Gasdermins as biomarker of psoriasis and psoriasis- associated disease

Abstract

The present invention provides a method for the in vitro diagnosis of psoriasis and / or psoriasis-associated disease in an individual, which method comprises the steps wherein : (a) a level of a gasdermin in a biological sample from an individual is determined; and (b ) the level of the gasdermin in the test sample from the individual obtained in step (a) is compared with the level of the gasdermin in a reference sample, wherein the reference sample is a sample from a healthy control individual; wherein the level of the gasdermin higher than in the reference sample indicates that the individual suf fers from psoriasis and / or psoriasis- associated disease. The present invention provides also a gasdermin for use as a diagnostic biomarker for psoriasis, as well as preferably a diagnostic biomarker for psoriasis- associated disease. The present invention provides also a test kit for determining a level of a gasdermin in a biological sample from an individual and / or for diagnosing psoriasis, wherein the kit comprises means for determining the level of the gasdermin and optionally detection reagents and possibly instructions for carrying out the determination of the level of the gasdermin.

Description

[0001] Gasdermins as biomarker of psoriasis and psoriasis- associated disease

[0002] Field of invention

[0003] The present invention relates generally to the field of medicine , more speci fically to the diagnosis of autoimmune diseases , even more speci fically to the diagnosis of psoriasis and psoriasis-associated disease . The invention provides a method for the in vi tro diagnosis of psoriasis and psoriasis-associated disease and the use of a gasdermin as a biomarker for the diagnosis of psoriasis , and as a biomarker for the diagnosis of psoriasis-associated disease . The invention also provides a test kit for the diagnosis of psoriasis and / or psoriasis-associated disease .

[0004] Background art

[0005] Psoriasis is one of the most common dermatoses , af fecting around 125 million people worldwide , and the prevalence of psoriasis in the world population is estimated to be around 3% on average .

[0006] Psoriasis is a chronic and incurable skin disease , the pathogenesis of which is not yet fully understood . Psoriasis is undoubtedly not only a medical , but also a social and economic problem . Its hallmark is a chronically sustained inflammation that leads to uncontrolled proli feration of keratinocytes and their abnormal di f ferentiation, which clinically mani fests itsel f in characteristic skin lesions . The immunological abnormalities characteristic of psoriasis includes the involvement of dendritic cells , Thl 7 lymphocytes , TNF a and IL- 17 , 22 and 23 . Moreover, an important element is the involvement of protein complexes called inf lamasomes , which exert their action through transmission using IL- l p and IL- 18 , subsequently leading to increased secretion of IL- 17 and 23 . According to recent knowledge , the disease is perceived as a systemic disorder, closely associated with many other diseases .

[0007] To date , the co-morbidity of psoriasis (psoriatic comorbidities ) consisting of cardiometabolic disorders , j oint disease , bowel disease , kidney disease and psychiatric disorders has been demonstrated . Precisely because of this widespread comorbidity, patients with psoriasis live an average of five years less than those without the disease , especially due to cardiovascular incidents . Because of the increased risk of cardiovascular disease , patients require appropriate health-promoting education and special medical care , such as periodic simple screening tests for risk factors for cardiovascular complications . Quick and accurate diagnosis and the inclusion of appropriate treatment for psoriasis and psoriasis-associated diseases are crucial . Given the complications patients face , it is important to find markers that detect the disease early or allow diagnosis in doubtful cases . However, there are currently no sensitive and speci fic biomarkers that al low a minimally invasive diagnosis of psoriasis , especially at an early stage when there are few skin lesions or when the clinical picture is uncharacteristic .

[0008] Currently, psoriasis is diagnosed on the basis of a clinical examination after the characteristic morphology and location of the skin lesions are noted . I f the clinical picture is uncharacteristic, making it impossible to diagnose the disease based of the clinical picture , dermatologist may perform a dermatoscopic examination or biopsy of the lesion for histopathological examination . In such cases , it is necessary to take a section of skin for microscopic evaluation . So far, with regard to psoriasis, only the expression of gasdermin genes in tissues in this group of patients has been determined. However, this is invasive and very often not accepted by patients. As such, it is not suitable for routine use in screening tests.

[0009] The currently available diagnostic measures and methods are therefore insufficient. The search for new, more effective and minimally invasive diagnostic methods and biomarkers for this dermatosis and psoriasis-associated disease (its complications) is therefore justified. There is therefore a need for minimally invasive, specific and sensitive means for the in vitro diagnosis of psoriasis, and psoriasis-associated disease. There is, furthermore, a need to provide sensitive and specific tools, including biomarkers and diagnostic kits, for the minimally invasive, specific and sensitive diagnosis of psoriasis and psoriasis- associated diseases, allowing early diagnosis of the disease, as well as allowing diagnosis of the disease when the clinical picture is equivocal.

