saliva of controlsubjects and IBD patients classified according to the pharmacological treatment received (CC:corticosteroids, biological, mesalazine and immunosuppressors). Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one- way ANOVA were used and *P<0.05 and ***P<0.001 indicate the statistical significance vs controls, while #P<0.05 vs IBD patients with biological treatment. Table on the left express mean±SEM and table on the right express the 95% CI of the median. Figure 24. Analysis of IL1β and calprotectin protein levels in Crohn disease (CD) andUlcerative colitis (UC) patients. Graphs show protein levels of A) IL1β and B) calprotectinlevels in saliva of control subjects and patients with Crohn´s disease (CD) or Ulcerative colitis (UC). Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann- Whitney U test and one-way ANOVA were used and ***P<0.001 indicate the statisticalsignificance vs controls. Table on the left express mean±SEM and table on the right expressthe 95% CI of the median.Figure 25. Increased protein expression of IL1β and calprotectin in rheumatoid arthritis-psoriasis (RAP) patients. A, B) Protein expression levels in saliva of RAP patients and controlindividuals of IL1β and calprotectin, respectively. Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA wereused and ***P<0.001 indicate the statistical significance vs controls. C, D) Area under thereceiver operating characteristic (ROC) curve of IL1β and calprotectin, respectively in saliva to discriminate between individuals with RAP patients and control subjects. Table on the left express mean±SEM and table on the right express the 95% CI of the median.Figure 26. Increased protein expression of inflammatory biomarkers in saliva of both,active and remission in rheumatoid arthritis-psoriasis (RAP) patients. A) Proteinexpression of IL1β and B) protein expression of calprotectin in saliva from both remission andactive RAP patients and healthy controls. Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one-way ANOVA were used and***P < 0.001, indicate the statistical significance vs controls. Table on the left expressmean±SEM and table on the right express the 95% CI of the median. C, D, E, F) Area underthe receiver operating characteristic (ROC) curve of IL1β and calprotectin, respectively in saliva to discriminate between individuals from both remission and active RAP patients and control subjects.Figure 27. Comparison of salivary concentrations of biomarkers in saliva of rheumatoidarthritis-psoriasis (RAP) patients depending on the pharmacological treatment received.Graphs show protein levels of A) IL1β and B) calprotectin levels in saliva of control subjectsand RAP patients classified according to the pharmacological treatment received (CC:corticosteroids, biological, immunosuppressors and s / drugs). Data show median and the interquartile range of protein expression; each dot represents the value of an individual expressed as fold induction vs the mean of control samples. Mann-Whitney U test and one- way ANOVA were used and **P<0.01 and ***P<0.001 indicate the statistical significance vs controls, while #P<0.05 vs IBD patients with immunosuppressors treatment. Table on the left express mean±SEM and table on the right express the 95% CI of the median. Figure 28. Correlation between protein levels of IL1β and calprotectin in saliva of CID,IBD and RAP patients. A,B,C) A positive and significant correlation is detected between theprotein expression of calprotectin and IL1β in saliva of CID, IBD and RAP patients. TheSpearman’s correlation coefficient (R) and p value are also shown. Detailed description of the invention The present invention is illustrated by means of the Examples set below without the intention of limiting its scope of protection. DISCOVERY PHASE Example 1. Material and methods Example 1.1. PatientsA bulk saliva sample from all control and patients included in Table 2 was obtained. Controlpatients were healthy subjects who fail to suffer any inflammatory pathology. IBD patients were classified by healthcare professional as active (those who present >200µg / ml calprotectin in feces) or remission (those who present ˂200µg / ml calprotectin in feces). Rheumatoid arthritis and Psoriasis patients were classified between active or remission according with the