Biomarkers for the progression of acute chronic liver failure (ACLF)

Abstract

The present invention relates to the field of liver disease, and more particularly to a method for determining the presence of acute chronic liver failure, or a method for determining the risk of developing ACLF in patients with cirrhosis or late-onset pre-ACLF. The present invention also relates to a method for distinguishing patients with compensated cirrhosis, acute decompensated cirrhosis (ADC), or late-onset ACLF from patients with early ACLF or ACLF 1, 2, or 3, a method for determining the degree of systemic inflammation, and a method for determining the presence of a bacterial infection with sepsis.

Description

Technical Field

[0001] The present invention relates to the field of liver diseases, and more particularly to a method for determining the presence of acute and chronic liver failure, or a method for determining the risk of developing acute-on-chronic liver failure (ACLF) in patients suffering from cirrhosis or pre-ACLF. The present invention also relates to a method for discriminating patients suffering from compensated cirrhosis, acute decompensated cirrhosis (ADC) or late ACLF from patients suffering from early ACLF or ACLF 1, 2, 3, a method for determining the degree of systemic inflammation, and a method for determining the presence of a bacterial infection associated with sepsis.

Background Art

[0002] The appearance of liver failure in cirrhotic patients represents a critical point in both medical management and prognosis, as this condition is often associated with rapidly progressing multiple organ failure. Among liver diseases, cirrhosis is clearly the most frequent cause of hospitalization or liver transplantation. Due to the large imbalance between supply and potential recipients, liver transplantation worldwide can only be received by 20% of advanced cirrhotic patients.

[0003] Cirrhosis progresses over several years. Initially compensated, the disease has no functional symptoms or complications. The occurrence of complications represents the so-called transition to decompensation, in which complications such as ascites, gastrointestinal bleeding, renal failure, bacterial infection or hepatic encephalopathy appear.

[0004] Acute decompensated cirrhosis (ADC) is a syndrome characterized by the development of ascites, encephalopathy, and / or gastrointestinal bleeding secondary to portal hypertension and liver failure, and its pathophysiology is in need of re-examination through recent large-scale observational studies combined with omics techniques. A new syndrome, acute chronic liver failure (ACLF) characterized by single or multiple organ failure and a high 28-day mortality risk, other phenotypes in ADC patients without ACLF, the identification of systemic inflammation as a central pathophysiological mechanism, and findings in Latin American patients where the severity of systemic inflammation is influenced by genetic lineage, move ADC into a new scenario requiring further investigation.

[0005] While the precise pathophysiology of ACLF development is still being elucidated, uncontrolled inflammation is considered a major contributing factor. Unique characteristics of ACLF include its rapid progression, the need for multi-organ support, and a high short- and medium-term mortality rate of 50–90%. Unfortunately, available treatment options for ACLF are limited. The only curative treatment for ACLF patients remains liver transplantation, but as mentioned above, the limited availability of donor organs restricts its usefulness in treating ACLF patients.

[0006] Theoretically, it would seem to directly use the currently used practical definition of ACLF, but medical practice is frequently hampered by the complex clinical picture, the unclear distinction from patients with end-stage hepatitis, and problems in determining the ideal timing and / or necessity or usefulness of specific therapeutic interventions. In terms of prognosis, ACLF also presents problems because it combines both potentially life-threatening acute trauma and severe chronic underlying disease.

[0007] The few prognostic models available for determining whether to pursue conventional treatment or liver transplantation in ACLF are generally far from satisfactory. The Model of End-Stage Liver Disease (MELD) is a scoring system for assessing the severity of chronic liver disease that is useful for prognosis assessment and prioritizing liver transplantation. However, the MELD score has several limitations, including inter-laboratory variability in creatinine measurements and the internationally normalized ratio of prothrombin time, as well as a sex bias that is dominance of women.