[0010] The objective of the invention is therefore to provide methods and means for the quick, minimally invasive, specific and sensitive diagnosis of psoriasis and psoriasis- associated diseases, especially at an early stage, such as specific and sensitive biomarkers and diagnostic methods and diagnostic kits using them.

[0011] Brief description of the invention

[0012] The above objectives have been achieved by the solutions claimed in the attached patent claims. Preferable variants of the invention are defined in the dependent claims . The present invention provides a method for the in vi tro diagnosis of psoriasis and / or psoriasis-associated disease in an individual , wherein the method comprises steps wherein :

[0013] ( a ) a level of a gasdermin in a biological sample from an individual is determined; and

[0014] (b ) the level of the gasdermin in the test sample from the individual obtained in step ( a ) is compared with the level of the gasdermin in a reference sample , wherein the reference sample is a sample from a healthy control individual ; wherein a level of gasdermin higher than in the reference sample indicates that the individual suf fers from psoriasis and / or psoriasis-associated disease .

[0015] Preferably, in the method according to the invention, at least one gasdermin selected from gasdermin A, gasdermin B, gasdermin C, gasdermin D and gasdermin E is determined as a gasdermin .

[0016] Preferably, in the method according to the invention, as the gasdermin, gasdermin A and / or gasdermin C is determined .

[0017] Preferably, in the method according to the invention, as the gasdermin, a pair of gasdermins selected from gasdermin A and B, gasdermin A and C, gasdermin A and D, gasdermin, A and E , gasdermin B and C, gasdermin B and D, gasdermin B and E , gasdermin C and D, gasdermin C and E , gasdermin D and E , is determined, more preferably a pair of gasdermin A and C .

[0018] Preferably, in the method according to the invention, as the gasdermin, gasdermins A to E are determined . Preferably, in the method according to the invention, the psoriasis-associated disease is overweight / obesity, hypertriglyceridemia, hyperglycemia and / or hyperuricemia . In a preferable embodiment , gasdermin E is determined in this case and the psoriasis-as sociated disease is overweight / obesity, hypertriglyceridemia, hyperglycaemia and / or hyperuricemia .

[0019] Preferably in the method according to the invention, the determination of the gasdermin is carried out in a sample of plasma or serum, preferably serum .

[0020] Preferably in the method according to the invention, the individual is a human being .

[0021] Preferably in the method according to the invention, the level of the gasdermin is determined by an ELISA method, more preferably by a sandwich ELISA.

[0022] The present invention provides furthermore a gasdermin for use as a diagnostic biomarker for psoriasis .

[0023] Preferably, as the gasdermin for use according to the invention at least one gasdermin selected from gasdermin A, gasdermin B, gasdermin C, gasdermin D and gasdermin E is used .

[0024] Preferably, as the gasdermin for use according to the invention gasdermin A and / or gasdermin C are used .

[0025] Preferably, as the gasdermin for use according to the invention a pair of gasdermin selected from gasdermin A and B, gasdermin A and C, gasdermin A and D, gasdermin, A and E , gasdermin B and C, gasdermin B and D, gasdermin B and E , gasdermin C and D, gasdermin C and D, gasdermin C and E , gasdermin D and E , more preferably gasdermin A and C, is used . Preferably, as the gasdermin for use according to the invention gasdermins A to E are used .

[0026] Preferably, the gasdermin for use according to the invention is also used as a diagnostic biomarker for psoriasis-associated disease .

[0027] Preferably, psoriasis-associated disease is selected from overweight / obesity ; diabetes ( or hyperglycaemia >100 mg / dl fasting) ; hypertriglyceridaemia and hyperuricaemia .

[0028] Preferably, as the gasdermin for use according to the invention gasdermin E is used and psoriasis-associated disease is overweight / obesity, hypertriglyceridemia, hyperglycaemia and / or hyperuricemia .

[0029] The present invention provides furthermore a test kit for determining a level of a gasdermin in a biological sample from an individual and / or for diagnosing psoriasis , which kit comprises means for determining the level of the gasdermin and optionally detection reagents and possibly instructions for carrying out the determination of the level of gasdermin .

[0030] Preferably, in the test kit according to the invention, the means for determining the gasdermin level are means for determining the gasdermin level by an ELISA method .

[0031] Preferably, in the test kit according to the invention, the means for determining the gasdermin level are means for determining the level of gasdermins A, B, C, D and / or E .

[0032] Detailed description of the invention

[0033] Gasdermins are a relatively newly identi fied family of proteins . There are six gasdermin types : A to F, of which the first five (A to E ) present a speci fic classical structure . They are involved in processes of cell proli feration, di f ferentiation, inflammation and death, especially the process of pyroptosis - caspase-dependent cell death, during which gasdermins produce pores in cell membranes . Such a process plays an important role in infections , neoplasms but , importantly, also in autoimmune disorders , which is undoubtedly what psoriasis is .