symptoms they present inflammation joint deformity articular and psoriatic plaque. Theinformation on all the patients analysed in this study is summarized in Table 2. The study wasapproved by the Institutional Review Board of the University Clinical Hospital (Valencia, Spain). Written informed consent was obtained from all participating patients. Table 2 Controls Inflammatory BowelRheumatoid Psoriasis (P) p 1>I Clinical Remission=62 Remission=2 Remission=3 F Example 1.2. RNA isolation and Real-Time Quantitative PCR (RT-pPCR) Total RNA from saliva was isolated using direct RNAspin 96 isolation kit from Cytiva AmershamTMaccording to the manufacturer’s instructions. cDNA was obtained from previously isolated RNA by reverse transcription PCR using the High-capacity cDNA RT reagent Kit (appliedbiosystems, Baltics, UAB). Gene expression was analyzed by real-time Quantitative PCR using SYBR®Ex Taq (Takara Bio Inc., Saint-Germain-en-Laye, France) in LightCycler thermocycler (Roche Diagnostics, Mannheim, Germany). Specificoligonucleotides detailed in Table 3 were designed to perform the analysis. The relative geneexpression, as fold increase vs control, was expressed as follows: change in expression (fold) = 2 − Δ(ΔCT) where ΔCT = CT (target) − CT (housekeeping) and Δ(ΔCT) = ΔCT (treated) − ΔCT (control), where β-actin was the housekeeping gene used. Table 3 Fragment’s G′ ′ ′ ′Il s si 1 Example 1.3. Protein quantification and Western blot Analysis Protein was isolated from bulk saliva. Western Blot was performed to analyze proteinexpression. SDS-PAGE gels were used, and equal amounts of protein were loaded. Then,proteins were transferred to nitrocellulose membranes, which were further incubated withspecific primary antibodies (detailed in Table 4 as well as the secondary antibodies peroxidase-conjugated anti-rabbit IgG (Thermo Scientific, Waltham, MA, USA, 1:5000). Protein bands were detected with Immobilon®West Femto Western HRP Substrate (Thermofisher Scientific Vienna, Austria) in AMERSHAM ImageQuant 800 (GE lifescience, Cornellà de Llobregat, Spain). To normalize protein bands, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as housekeeping. The densitometry of the bands was quantified using the software ImageJ. Table 4 Antibody Supplier DilutionSGPrimary a alysis Example 1.4. ELISA Example 1.4.1 IL1βSecreted protein levels of IL-1β from bulk saliva were quantified by ELISA using the humanIL-1β high sensitivity ELISA KIT (Invitrogen, Thermofisher Scientific Vienna, Autria)following manufacturer’s instructions. Bulk saliva sample of IBD-RAP patients and healthy controls were diluted 1:50 with PBS, then biotin conjugate is added to all wells. Samples were incubated the dark overnight at room temperature in the precoated 96-well strip plate. After, streptavidin-HRP was added during 1 hour at 18-25 °C. Then amplification solution I was incubated for 15 min at 18-25 °C and that, amplification solution II was then added at 18-25 °C during 30 min. TMB Substrate Solution was then added at 18-25 °C during 10-20 min. Finally, Stop Solution was added, and absorbance was measured at 450 nm with the microplate reader SpectaMax Plus 384 (Molecular Devices, San Jose, CA, USA). Example 1.4.2. Calprotectin Secreted protein levels of calprotectin from bulk saliva were quantified by ELISA using the human calprotectin L1 / S100-A8 / A9 complex (Invitrogen, Thermofisher Scientific Vienna, Autria) following manufacturer’s instructions. Bulk saliva sample of IBD-RAP patients and healthy controls were diluted 1:20000 with PBS. Samples were incubated 2.5 hours at room temperature in the precoated 96-well strip plate. Then, biotin conjugate is added to all was then added a room temperature for 1 hour and that, streptavidin-HRP was added during 45 min at room temperature. TMB Substrate Solution was then added at room temperature during 30min. Finally, Stop Solution was added, and absorbance was measured at 450 nm with themicroplate reader SpectaMax Plus 384 (Molecular Devices, San Jose, CA, USA). Example 1.5. Statistical Analysis Data were represented as individual values and median ± interquartile range were analysed using GraphPad Prism V.8 San Diego, CA, USA and were compared by a t-test forcomparisons between two groups with Mann-Whitney U test post hoc correction was