[0008] ACLF patients are stratified into three severity stages (ACLF-1, ACLF-2, ACLF-3) according to the number of organ failures (1, 2, 3, or more), which correlates with the severity of systemic inflammation. Systemic inflammation, estimated by C-reactive protein (CRP), cytokines, or white blood cell count (WBC), also correlates with the patient's clinical course. Among hospitalized ADC patients without ACLF, who show lower systemic inflammation than ACLF patients, there is a group known as pre-ACLF, so named because they develop ACLF during hospitalization ("early" pre-ACLF) or later during the 90-day follow-up period ("late" pre-ACLF). The remaining ADC patients are stratified into "unstable" or "stable" decompensated cirrhosis depending on whether they require further hospitalization during the 90-day follow-up. Patients with unstable cirrhosis exhibit systemic inflammation of a similar grade to those with stable decompensated cirrhosis, but have a higher prevalence of events associated with severe portal hypertension and a lower one-year survival rate.

[0009] Current research in patients with ADC and ACLF is limited by the low precision of standard inflammatory biomarkers in cirrhosis. Well-specified predictive models are needed to establish optimal assessment and management of ACLF, as well as to more accurately distinguish between high and low severity ACLF phenotypes. [Overview of the project] [Problems that the invention aims to solve]

[0010] The authors of this invention have developed a novel scoring system that can more accurately distinguish between high-severity and low-severity ADC phenotypes than current biomarkers, based on genes related to innate immunity and B cells, and have identified new important aspects of the pathophysiology. In particular, the authors of this invention have found that patients with compensated cirrhosis (phenotype 1), ADC (phenotype 2), or late-onset ACLF (phenotype 3) can be distinguished from patients with early ACLF (phenotype 4) or ACLF 1, 2, and 3 (phenotypes 5, 6, and 7) based on the expression levels of different marker genes. They also showed that the levels of marker genes change as ACLF progresses.

[0011] The authors of this invention have also found that the same scoring system can be used to determine the degree of systemic inflammation in a subject and to determine the presence of a bacterial infection with sepsis in a subject. [Means for solving the problem]

[0012] Therefore, in a first aspect, the present invention relates to an in vitro method for determining the presence of acute chronic liver failure (ACLF) in subjects suffering from cirrhosis or late-onset pre-ACLF, wherein the method is a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, The fact that the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are greater than the reference values ​​for these genes indicates that the subject has ACLF.

[0013] In another aspect, the present invention relates to an in vitro method for determining the risk of developing ACLF in subjects suffering from cirrhosis or late-onset pre-ACLF, the method being: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, The fact that the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are higher than the reference values ​​for these genes indicates that the subject is at high risk of developing ACLF.

[0014] In another aspect, the present invention relates to an in vitro method for monitoring the progression of ACLF in a subject, the method being: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject. b) Comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point after the onset of ACLF, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes are lower than the aforementioned reference values, it indicates ACLF resolution.

[0015] In another aspect, the present invention relates to an in vitro method for monitoring the effects of a therapy in a subject suffering from ACLF and being treated with the said therapy, the method being a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time, If the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are lower than the reference values, it indicates that the therapy is effective.

[0016] In another aspect, the present invention relates to an in vitro method for determining the degree of systemic inflammation in a subject, the method being: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, The fact that the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are higher than the reference values ​​for these genes indicates that the subject has severe systemic inflammation.

[0017] In another aspect, the present invention relates to an in vitro method for determining the presence of a bacterial infection with sepsis in a subject, wherein the method is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, The fact that the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are higher than the reference values ​​for these genes indicates that the subject has a bacterial infection accompanied by sepsis.

[0018] In another aspect, the present invention relates to a kit or assay device comprising reagents suitable for determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes, hereinafter referred to as the first kit or assay device of the present invention.

[0019] In another aspect, the present invention relates to the use of reagents specific for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, or a first kit of the present invention, for determining the presence of ACLF in a subject, determining the risk of developing ACLF, monitoring the progression of ACLF, monitoring the effectiveness of therapy, selecting patients with ACLF for therapy, or determining the degree of systemic inflammation.