[0034] Serum concentrations of gasdermins A to E have never before been studied in this disease .

[0035] A biomarker, otherwise known as a biological marker, is a biological indicator that allows qualitative and / or quantitative assessment of various medical , pathological , disease conditions and / or biological phenomena or characteristics . In modern medicine , biomarkers play an invaluable role , allowing, among other things , a quick, precise , speci fic and sensitive diagnosis of various diseases or disorders . Biomarkers found in blood, e . g . in plasma or serum, allow such diagnostics to be carried out in a minimally invasive manner, requiring only a collection of a biological sample for examination, wherein such a sample is whole blood or plasma or serum isolated therefrom .

[0036] The present inventors have identi fied and developed a novel biomarker that allows speci fic and sensitive diagnosis of psoriasis , as well as psoriasis-associated disease , in a minimally invasive manner . This biomarker indicates a highly speci fic diagnostic association of gasdermins with psoriasis and psoriasis-associated diseases . In the context of the present invention, a psoriasi s-associated disease is understood, according to the literature , to be certain components of the metabolic syndrome . These components are strictly defined and include : type 2 diabetes mellitus , or fasting hyperglycaemia >100 mg / dL ; dyslipidaemia, i . e . fasting triglyceride levels >150 mg / dL (hypertriglyceridaemia ) , furthermore obesity / overweight , which is defined according to BMI values . These disorders have a two-way reciprocal ef fect with psoriasis - one disorder exacerbates the other .

[0037] The present inventors have shown that a gasdermin, such as gasdermin A, B, C, D and E , is a speci fic and sensitive biomarker for psoriasis , and it can also be used as a biomarker for psoriasis-associated disease . For example , gasdermin E , as it is negatively correlated with BMI and triglycerides , its reduced concentration here indicates hypertriglyceridaemia and overweight / obesity .

[0038] In the first aspect of the present invention, a method for in vi tro diagnosis of psoriasis in an individual is provided . In step a ) of the above-mentioned method, a level of a gasdermin in a biological sample from an individual is determined; and then in step b ) the level of the gasdermin in the test sample from the individual obtained in step a ) is compared with the level of the gasdermin in a reference sample , wherein the reference sample is a sample from a healthy control individual (without psoriasis and with a negative family history of psorias is ) ; wherein the level of the gasdermin higher than in the reference sample indicates that the individual suf fers from psoriasis .

[0039] In one embodiment , as a gasdermin at least one gasdermin selected from gasdermin A, gasdermin B, gasdermin C, gasdermin D and gasdermin E , in particular gasdermin A and / or C, is determined . This allows a quick, sensitive and speci fic diagnosis of psoriasis by the method according to the invention . In another embodiment , a combination of biomarkers according to the invention, i . e . more than one gasdermin selected from gasdermins A to E , is determined, which allows an additional increase in the ef ficiency of psoriasis diagnosis . In another embodiment , a pair of gasdermins selected from gasdermins A and B, gasdermins A and C, gasdermins A and D, gasdermins , A and E , gasdermins B and C, gasdermins B and D, gasdermins B and E , gasdermins C and D, gasdermins C and E , gasdermins D and E is determined, more preferably a pair of gasdermins A and C for which a particular increase in the sensitivity and speci ficity for the diagnosis of psoriasis according to the invention is obtained .

[0040] In another embodiment , gasdermins A to E are determined, which allows for even greater ef ficiency in diagnosing psoriasis by the method according to the invention .

[0041] The method according to the invention also allows in vi tro diagnosis of psoriasis-associated disease . Psoriasis- associated disease is selected from overweight / obesity, diabetes mellitus ( or fasting hyperglycaemia >100 mg / dL ) ; hypertriglyceridaemia, speci fically hypertriglyceridaemia ; hyperuricaemia . In the preferable embodiment , gasdermin E is determined, and the comorbidities are overweight / obesity, hypertriglyceridaemia, hyperglycaemia and hyperuricaemia .

[0042] In the first step ( a ) of the method according to the invention, a biological sample from a test individual (patient ) is used . Such a sample i s preferably a whole blood s amp 1 e .

[0043] Collection of venous blood from a patient is performed in a standard manner, such as during routine blood tests . The use of blood as a test sample has a number of advantages . For example , its collection is generally minimally invasive ( only a standard venous blood draw is required) , which is more convenient for the patient , as well as allowing early diagnosis of the disease (before the full characteristic clinical picture has developed) and use in screening tests for associated diseases . In addition, due to the small volume of the sample , it is easy to store and manipulate , which also reduces the risk of contamination of the test sample .