employedfor clinical data analysis by analysis of variance (one-way ANOVA) with Tukey post hoc correction where appropriate for multiple comparisons. Statistical significance was consideredwith a p-value < 0.05. Correlations from data obtained were analysed using Spearman’scorrelation coefficient. Example 2. Results Example 2.1. mRNA expression of inflammatory biomarkers in saliva Example 2.1.1. Chronic inflammatory disease (CID) patients Our study aim to analyse the mRNA expression of pro-inflammatory markers in saliva of control subjects and CID patients, which include inflammatory bowel diseases (IBD) and rheumatoid arthritis-psoriasis (RPA) patients. The analysis of the mRNA expression of Il1βand s100a9 calprotectin revealed a significant increase in the expression of these genes, in CIDcompared with levels detected in controls subjects (Fig.1A, B). As show in Fig. 1C, it isobserved a decrease in the expression of sirt1 in CID patients compared to controles subjects.Example 2.1.2. Inflammatory bowel diseases (IBD) patients Next, we classified patients according with the specific pathology and results reveal asignificant increase in the mRNA expression of both Il1β and s100a9 calprotectin (Fig.2A, B)in saliva from IBD patients compared with that of controls individuals. Different investigationsindicate that the sirt1 molecule is involved in different biological processes, including inflammation, and its main function is to block the NFκB factor. The results show that patients with IBD experience a decrease in SIRT1 mRNA expression levels compared with levels detected in controls subjects (Fig.2C). Example 2.1.3. Active vs remission Inflammatory bowel diseases (IBD) patients Next, we classified IBD patients in those that had active disease and those who were in clinical remission, according with the clinical criteria and results are shown in different colours,depending on the pharmacological treatment received by patients. As shown in Fig. 3, IBDpatients in remission exhibited Il1β mRNA levels non-significantly different to those detectedin control samples while levels of this cytokine in active IBD were significantly higher than both, those obtained in patients with clinical remission and those obtained in control subjects. In relation to the mRNA expression of s100a9 calprotectin, levels in IBD patients in remission were significantly lower than those in control patients while levels in active IBD weresignificantly higher than those in patients with clinical remission. In all cases data show thatthe drug does not modulate the expression of these molecules. Example 2.1.4. Rheumatoid arthritis-psoriasis (RPA) patients The reduced number of samples obtained from patients with arthritis or psoriasis led us to analyse samples from all of them together (RPA), and results show a non-significant increasein the mRNA expression of Il1β in patients compared with healthy controls (Fig.4A). Incontrast, the mRNA expression of s100a9 calprotectin and sirt1 was significantly decreased insaliva from RPA patients compared with that of control subjects (Fig.4B, C).Example 2.1.5. Active vs remission Rheumatoid arthritis-psoriasis (RPA) patientsAs shown in Fig 5A, the mRNA expression of Il1β did not significantly differ between controlsubjects and patients with ARP, irrespective of the activity or the remission of the disease. Incontrast, the mRNA expression of s100a9 calprotectin (Fig. 5B) was significantly reduced inboth remission and active ARP patients, compared with levels in control samples. Example 3. Protein expression of inflammatory biomarkers in saliva Example 3.1. Chronic inflammatory disease (CID) patients The analysed the protein levels of IL1β, calprotectin and SIRT1 in saliva samples. As show in results, protein expression of IL1β and calprotectin (Fig.6A, B) was significantly increase in CID patients than in control subject. In addition, the level of Sirt1 was significantly lower insamples of CID patients compared with levels detected in controls subjects (Fig. 6C).Subsequently, we analyse the ROC curve of these proteins in samples and results show proteinsexpression was analysed ROC curve, CID patients salivary IL1β (AUC-ROC = 0.8534) andcalprotectin (AUC-ROC = 0.8335) had the highest AU-ROC for distinguishing patients withIBD from control individuals (Fig. 6D, E). Supporting this result, we observed that the Sirt1 protein (AUC-ROC = 0.7462) (Fig.6F), which has an important exalt role in regulating inflammatory processes by inhibiting NFκB. Example 3.2. Inflammatory bowel disease (IBD) patients Next, we classified patients according with the pathology and results reveal an increase in protein expression of both, IL1β and calprotectin in saliva of IBD patients when compared withlevels in control samples (Fig. 7A, B). Unlike these, Sirt1 shows levels lower in IBD groupfrom control individuals (Fig. 7C).Subsequently, proteins expression was analysed ROC curve, IBD patients salivary IL1β (AUC-ROC = 0.8604) and calprotectin (AUC-ROC = 0.8316) had the highest AU-ROC fordistinguishing patients with IBD from control individuals (Fig.7D, E). Supporting this result,we have la sirt1 protein (AUC-ROC = 0.7716) (Fig.7F).Example 3.3. Protein expression of salivary biomarkers depending on the pharmacological treatment received by IBD patients IBD patients were classified according to the pharmacological treatment received (Fig.8) andresults show that patients receiving biological treatment exhibited a significant increase in theprotein expression of the cytokine IL1β and calprotectin, compared with control samples while non-significant differences were detected in the expression of Sirt1. In contrast, patients receiving corticosteroids exhibited protein levels of IL1β, calprotectin and Sirt1 that were non- significantly different to that detected in control subjects. Finally, in receiving immunosuppressors protein levels of IL1β and calprotectin were non-significantly different than those obtained in control samples while Sirt1 levels were significantly lower. Example 3.4. Protein expression of salivary biomarkers in Crohn´s disease and Ulcerative colitis Finally, we classified IBD patients according to the specific clinical entity, Crohn´s disease and Ulcerative Colitis. Results show a significant increase in protein levels of IL1β, and calprotectin while significant lower levels of Sirt1 in saliva of CD patients compared with control samples (Fig. 9A, B, C). We also detected a significant increase in the protein levels of both IL1β and calprotectin in samples from UC patients compared with control samples while a significant diminution of Sirt1 levels was observed in UC patients compared with both control and CD patients (Fig 9A, B, C). Example 3.4.1. Rheumatoid arthritis-psoriasis (RPA) patients Additionally, when analysed protein expression in ARP patients results show higher levels of IL1β in ARP than in control group (Fig.10A). However, the protein expression of both calprotectin and Sirt1, was not significant different between ARP patients and control subjects(Fig.10 B, C).Example 4. Correlation of inflammatory biomarkers in saliva of IBD patients Data show a positive and significant correlation, by the Spearman rank test, between the mRNA of IL1β and the mRNA expression of S100A9 calprotectin in saliva of IBD patients (r = 0.4061, P = ˂0,0001) (Fig.11). The analysis of the correlation between protein levels of IL1β and S100A9 calprotectin also reveal a positive and significant correlation (Fig. 12A). Finally, protein levels of IL1β and SIRT1 also exhibited a positive no significant correlation (Fig.12B). VALIDATION PHASE Example 5. Material and methods Example 5.1 PatientsA bulk saliva sample from all control and patients included in Table 5 was obtained. Controlpatients were healthy subjects who fail to suffer any inflammatory pathology. IBD patients were classified by healthcare professional as active (those who present >200µg / ml calprotectin in feces) or remission (those who present ˂200µg / ml calprotectin in feces). Rheumatoid arthritis and Psoriasis (RAP) patients were classified between active or remission according with the symptoms they present: inflammation joint deformity articular and psoriatic plaque.The information on all the patients analysed in this study is summarized in Table 5. The studywas approved by the Institutional Review Board of the Hospital Clínico Universitario (Valencia, Spain). Written informed consent was obtained from all participating patients. Table 5. Patients characteristics Controls Inflammatory Bowel Disease (IBD) Rheumatoid arthritis and Psoriasis (RAP)N Example 