[0020] In another aspect, the present invention relates to a kit or assay device comprising reagents suitable for determining the expression levels of at least the HMGB2, RETN, ZNF608, and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28, hereinafter the second kit or assay device of the present invention.

[0021] In another aspect, the present invention relates to the determination of the expression levels of at least the HMGB2, RETN, ZNF608, and PYCARD genes, and to reagents specific for the determination of the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28, or of the second kit of the present invention, for determining the presence of ACLF in a subject, for determining the risk of developing ACLF, for monitoring the progression of ACLF, for monitoring the efficacy of a therapy, for selecting a patient suffering from ACLF for therapy, or for determining the degree of systemic inflammation.

[0022] In another aspect, the present invention relates to a method of treating a patient by determining the presence of acute-on-chronic liver failure (ACLF) in a subject suffering from cirrhosis or late pre-ACLF, the method comprising a) determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the patient, and b) comparing the expression levels of the genes with the respective reference values for each gene, where the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the patient are each greater than their respective reference values, the patient is treated with an appropriate therapy including antibiotics, diuretics, albumin, lactulose, β - adrenergic blockers, vasoconstrictors, sclerosing agents, intrahepatic portosystemic shunts, plasma exchange, mechanical ventilation support, renal hemodialysis, albumin dialysis, liver transplantation, and / or combinations thereof.

[0023] In another aspect, the present invention relates to a method of determining the risk of developing ACLF in a subject suffering from cirrhosis or late - onset pre - ACLF and treating the patient, the method comprising: a) determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject, and b) comparing the expression levels of the genes in the sample with their reference values, where the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject are greater than the reference values for the genes, the patient is treated with a therapy suitable for the prevention of ACLF, including antibiotics, diuretics, albumin, lactulose, β - adrenergic blockers, terlipressin, sclerosing agents, intrahepatic portosystemic shunts, and / or combinations thereof.

[0024] In another aspect, the present invention relates to a method of determining the degree of systemic inflammation in a subject and treating the subject, the method comprising: a) determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject, and b) comparing the expression levels of the genes in the sample with their reference values, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are greater than the reference values ​​for those genes, the patient is treated with a therapy appropriate for severe systemic inflammation, including antibiotics, corticosteroids, albumin, plasmapheresis, and / or a combination thereof.

[0025] In another aspect, the present invention relates to a method for determining the presence of a bacterial infection accompanied by sepsis in a subject and for treating the subject, wherein the method is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are greater than the reference values ​​for those genes, the patient will be treated with appropriate therapy for bacterial infection and / or sepsis, including antibiotics, vasoconstrictors, and organ support including renal hemodialysis and / or mechanical ventilation. [Brief explanation of the drawing]