[0044] Plasma or serum is then isolated from the collected venous whole blood for testing . This can be conveniently accomplished in a standard manner, e . g . by allowing the whole blood sample to clot and then centri fuging it . Preferably, in step a ) of the method according to the invention, a serum sample ( serum biomarker ) is used . This allows quick, sensitive and speci fic diagnosis o f psoriasis by the method according to the invention .

[0045] In the second step (b ) of the method according to the invention, a comparison is carried out between the level of the gasdermin in the test sample and a reference level of the gasdermin, i . e . in a sample from a healthy control individual .

[0046] In a preferable embodiment , the individual is a human being .

[0047] In the method according to the invention, the level of the gasdermin in the biological sample can be determined by any measurement method known in the field that allows a quantitative evaluation of the level of a tested biomarker in a biological sample such as blood, especially serum .

[0048] In a preferable embodiment , the gasdermin level is determined by an ELISA method . Determination of gasdermin levels by the ELISA method is particularly advantageous as it facilitates and accelerates the diagnosis , especially of di f f icult-to-diagnose cases of psoriasis and its complications , providing a quick and simple measurement method suitable for use in everyday clinical practice , especially in diagnostic laboratories .

[0049] In a further aspect of the present invention, a gasdermin for use as a diagnostic biomarker for psoriasis is provided . In a preferable embodiment , the gasdermin for use according to the invention is selected from gasdermin A, gasdermin B, gasdermin C, gasdermin D and gasdermin E , in particular gasdermins A or C . The indicated gasdermins are particularly preferable biomarkers according to the invention, providing quick, sensitive and speci fic diagnosis of psoriasis and psoriasis-associated disease . In another embodiment , the gasdermin for use according to the invention is a pair of gasdermins selected from gasdermins A and B, gasdermins A and C, gasdermins A and D, gasdermins , A and E , gasdermins B and C, gasdermins B and D, gasdermins B and E , gasdermins C and D, gasdermins C and E , gasdermins D and E , more preferably a pair of gasdermins A and C which constitutes a particularly ef fective diagnostic biomarker for psoriasis according to the present invention . In another embodiment , the gasdermin for use according to the invention are gasdermins A to E , which provide an even more ef fective biomarker for use according to the invention .

[0050] In another embodiment , the gasdermin for use according to the invention is also used as a diagnostic biomarker for psoriasis-associated disease . In a preferable embodiment , psoriasis-associated disease is selected from overweight / obesity, diabetes mellitus ( or fasting hyperglycaemia >100 mg / dL ) ; hypertriglyceridaemia and hyperuricaemia . In a preferable embodiment , gasdermin E is used as the biomarker of psoriasi s-associated disease , and psoriasis-associated disease is overweight / obesity, hypertriglyceridaemia, hyper glycaemia and / or hyperuricaemia .

[0051] In a further aspect of the present invention, there is provided a test kit for determining a gasdermin level in a biological sample from an individual and / or for diagnosing psoriasis , which test kit comprises means for determining the gasdermin level and optionally detection reagents and possibly instructions for carrying out the determination of the gasdermin level .

[0052] In preferable embodiment , the kit according to the invention comprises means for determining the level of the gasdermin by an ELISA method . The determination of the level of the gasdermin by the ELISA method is particularly advantageous as it facilitates and accelerates the diagnosis , especially of di f f icult-to-diagnose cases of psoriasis and its complications , by providing a quick and simple method for measuring the level of the biomarker according to the invention . It is also possible to use other methods for this purpose that allow quantitative assessment of the level of the tested biomarker in a biological sample such as blood, especially serum .

[0053] As a result of the studies carried out by the present inventors on the evaluation of gasdermin levels in patients with psoriasis relative to healthy subj ects , signi ficantly higher serum gasdermin levels in patients with psoriasis relative to healthy subj ects were found . It was therefore shown that the level of the gasdermin in body fluids , such as blood, in particular serum, higher than the level of the gasdermin in a healthy person ( representing a reference level ) is a useful biomarker of psoriasis , as well as of psoriasis-associated diseases in light of the correlation between the level of the biomarker ( gasdermin) in blood, in particular serum ( serum concentration) and blood laboratory parameters indicative of the presence of psoriasis- associated diseases .

[0054] More speci fically, the study underlying the invention reported signi ficantly higher serum concentrations of gasdermin A, B, C and E in patients with psoriasis compared to the control group . A non-signi f icantly higher serum concentration of gasdermin D was also noted . Furthermore , negative correlations of gasdermin E with BMI , ALT activity, with triglyceride , glucose and uric acid concentrations ; of gasdermin B with erythrocyte count ; and of gasdermin C with erythrocyte count , haemoglobin and uric acid concentrations were observed . Given the negative correlation between gasdermin E and triglycerides , glycaemia and uric acid, this indicates that gasdermin E may have a protective function against metabolic disorders in psoriasis and its elevated serum levels may indicate the presence of such disorders , in this case speci fically overweight / obesity, hypertriglyceridaemia, hyperglycaemia and / or hyperuricaemia .