5.2. RNA isolation and Real-Time Quantitative PCR (RT-pPCR) Total RNA from saliva was isolated using direct RNAspin 96 isolation kit from Cytiva AmershamTMaccording to the manufacturer’s instructions. cDNA was obtained from previously isolated RNA by reverse transcription PCR using the High capacity cDNA RT reagent Kit (appliedbiosystems, Baltics, UAB). Gene expression was analyzed by real-timeQuantitative PCR using SYBR® Ex Taq (Takara Bio Inc., Saint-Germain-en-Laye, France) inLightCycler thermocycler (Roche Diagnostics, Mannheim, Germany). Specificoligonucleotides detailed in Table 6 were designed to perform the analysis. The relative geneexpression, as fold increase vs control, was expressed as follows: change in expression (fold) = 2 − Δ(ΔCT) where ΔCT = CT (target) − CT (housekeeping) and Δ(ΔCT) = ΔCT (treated) − ΔCT (control), where β-actin was the housekeeping gene used. Table 6. Sequences of human primers used in real-time PCR. Fragment’s G′ ′ ′ ′Ils1 Example 5.3. ELISA IL1βSecreted protein levels of IL-1β from bulk saliva were quantified by ELISA using the humanIL-1β high sensitivity ELISA KIT (Invitrogen, Thermofisher Scientific Vienna, Autria)following manufacturer’s instructions. Bulk saliva sample of IBD-RAP patients and healthycontrols were diluted 1:50 with PBS, then biotin conjugate is added to all wells. Samples wereincubated the dark overnight at room temperature in the precoated 96-well strip plate. After, streptavidin-HRP was added during 1 hour at 18-25 °C. Then amplification solution I was incubated for 15 min at 18-25 °C and that, amplification solution II was then added at 18-25 °C during 30 min. TMB Substrate Solution was then added at 18-25 °C during 10-20 min. Finally, Stop Solution was added, and absorbance was measured at 450 nm with the microplate reader SpectaMax Plus 384 (Molecular Devices, San Jose, CA, USA). Calprotectin Secreted protein levels of calprotectin from bulk saliva were quantified by ELISA using the human calprotectin L1 / S100-A8 / A9 complex (Invitrogen, Thermofisher Scientific Vienna, Autria) following manufacturer’s instructions. Bulk saliva sample of IBD-RAP patients and healthy controls were diluted 1:20000 with PBS. Samples were incubated 2.5 hours at room temperature in the precoated 96-well strip plate. Then, biotin conjugate was added to everything a room temperature for 1 hour and that, streptavidin-HRP was added during 45 min at room temperature. TMB Substrate Solution was then added at room temperature during 30 min. Finally, Stop Solution was added and absorbance was measured at 450 nm with the microplate reader SpectaMax Plus 384 (Molecular Devices, San Jose, CA, USA). Example 5.4. Statistical Analysis Data were represented as individual values and median ± interquartile range were analysed using GraphPad Prism V.8 San Diego, CA, USA and were compared by a t-test or Mann- Whitney U test when appropriate. Analysis of variance (one-way ANOVA) with Tukey post hoc correction was used for multiple comparisons. Statistical significance was considered witha p-value <0.05. Correlations from data obtained were analysed using Spearman’s correlationcoefficient.Example 6. ResultsExample 6.1. mRNA expression of inflammatory biomarkers in saliva ^Chronic inflammatory disease (CID) patientsOur study aim to analyse the mRNA expression of pro-inflammatory markers in saliva of control subjects and CID patients, which include inflammatory bowel diseases (IBD) and rheumatoid arthritis-psoriasis (RAP) patients. The analysis of the mRNA expression of Il1βand s100a9 calprotectin revealed a significant increase in the expression of these genes, in CIDcompared with levels detected in controls subjects (Figure 13A, B). ^Active vs remission Chronic Inflammatory diseases (CID) patientsNext, we classified CID patients in those that had active disease and those who were in clinical remission, according with the clinical criteria and results are shown in different colours, depending on the pharmacological treatment received by patients. As shown in Figure 14A,CID patients, both in remission and active exhibited Il1β mRNA levels significantly higherthan those detected in control samples. In turn, active CID patients exhibited higher Il1β mRNAlevels than those detected in patients with clinical remission. In relation to the mRNAexpression of s100a9 calprotectin (Figure 14B), levels in CID patients in remission exhibitedIl1β mRNA levels non-significantly different to those detected in control samples while levelsof this cytokine in active CID were significantly higher than those obtained in control subjects.