[0026] [Figure 1A] The relative contribution of each of the 28 selected genes to the CLIF-SIG score. [Figure 1B] Comparison of CLIF-SIG scores at T1 between healthy subjects (HS) and patients in the low-severity and high-severity groups. Notably, the high-severity group is divided into "early" pre-ACLF and ACLF, illustrating the different CLIF-SIG scores shown by these subgroups. [Figure 1C] Comparison of CLIF-SIC scores at T1 between healthy subjects (HS) and patients with low and high severity. [Figure 1D]ROC curves containing complete records of CLIF-SIC scores, distinguishing between high-severity and low-severity groups by CLIF-SIG scores (black line) and CLIF-SIC scores (gray line) at T1 in the training set (solid line) and test set (dashed line). Asterisks indicate p < 0.001 between scores in each set. [Figure 1E] Correlation between individual CLIF-SIG and CLIF-SIC scores at T1. [Figure 2A] Comparison of CLIF-SIG scores at T1 across different phenotypes of ADC in Predict study patients from Cohort 1; HS: healthy subjects, SDC: stable decompensated cirrhosis, UDC: unstable decompensated cirrhosis. [Figure 2B] Comparison of CLIF-SIC scores at T1 across different phenotypes of ADC in Predict study patients from Cohort 1; HS: healthy subjects, SDC: stable decompensated cirrhosis, UDC: unstable decompensated cirrhosis. [Figure 2C] Comparison of CRP levels at T1 across different phenotypes of ADC in Predict study patients from Cohort 1; HS: healthy subjects, SDC: stable decompensated cirrhosis, UDC: unstable decompensated cirrhosis. [Figure 2D] Comparison of WBC counts at T1 across different phenotypes of ADC in Predict study patients from Cohort 1; HS: healthy subjects, SDC: stable decompensated cirrhosis, UDC: unstable decompensated cirrhosis. [Figure 3A] Density curves of patients capturing upregulation, minor change, and downregulation of CLIF-SIG scores in subcohort 1a. Stratification was based on a symmetric cutoff selected to maintain the minimum proportion of patients greater than 20% exhibiting upregulation or downregulation. T1 values ​​were weighted to increase sensitivity for patients at the extrema of the distribution. [Figure 3B] Individual changes in CLIF-SIG scores in subcohort 1a. [Figure 3C]ROC curves of CLIF-SIG scores that differentiate patients from low-severity and high-severity groups, as well as the critical score level (CSL, 0.386) that better identifies these groups in Cohort 1. [Figure 3D] Progression of CLIF-SIG scores in subcohort 1b. [Figure 3E] Within the first three weeks after hospitalization, all or part of the three phases of systemic inflammatory response were detected in only four of the 59 patients included in subcohort 1b. [Figure 3F] Representative examples of systemic inflammation curves in patients with only the AD phase and those with the ITSI phase plus the AD phase. [Figure 3G] Representative examples of systemic inflammation curves in patients with only the AD phase and those with the ITSI phase plus the AD phase. [Figure 3H] The figure shows CLIF-SIG score curves (gray) corresponding to three patients who experienced recurrent bursts of systemic inflammation. Following the decline-recovery phase of the previous burst of systemic inflammation, the patients developed a severe reactivation of the systemic inflammatory response. The figure shows three patients who had normal CLIF-SIG scores at T1 and developed ADC in the absence of a detectable systemic inflammatory response. Note that the estimated portion of each curve is represented by a dashed line. The shaded area represents the period before T1. [Figure 4A] Individual changes in CLIF-SIG scores in the low-severity and high-severity groups of patients included in subcohort 1a. The upper interrupted horizontal line represents CSL-specific patients with moderate (gray) and severe (black) systemic inflammation at T2. The lower interrupted horizontal line represents the median level CLIF-SIG score in healthy subjects (HS). The main results are shown in the figure below. [Figure 4B]In subcohort 1b, the clinical course of most patients who developed ACLF during hospitalization occurred above a CSL ≥ 0.386. Notably, the horizontal axis is interrupted to reduce the impact of the prolonged hospitalizations of four patients on the length of the figure. To reduce the density of the lines during this period, the initial courses of these four patients are also not shown on the left side of the figure. The interrupted vertical line corresponds to the first three weeks after T1. Diamonds represent the time of ACLF detection. Black and gray lines represent the courses of patients who developed ACLF and those who did not, respectively. [Figure 5A] External validation of the CLIF-SIG score gene. Results of analysis using a publicly available RNA-seq dataset (Monaco et al. Cell Rep. 2019;26:1627-1640). The dataset consisted of three groups: healthy subjects (HS) (n=40), non-cirrhotic patients with bacterial infections (n=32, including 20 with sepsis and 12 with septic shock). (A) Heatmap showing CLIF-SIG score gene expression across the three groups. [Figure 5B] External validation of the CLIF-SIG score gene. Results of analysis using a publicly available RNA-seq dataset (Monaco et al. Cell Rep. 2019;26:1627-1640). The dataset consisted of three groups: healthy subjects (HS) (n=40), non-cirrhotic patients with bacterial infections (n=32, including 20 with sepsis and 12 with septic shock). (B) Principal component analysis (PCA) plot across the three study groups. [Figure 5C] External validation of the CLIF-SIG score gene. Results of analysis using a publicly available RNA-seq dataset (Monaco et al. Cell Rep. 2019;26:1627-1640). The dataset consisted of three groups: healthy subjects (HS) (n=40), non-cirrhotic patients with bacterial infections (n=32, including 20 with sepsis and 12 with septic shock). (C) Box plot identifying HS and non-cirrhotic patients with or without septic shock based on the first principal component score. [Figure 6A] Results derived from a score composed of four genes. Comparison of four-gene CLIF-SIG scores at T1 between healthy subjects (HS) and patients in low-severity and high-severity groups. Notably, the high-severity group is divided into "early" pre-ACLF and ACLF, and the different four-gene CLIF-SIG scores shown for these subgroups are presented. [Figure 6B] Results derived from scores composed of four genes. ROC curves distinguishing between high-severity and low-severity groups by CLIF-SIG scores (black line) and CLIF-SIC scores (gray line) at T1 in a training set (solid line) and a test set (dashed line) containing complete records of CLIF-SIC scores. Asterisks indicate p<0.001 between scores in each set. [Figure 6C] Results derived from a score composed of four genes. Correlation between individual CLIF-SIG scores and CLIF-SIC scores in T1. [Figure 7] Correlation between the gene expression of 28 genes that constitute the CLIF-SIG score, as determined by RNA-seq, and their gene expression as determined by Nanostring and Fluidigm techniques. [Modes for carrying out the invention]