[0055] Given the proven role of gasdermins in cell proli feration and death, the elevated levels of gasdermins in patients with psoriasis compared to controls ( reference level in a healthy individual without psoriasis ) demonstrate the involvement of these proteins in the pathogenesis of psoriasis . Gasdermins may represent a new link to it , where the activation of inflamasomes and then caspases results in a cleavage of gasdermins , resulting in a release of pro- inflammatory cytokines and epidermal hyperproli feration . It is most likely that pyroptosis , in addition to inducing cell death, potentiates the inflammation which, chronically sustained, is a feature of the psoriatic epidermis . The results obtained according to the invention indicate that gasdermin represents a new, minimally invasive diagnostic biomarker for psoriasis . The method according to the invention allows a quick, simple , sensitive and specific diagnosis of this disease , as well as possibly of psoriasis-associated disease . In addition, gasdermin provides a new, minimally invasive diagnostic biomarker for psoriasis-associated disease , especially for gasdermin E in terms of metabolic disorders , especially overweight / obesity, hypertriglyceridemia, hyperglycaemia and hyperuricaemia , which is particularly important considering the high ris k of complications , especially cardiometabolic complications in patients with psoriasis , where early detection and prevention of such disorders is very important . The present invention also allows a screening test for psoriasis-associated diseases , improving patient care , allowing early detection of complications and treatment of the associated diseases .

[0056] The greatest advantage of the present invention is the low invasiveness of the method for the diagnosis . Indeed, the collection of a biological sample for examination by the method according to the invention only requires a collection of a blood sample , e . g . when drawing blood for routine screening test . The present invention can therefore be used on a large scale for the diagnosis of psoriasis and its complications , e . g . for screening tests identi fying a group of people at increased risk of disease or complications , and in particular for the diagnosis o f cases of psoriasis that are unclear for diagnosis .

[0057] More speci fically, the present invention allows :

[0058] • accurate diagnosis of psoriasis even at an early stage ;

[0059] • early inclusion of treatment and providing patients with special medical care ; • early inclusion of periodic screening tests for metabolic disorders in patients diagnosed with psoriasis at an early stage;

[0060] • minimally invasive collection of material for test;

[0061] • high availability of the material collected;

[0062] • simple and relatively inexpensive biomarker determination .

[0063] The present invention will now be illustrated in the following example and figures, which, however, are not intended to limit in any way the scope of the invention as defined in the patent claims.

[0064] Brief description of the figures

[0065] Fig. 1 shows the serum concentration of gasdermin A in patients with psoriasis compared to the control group. * denotes a statistically significant difference at p<0.05.

[0066] Fig. 2 shows the correlations between serum gasdermin A concentration and blood laboratory parameters (TGs, triglycerides; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GLU, fasting glucose; Choi, total cholesterol; HDL, high-density lipoprotein; LDL, low-density lipoprotein; RBC, erythrocytes; WBC, leukocytes; PLT, platelets; HGB, haemoglobin; UAC, uric acid; CR, creatinine; GRP, C-reactive protein) .

[0067] Fig. 3 shows serum level of gasdermin B in patients with psoriasis compared to the control group. * denotes a statistically significant difference at p<0.05.

[0068] Fig. 4 - correlations between serum gasdermin B concentration and laboratory parameters. Fig. 5 shows the serum concentration of gasdermin C in patients with psoriasis compared to controls. * denotes a statistically significant difference at p<0.001.

[0069] Fig. 6 shows the correlations between serum gasdermin C concentration and laboratory parameters.

[0070] Fig. 7 shows serum gasdermin D level in patients with psoriasis compared to controls.

[0071] Fig. 8 shows the correlations between serum gasdermin D concentration and laboratory parameters.

[0072] Fig. 9 shows the serum concentration of gasdermin E in patients with psoriasis compared to controls. * denotes a statistically significant difference at p<0.05.

[0073] Fig. 10 shows the correlations between serum gasdermin E concentration and laboratory parameters.

[0074] Example 1 - study of the effectiveness of psoriasis diagnosis using the psoriasis biomarker according to the invention

[0075] All the procedures, tests and experimental analyses described below were carried out using commercially available test kits, reagents and apparatus, following the recommendations of the manufacturers of the kits, reagents and apparatus used, unless expressly indicated otherwise here, and using standard, well-known methods used in the field to which the present invention belongs.