^ Inflammatory bowel diseases (IBD) patientsNext, we classified patients according with the specific pathology and results reveal asignificant increase in the mRNA expression of both Il1β and s100a9 calprotectin (Figure 15A,B) in saliva from IBD patients compared with that of controls individuals. ^Active vs remission Inflammatory bowel diseases (IBD) patientsNext, IBD patients were classified in those that had active disease and those who were in clinical remission, according with the clinical criteria. Results are shown in different colours (Figure 16A), depending on the pharmacological treatment received by patients and reveal thatboth IBD patients in remission and active exhibited Il1β mRNA levels significantly higher thanthose detected in control samples. Furthermore, it was observed that patients with active IBDexhibited a significantly higher expression of Il1β than patients in remission. In relation to themRNA expression of s100a9 calprotectin (Figure 16B), levels in IBD patients weresignificantly higher than those in control patients, irrespective of the clinical condition of the patient. In all cases data show that the expression of these molecules does not seem to be differentially modulated by any of the pharmacological treatment received by patients. ^Rheumatoid arthritis-psoriasis (RAP) patientsThe reduced number of samples obtained from patients with rheumatoid arthritis or psoriasis led us to analyse all of them together (RAP), and results show a significant increase in themRNA expression of Il1β in patients compared with healthy controls (Figure 17A). In contrast,the mRNA expression of s100a9 calprotectin was not significantly altered in saliva from RAPpatients compared with that of control subjects (Figure 17B). ^Active vs remission Rheumatoid arthritis-psoriasis (RAP) patientsAs shown in Figure 18A, the mRNA expression of Il1β did not significantly differ betweencontrol subjects and patients with RAP in clinical remission, while levels of this cytokine in active RAP were significantly higher than both. In contrast, the mRNA expression of s100a9calprotectin (Figure 18B) was not significantly altered in both remission and active RAPpatients, compared with levels in control samples. Example 6.2. Protein expression of inflammatory biomarkers in saliva ^Chronic inflammatory disease (CID) patientsNext, we analysed the protein levels of IL1β and calprotectin in saliva samples. As shown inFigure 19A, B protein expression of both IL1β and calprotectin was significantly increased inCID patients compared with control subjects. Subsequently, we analyse the area under the ROC curve of these proteins in salivary samplesof control subjects and CID patients and results show for IL1β an AUC-ROC = 0.8800 and forcalprotectin an AUC-ROC = 0.8429 (Figure 19C, D).^ Active vs remission Chronic Inflammatory diseases (CID) patientsNext, we classified CID patients in those that had active disease and those who were in clinical remission, according with the clinical criteria and results are shown in different colours,depending on the pharmacological treatment received by patients. As shown in Figure 20A,CID patients in both clinical remission and active exhibited IL1β protein levels significantly higher than those detected in control samples. In relation to the protein expression ofcalprotectin levels (Figure 20B) in CID patients, both in remission and active exhibitedsignificantly higher levels than those in control patients. Besides, it was observed that patients with active CID exhibited a significantly higher expression of IL1β and calprotectin protein levels than patients in remission. In all cases, data show that the expression of these moleculesdoes not seem to be differentially modulated by any of the pharmacological treatment receivedby patients. Subsequently, we analyse the area under the ROC curve of these proteins insalivary samples and results show for IL1β an AUC-ROC = 0.7734 between control andremission (Figure 20C) and an AUC-ROC= 0.8036 between