[0027] The authors of this invention have discovered a novel scoring system that more accurately identifies high-severity and low-severity ADC phenotypes than current biomarkers, based on genes related to innate immunity and B cells, and have identified new important aspects of the pathophysiology.

[0028] Method 1 of the present invention In a first aspect, the present invention relates to an in vitro method for determining the presence of acute chronic liver failure (ACLF) in subjects suffering from cirrhosis or late-onset pre-ACLF, hereinafter referred to as the first method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, The fact that the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are greater than the reference values ​​for these genes indicates that the subject has ACLF.

[0029] In a particular embodiment, the first method of the present invention is a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression levels of the genes with their reference values, The expression level of the gene changes compared to the reference value of the gene, and the change in the expression level is - Increased expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - Decreased expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes. If so, it indicates that the subject has ACLF.

[0030] Therefore, in certain embodiments, the first method of the present invention is the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes, and at least two, at least three, at least four genes, selected from the group consisting of IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes. Each method includes determining the expression levels of 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes, or at least 34 genes.

[0031] As used herein, the terms “acute chronic liver failure” or “ACLF” refer to a syndrome characterized by acute deterioration of liver function in patients with compensated or decompensated cirrhosis who have failure of one or more organs and a low short-term survival rate, as described below. According to the CANONIC study, the syndrome is classified into three grades of severity based on organ failure and mortality data.

[0032] - ACLF Grade 1: This grade includes three subgroups of patients: (1) patients with a single renal failure; (2) patients with a single hepatic, coagulation, circulatory, or respiratory failure with serum creatinine levels in the range of 1.5–1.9 mg / dL and / or mild to moderate hepatic encephalopathy; and (3) patients with a single encephalopathy with serum creatinine levels in the range of 1.5–1.9 mg / dL. The 28-day and 90-day mortality rates are approximately 22.1% and 40.7%, respectively.

[0033] -ACLF Grade 2: This group includes patients with two organ failures. The 28-day and 90-day mortality rates are approximately 32% and 52.3%, respectively.

[0034] - ACLF Grade 3: This group includes patients with three or more organ failures. The 28-day and 90-day mortality rates are approximately 76.7% and 79.1%, respectively.