[0076] The study included 60 patients with plaque psoriasis and 30 volunteers without skin disease and with a negative family history of psoriasis.

[0077] A biological sample for the study was collected from all the study participants in the standard way, with the sample being whole venous blood. The fasting blood sample was collected and allowed to clot for 30 minutes . The sample was then centri fuged to obtain serum ( 10 min, 2000 g) . Subsequently, the determination of gasdermin A, B, C, D and E levels in the obtained serum from all the study participants was carried out by the ELISA method .

[0078] Determination of gasdermin A, B, C, D and E proteins was performed by immunoenzymatic ELISA method, using a commercially available kit for humans :

[0079] Human gasdermin-A ELISA Kit (EIAABSCIENCE INC,

[0080] Wuhan) , No . E11790h

[0081] • Human gasdermin-B ELISA Kit (EIAABSCIENCE INC, Wuhan) , No . E15865h

[0082] • Human gasdermin-C ELISA Kit (EIAABSCIENCE INC, Wuhan) , No . E1646h

[0083] • Human gasdermin-D ELISA Kit (EIAABSCIENCE INC, Wuhan) , No . E15480h

[0084] • Human gasdermin-E ELISA Kit (EIAABSCIENCE INC,

[0085] Wuhan) , No . E2649h

[0086] The principle of measurement was based on the sandwich reaction - in the first step, a speci fic antibody coated on a solid phase (plate ) reacted with the antigen present in the test serum . In the next step, a reaction took place between the bound antigen and the enzyme-conj ugated speci fic antibody, which was added after washing away the unbound substances . The addition of the chromogenic substrate led to a colorimetric reaction, after which the absorbance was measured spectrophotometrically . The absorbance reading was plotted on a standard curve , which was a function of the known concentrations of the antigen under test and their corresponding absorbances . The standard ( Stock solution) was dissolved in 1 mL Sample Diluent (Gasdermin A, B, C, D) or in 2 mL in the case of Gasdermin E. The test samples were not diluted. a) gasdermins A, B, D, E

[0087] In the initial step, 100 pL of standards and test samples, respectively, were added to each well of a plate coated with a monoclonal antibody directed against gasdermin A, B, D and E and incubated for 120 minutes at 37°C. Then 100 pL of Detection Reagent A was added and incubated for 60 minutes at 37°C. In the next step, the plate was rinsed three times and then 100 pL Detection Reagent B was added to each well and incubated again for 60 minutes at 37°C. The plate washing steps were carried out using an Elx-50 automated microplate washer (BioTEK®) . After the incubation period, the plate was washed five times. In the next step, 90 pL of Substrate was added to each well and a 20-minute incubation was carried out at 37°C, protecting the plate from light. The enzymatic reaction was stopped by adding 50 pL of STOP Solution. The activity of gasdermin A, B, D and E was measured photometrically at 450 nm wavelength using a Multiskan FC microplate reader (ThermoScientif ic®) . Standard sera having the following concentrations were used to prepare the standard curve:

[0088] • Gasdermin A: 10; 5.0; 2.5; 0.625; 0.312; 0

[0089] • Gasdermin B: 20; 10; 5; 1.25; 0.312; 0

[0090] • Gasdermin D: 20;10; 5; 1,25; 0,156; 0

[0091] • Gasdermin E: 10; 5; 2.5; 0.625; 0.312; 0.

[0092] Gasdermin A, B, D and E concentrations were expressed in units of ng / mL. b) Gasdermin C

[0093] In the initial step, 50 pL of standards and test samples, respectively, were added to each well of a plate coated with a monoclonal antibody directed against gasdermin C, followed by the addition of 50 pL of Detection Reagent A and incubated for 60 minutes at 37 °C. In the next step, the plate was rinsed three times, then 100 pL Detection Reagent B was added to each well and incubated again for 45 minutes at 37°C. The plate washing steps were carried out using an Elx-50 automated microplate washer (BioTEK®) . After the incubation period, the plate was washed five times. In the next step, 90 pL of substrate was added to each well and a 20-minute incubation was carried out at 37°C, protecting the plate from light. The enzymatic reaction was stopped by adding 50 pL of STOP Solution. The activity of gasdermin C was measured photometrically at 450 nm wavelength using a Multiskan FC microplate reader (ThermoScientif ic®) . Standard sera with the following concentrations were used to prepare the standard curve:

[0094] Gasdermin C: 40; 20; 10; 5; 1.25; 0

[0095] The concentration of Gasdermin C was expressed in units of ng / mL.