control and active CID (Figure20D). In relation to calprotectin results reveal an AUC-ROC = 0.8129 between control andremission (Figure 20E) and an AUC-ROC = 0.8696 between control and active CID (Figure20F). ^Inflammatory bowel disease (IBD) patientsNext, we classified patients according with the pathology and results reveal an increase in protein expression of both, IL1β and calprotectin in saliva of IBD patients when compared withlevels in control samples (Figure 21A, B). Subsequently, we analysed the area under the ROC curve, and data show for IL1β an AUC-ROC = 0.8702 and calprotectin (AUC-ROC = 0.8364) between control and IBD patients(Figure 21C, D).^ Active vs remission Inflammatory bowel diseases (IBD) patientsAs shown in Figure 22A, B, both IBD patients in remission and active exhibited IL1β protein levels and calprotectin levels significantly higher than those detected in control samples. In addition, it was observed that patients with active IBD exhibit a significantly higher expression of IL1β and calprotectin protein levels than those exhibited in IBD patients in remission. Subsequently, the area under the ROC curve was analysed and results for salivary IL1β showbetween IBD patients in remission and control subjects an AUC-ROC = 0.8604 and betweenactive IBD and control an AUC-ROC = 0.8811. Regarding salivary calprotectin in patientscontrol subjects vs remission exhibited an AUC-ROC = 0.8004 and active IBD vs control anAUC-ROC = 0.8769.^ Protein expression of salivary biomarkers depending on the pharmacologicaltreatment received by IBD patients IBD patients were classified according to the pharmacological treatment received (Figure 23A, B) and results show that irrespective of the pharmacological treatment received, patients exhibited a similar level of both IL1β and calprotectin with the only exemption that IBD patients receiving biological treatment showed significant higher levels of IL1B protein than patients receiving corticosteroid treatment. ^Protein expression of salivary biomarkers in Crohn´s disease and Ulcerative colitisFinally, we classified IBD patients according to the specific clinical entity, Crohn´s disease and Ulcerative Colitis. Results show a significant increase in protein levels of both IL1β, andcalprotectin in saliva of CD and UC patients compared with control samples (Figure 24A, B).^ Rheumatoid arthritis-psoriasis (RAP) patientsAdditionally, when we analysed protein expression in RAP patients results show higher levels of IL1β and calprotectin in RAP patients than in control group (Figure 25A, B). Subsequently, the area under the ROC curve was analysed in saliva from control and RAPpatients and results show for IL1β an AUC-ROC = 0.9144) and for calprotectin an AUC-ROC = 0.8732 (Figure 25C, D).^ Active vs remission Rheumatoid arthritis-psoriasis (RAP) patientsAs shown in Figure 26A, B both active and remission RAP patients exhibited significantlyhigher protein levels of Il1β and calprotectin than those detected in control samples. Subsequently, the area under the ROC curve for IL1β between remission RAP patients andcontrol subjects (Figure 26C) was AUC-ROC = 0.9005 and between active RAP patients andcontrol (Figure 26D) was AUC-ROC = 0.9420). Regarding salivary calprotectin between RAPpatients in remission and control subjects (Figure 26E) was AUC-ROC = 0.8382 and betweenactive RAP patients and controls (Figure 26F) was AUC-ROC = 0.9453.^ Protein expression of salivary biomarkers depending on the pharmacologicaltreatment received by RAP patients RAP patients were classified according to the pharmacological treatment received (Figure 27A,B) and results show that irrespective of the pharmacological treatment received, the protein levels of IL1β and calprotectin did not significantly differ. However, RAP patientsreceiving immunosuppressors treatment exhibited a significant difference in the expression ofthe IL1B protein compared to patients receiving biological treatment.Example 6.3 Correlation of inflammatory biomarkers in saliva of IBD patientsThe analysis of the correlation between protein levels of IL1β and calprotectin also reveal apositive and significant correlation (Figure 28A, B, C) in saliva of CID (r = 0.4584, P =˂0,0001), IBD (r = 0.4569, P = ˂0,0001) and RAP patients (r = 0.5330, P = ˂0,0001), respectively.