[0035] As used herein, the term “cirrhosis” refers to a condition characterized by the replacement of liver tissue with fibrosis and regenerative nodules, resulting in loss of liver function. Ascites (accumulation of fluid in the abdominal cavity) is the most common complication of cirrhosis. It is associated with a reduced quality of life, an increased risk of infection, and a worsening of long-term outcomes. Other potentially life-threatening complications include hepatic encephalopathy (confusion and coma) and bleeding from esophageal varices. There are many possible signs of cirrhosis. These signs and symptoms may be either a direct result of hepatocyte failure or secondary to the resulting portal hypertension. Effects of portal hypertension include splenomegaly, gastroesophageal varices, and portocollateral circulation as a result of portal hypertension, which is the formation of venous collateral circulation between the portal system and the periumbilical veins.

[0036] Cirrhosis is classified into two clinical categories: compensated cirrhosis and decompensated cirrhosis. As used herein, the term “compensated cirrhosis” means that the liver is severely scarred but can still perform many important bodily functions. Patients with compensated cirrhosis may experience few or no symptoms and may survive without serious clinical complications. Patients in the early stages of compensated cirrhosis are characterized by a low level of portal hypertension and the absence of esophageal varices. Patients with advanced stages of compensated cirrhosis are characterized by a high level of portal hypertension and the presence of esophageal varices, but without ascites and hemorrhage. As used herein, the terms “decompensated cirrhosis” or “acute decompensated cirrhosis” mean that the liver is extensively scarred and unable to function properly. Patients with decompensated cirrhosis develop a variety of symptoms, including fatigue, loss of appetite, jaundice, weight loss, ascites and / or edema, hepatic encephalopathy and / or hemorrhage. Patients in the early stages of decompensated cirrhosis are characterized by the presence of ascites, with or without esophageal varices, in patients who have not experienced bleeding. Patients in the advanced stages of compensated cirrhosis are characterized by more severe ascites, either isolated or associated with hemorrhage, bacterial infection and / or hepatic encephalopathy.

[0037] As used herein, the terms “subject” or “patient” refer to any animal classified as a mammal, including but not limited to domestic animals and livestock, primates and humans, such as humans, non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents. The subject is preferably a human male or female of any age or race.

[0038] In certain embodiments, the subject suffers from unstable or stable acute decompensated cirrhosis of the liver.

[0039] As used herein, the term “pre-ACLF” refers to subjects who develop ACLF during hospitalization (“early” pre-ACLF) or subjects who develop ACLF during a subsequent 90-day follow-up period (“late” pre-ACLF).

[0040] As used herein, the terms “marker” or “marker gene” refer to a gene that is differentially expressed within a population exhibiting a different phenotype, and that differential expression, either alone or in combination with other genes, correlates more strongly with a particular phenotype than would be expected randomly.

[0041] As used herein, the term “sample” refers to biological material isolated from a subject, and therefore includes biological samples. The sample may contain any biological material suitable for detecting a desired marker and may include cellular and / or non-cellular material from the subject. Generally, the sample may be isolated from any suitable biological tissue or body fluid, but the sample is preferably a biological fluid from a subject under study for carrying out the present invention. The biological fluid sample may be a urine sample, a blood sample, a serum sample, etc., and may be obtained by any conventional method. In certain embodiments, the sample is a biological fluid, and in more specific embodiments, the biological fluid is blood.

[0042] In the first step, the first method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0043] As described above, in certain embodiments, the first method of the present invention further includes determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0044] As used herein, the term “expression level” refers to the value of a parameter that measures the degree of expression of a particular gene. In certain embodiments, the value may be determined by measuring the mRNA level of the gene or fragment thereof of interest, or by measuring the amount of protein encoded by the gene or variant thereof of interest.