[0096] In addition, baseline laboratory tests of blood parameters were performed, including determination of triglycerides, alanine aminotransferase, aspartate aminotransferase, fasting blood glucose, total cholesterol high-density lipoprotein (HDL) , low-density lipoprotein (LDL) , erythrocytes, leukocytes (WBC) , platelets (PLT) , haemoglobin (HGB) , uric acid (UAC) , creatinine (CR) , C- reactive protein (GRP) . In addition, an assessment of the severity of skin lesions using the Psoriasis Activity and Severity Index (PAST) was performed.

[0097] Statistical analysis of the results obtained was carried out using GraphPad Prism 9.4 software.

[0098] Table 1 shows the basic parameters of the study group (age, sex, height, weight, BMI) .

[0099] Table 1.

[0100] The study reported significantly higher serum levels of gasdermin A, B, C and E in patients with psoriasis compared to controls, and non-signif icantly higher levels of gasdermin D.

[0101] The sensitivity and specificity of the determination of the levels of individual gasdermins by the method according to the invention are shown in Table 2 below. Table 2.

[0102] The results obtained for the levels of the tested gasdermins A, B, C, D and E in the sera of the study participants are shown in Figures 1, 3, 5, 7 and 9, respectively. These results clearly indicate that there are significant differences in the levels of the tested gasdermin in samples from patients with psoriasis (especially gasdermin A, B, C, D) relative to the levels in samples from healthy subjects without psoriasis (reference level) . This means that the determination of gasdermin levels according to the present invention allows an effective, sensitive, specific and minimally invasive diagnosis of psoriasis.

[0103] In addition, negative correlations of gasdermin E with BMI, ALT activity, triglyceride, glucose and uric acid concentrations; gasdermin B with erythrocyte count; and gasdermin C with erythrocyte count, haemoglobin and uric acid concentrations were observed. The correlation results obtained are shown in Figures 2, 4, 6, 8 and 10, respectively. Given the negative correlation between gasdermin E and triglycerides, glycaemia and uric acid, this indicates that gasdermin E may have a protective function against metabolic disorders in psoriasis and its elevated serum levels may indicate the presence of such disorders, in this case specifically obesity / overweight . hypertriglyceridaemia, hyperglycaemia and hyperuricaemia .

[0104] This also means that gasdermins are suitable for use as effective, sensitive, specific and minimally invasive biomarkers for the diagnosis of psoriasis-associated disease .

[0105] Example 2 - study of the effectiveness of psoriasis diagnosis using a combination of psoriasis biomarkers according to the invention

[0106] An analysis of the effecti eness of the use of the biomarker according to the invention in combinations of biomarkers according to the invention was also carried out. More specifically, the aforementioned study was carried out using regression analysis of representative examples of such combinations in the form of pairs of gasdermins A to E: gasdermins A and B, gasdermins A and C, gasdermins A and D, gasdermins, A and E, gasdermins B and C, gasdermins B and D, gasdermins B and E, gasdermins C and D, gasdermins C and E, gasdermins D and E.

[0107] The regression analysis of the gasdermin A and C pairs resulted in an R2 value (Cox & Snell) of 24%. This is the highest value of all pairs of proteins tested indicating the best fit of all models based on combinations of pairs of proteins. In this pair, both variables are statistically significant. This is the only pair for which both variables are statistically significant. The area under the ROC curve is 0.791. This is the best fit of all pairs indicating the highest quality of classification based on pair A and C among all protein pairs analysed.

[0108] Summary of results for the pair of gasdermins A and C:

[0109] AUG 0.791 Sensitivity for the A+C pair = 81.67%

[0110] Specificity for the A+C pair= 56.67%.

[0111] For the other gasdermin pairs tested, no such statistically significant results were obtained, most likely due to too few samples for the study. More precisely, for all other pairs the area under the ROC curve is higher than 0.5 representing the hypothesised random model. It can therefore be concluded that all of the analysed logistic models based on individual protein pairs have some discriminatory power.

[0112] Summary of results for other combinations (p>0.05) :

[0113] GASDERMINS A and B

[0114] AUG 0.766

[0115] Sensitivity for the A+C pair 86.67%

[0116] Specificity for the A+C pair 33.33%

[0117] GASDERMINS A and D

[0118] AUG 0.752

[0119] Sensitivity for the A+D pair = 85%

[0120] Specificity for the A+D pair = 33.33

[0121] GASDERMINS A and E

[0122] AUG 0.756

[0123] Sensitivity for the A+E pair = 88.33

[0124] Specificity for the A+E pair = 36.67 GASDERMINS B and C

[0125] AUG 0.746

[0126] Sensitivity for the B+C pair = 88.33%

[0127] Specificity for the B+C pair = 36.67%

[0128] GASDERMINS B and D

[0129] AUG 0.652

[0130] Sensitivity for the B+D pair = 100%

[0131] Specificity for the B+D pair = 0%.