Claims

CLAIMS 1. In vitro method for identifying biomarker signatures for the diagnosis or screening of achronic inflammatory disease selected from the group comprising: inflammatory bowel disease (IBD), arthritis or psoriasis in a subject, which comprises assessing theexpression level of the protein combination IL1B and calprotectin, or of the genesencoding those proteins, in a saliva sample obtained from the subject.

2. In vitro method for the diagnosis or screening of a chronic inflammatory diseaseselected from the group comprising: IBD, arthritis or psoriasis in a subject, which comprises assessing the expression level of the protein combination IL1B andcalprotectin, or of the genes encoding those proteins, in a saliva sample obtained from the subject.

3. In vitro method for monitoring patients suffering from a chronic inflammatory diseaseselected from the group comprising: IBD, arthritis or psoriasis; and / or for differentiating active patients suffering from a chronic inflammatory disease selectedfrom the group comprising: IBD, arthritis or psoriasis from those patients who are in remission, which comprises assessing the expression level of the protein combinationIL1B and calprotectin, or of the genes encoding those proteins, in a saliva sample obtained from the subject.

4. In vitro use of the protein combination IL1B and calprotectin, or of the genes encodingthose proteins, or of a kit for assessing the level expression of the proteins or the genes, in a saliva sample obtained from the subject, for identifying biomarker signatures for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis.

5. In vitro use of the protein combination IL1B and calprotectin, or of the genes encodingthose proteins, or of a kit for assessing the level expression of the proteins or the genes, in a saliva sample obtained from the subject, for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis.

6. In vitro use of the protein combination IL1B and calprotectin, or of the genes encodingthose proteins, or of a kit for assessing the level expression of the proteins or the genes,in a saliva sample obtained from the subject, for monitoring patients suffering from achronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; and / or for differentiating active patients suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis from those patients who are in remission.

7. In vitro method, or in vitro use, according to any of the previous claims, wherein adeviation of the expression level of the proteins or of the genes encoding those proteins, as compared to a pre-established reference value, is an indication that the biomarker signature may be used for the diagnosis or screening of a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis; or that the subject is suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis8. In vitro method, or in vitro use, according to any of the previous claims, wherein anincrease of the protein level of IL1B and calprotectin as compared to a pre-establishedreference value, is an indication that the biomarker signature may be used for the diagnosis or screening of a chronic inflammatory disease selected from the groupcomprising: IBD, arthritis or psoriasis; or that the subject is suffering from a chronic inflammatory disease selected from the group comprising: IBD, arthritis or psoriasis.

9. In vitro method, or in vitro use, according to any of the previous claims, wherein anincrease of the gene expression level of IL1B and calprotectin as compared to a pre-established reference value, is an indication that the biomarker signature may be used for the diagnosis or screening of IBD; or that the subject is suffering from IBD.

10. In vitro method, or in vitro use, according to any of the previous claims, wherein anincrease of the gene expression level of IL1B and a decrease of the gene expressionlevel of calprotectin as compared to a pre-established reference value, is an indicationthat the biomarker signature may be used for the diagnosis or screening of arthritis and / or psoriasis; or that the subject is suffering from arthritis and / or psoriasis.

11. In vitro method, or in vitro use, according to any of the previous claims, wherein themethod comprises the use of unstimulated saliva as biological sample.

12. In vitro method, or in vitro use, according to any of the previous claims, wherein theIBD disease is selected from: Chron´s disease or ulcerative colitis.

13. Anti-inflammatory drugs selected from the group comprising:e) Salicylates;f) Immunosuppressants selected from azathioprine, methotrexate andmercaptopurine;g) Biological treatments selected from adalimumab, golimumab, infliximab,risankisumab, upadicitinib, ustekinumab and vedolizumab;h) Corticosteroids selected from: prednisone, cortisone, betamethasone, budesonideand beclomethasone;for use in a method for the treatment of a chronic inflammatory disease selected fromthe group comprising: IBD, preferably selected from Chron’s disease or ulcerative colitis, arthritis or psoriasis, wherein the method comprises diagnosing patients that suffer from the chronic inflammatory disease by using the method of claims 1 to 12.

Images