[0132] GASDERMINS B and E

[0133] AUG 0.641

[0134] Sensitivity for the B+E pair = 100%

[0135] Specificity for the B+E pair = 0%.

[0136] GASDERMINS C and D

[0137] AUG 0.738

[0138] Sensitivity for the C+D pair = 88.33%

[0139] Specificity for the C+D pair = 40%

[0140] GASDERMINS C and E

[0141] AUG 0.748

[0142] Sensitivity for the C+E pair =86.67%

[0143] Specificity for the C+E pair = 40% GASDERMINS D and E

[0144] AUG 0.567

[0145] Sensitivity for the D+E pair = 100%

[0146] Specificity for the D+E pair = 0%.

Claims

Patent claims1 . A method for the in vi tro diagnosis of psoriasis and / or psoriasis-associated disease in an individual , which method comprises the steps wherein :( a ) a level of a gasdermin in a biological sample from an individual is determined; and(b ) the level of the gasdermin in the test sample from the individual obtained in step ( a ) is compared with the level of the gasdermin in a reference sample , wherein the reference sample is a sample from a healthy control individual ; wherein the level of the gasdermin higher than in the reference sample indicates that the individual suf fers from psoriasis and / or psoriasis-associated disease .2 . The method according to claim 1 , wherein as the gasdermin, at least one gasdermin selected from gasdermin A, gasdermin B, gasdermin C, gasdermin D and gasdermin E is determined .3 . The method according to claim 1 or 2 , wherein as the gasdermin, gasdermin A and / or gasdermin C is determined .4 . The method according to claim 1 or 2 , wherein as the gasdermin, a pair of gasdermins selected from gasdermin A and B, gasdermin A and C, gasdermin A and D, gasdermin, A and E , gasdermin B and C, gasdermin B and D, gasdermin B and E , gasdermin C and D, gasdermin C and E , gasdermin D and E is determined, preferably gasdermin A and C .5 . The method according to claim 1 or 2 , wherein as the gasdermin, gasdermins A to E are determined .6 . The method according to claim 1 , wherein psoriasis- associated disease is overweight / obesity,hypertriglyceridemia, hyperglycemia and / or hyperuricemia, wherein gasdermin E is preferably determined and psoriasis- associated disease is overweight / obesity, hypertriglyceridemia, hyperglycemia and / or hyperuricemia .7 . The method according to any one of claims 1 to 6 , wherein the determination is carried out in a plasma or serum sample , preferably serum sample .8 . The method according to any one of claims 1 to 7 , wherein the individual is a human being .9 . The method according to any one of claims 1 to 8 , wherein the gasdermin level is determined by an ELISA method, preferably a sandwich ELISA.10 . A gasdermin for use as a diagnostic biomarker for psoriasis .11 . The gasdermin for use according to claim 9 , wherein at least one gasdermin selected from A, gasdermin B, gasdermin C, gasdermin D and gasdermin E is used as the gasdermin .12 . The gasdermin for use according to claim 10 or 11 , wherein gasdermin A and / or gasdermin C is used as the gasdermin .13 . The gasdermin for use according to claim 10 or 11 , wherein as the gasdermin, a pair of gasdermins selected from gasdermin A and B, gasdermin A and C, gasdermin A and D, gasdermin, A and E , gasdermin B and C, gasdermin B and D, gasdermin B and E , gasdermin C and D, gasdermin C and E , gasdermin D and E , gasdermin D and E is used, preferably gasdermin A and C .14 . The gasdermin for use according to claim 10 or 11 , wherein as the gasdermin, gasdermins A to E are used .15 . The gasdermin for use according to any one of claims 10 to 14 , wherein the diagnostic biomarker for psoriasis is also a diagnostic biomarker for psoriasis- associated disease .16 . The gasdermin for use according to claim 15 , wherein psoriasis-associated disease is selected from overweight / obesity, diabetes mellitus ( or hyperglycaemia >100 mg / dL fasting) ; hypertriglyceridaemia ; hyperuricaemia .17 . The gasdermin for use according to claim 15 or 16 , wherein the gasdermin is gasdermin E and associated disease is overweight / obesity, hypertriglyceridemia, hyperglycemia and / or hyperuricemia .18 . A test kit for the determination of a level of gasdermin in a biological sample from an individual and / or for the diagnosis of psoriasis , which kit comprises means for determining a gasdermin level and optionally detection reagents and possibly instructions for carrying out the determination of gasdermin levels .19 . The kit according to claim 18 , wherein the means for determining the level of the gasdermin is a means for determining the level of the gasdermin by an ELISA method .20 . The kit according to claim 18 or 19 , wherein the means for determining the level o f the gasdermin are means for determining the level of gasdermin A, B, C, D, and / or E .

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