[0045] Substantially, any conventional method for detecting and quantifying gene expression levels can be used within the framework of the present invention for detecting and quantifying the expression levels of specific genes. As a non-limiting example, gene expression levels can be determined by quantifying the mRNA level of the gene or by quantifying the level of the protein encoded by the gene.

[0046] Methods for determining the amount of mRNA are well known in the latest technology. For example, nucleic acids contained in a sample, such as a biological fluid sample from a subject under study, are extracted according to conventional methods, for example, by using lytic enzymes, chemical solutions, or fixation resins. The extracted mRNA can be detected by hybridization (e.g., by converting mRNA to labeled cDNA and then by Northern blot analysis or DNA or RNA array (microarray)) and / or amplification by enzyme chain reaction. Generally, quantitative or semi-quantitative enzyme amplification methods are preferred. Polymerase chain reaction (PCR) or quantitative real-time RT-PCR or semi-quantitative RT-PCR techniques are particularly advantageous. It is preferable to design primer pairs with introns overlapping to distinguish cDNA amplification from contamination from genomic DNA (gDNA). Additional primers or probes, preferably fluorescently labeled, that specifically hybridize in a region located between two exons may be designed to distinguish cDNA amplification from contamination from gDNA. If desired, the primer may be designed such that approximately nucleotides contained in half of the total length from the 5' end of the primer hybridize with one of the target exons, and approximately nucleotides contained in half of the total length from the 3' end of the primer hybridize with the other target exon. Suitable primers can be easily designed by those skilled in the art. Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand-displacement amplification (SDA), and nucleic acid-based amplification (NASBA). The amount of mRNA is preferably measured quantitatively or semi-quantitatively. Relevant information on conventional methods for quantifying gene expression levels can be found, for example, in Sambrook et al., 2001 [Sambrook, J., et al., "Molecular cloning: a Laboratory Manual", 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3].

[0047] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0048] In another specific embodiment, the expression levels of one or more of the HMGB2, RETN, ZNF608, and PYCARD genes, as well as the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28, can also be determined by determining the expression levels of the proteins encoded by these genes, since increased gene expression usually results in increased amounts of the corresponding proteins. The determination of the amount of protein corresponding to the expression of a specific gene can be performed using any conventional method for protein detection and quantification, for example, by immunoassay. In non-limiting examples, the determination can be performed using an antibody having the ability to specifically bind to the protein to be determined (or a fragment thereof having an antigenic determinant), and subsequent quantification of the antigen-antibody complex derivative. The antibody may be, for example, polyclonal serum, hybridoma supernatant or monoclonal antibody, antibody fragments, Fv, Fab, Fab' and F(ab')2, scFv, diabodies, triabodies, tetrabodies, or humanized antibodies. The antibody may be labeled with a marker (or not). Exemplary, non-limiting examples of markers that can be used in the present invention include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme cofactors, enzyme substrates, and enzyme inhibitors. The well-known assays that can be used in the present invention are wide-ranging, including, for example, Western blotting or immunoblotting techniques, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), EIA (enzyme-linked immunoassay), DAS-ELISA (double antibody sandwich ELISA), and assays based on immunocytochemical or immunohistochemical techniques.Other methods for detecting and quantifying proteins include affinity chromatography, ligand-binding assays, and particle-enhanced turbidimetric immunoassays (PETIA).

[0049] A second step of the first method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0050] In certain embodiments, the first method of the present invention further comprises comparing the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 to the respective reference values ​​of each gene.

[0051] In some embodiments, the expression levels of the gene under consideration are normalized. To normalize mRNA expression values ​​between different samples, it is possible to compare the expression level of the target mRNA in the test sample with the expression level of a control RNA. As used herein, “control RNA” refers to RNA whose expression level in tumor cells is no different or only slightly different compared to non-tumorogenic cells. Preferably, the control RNA is a housekeeping gene-derived mRNA that is constitutively expressed and encodes a protein that performs an essential cellular function. Preferred housekeeping genes for use in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal protein, cyclophyllin, IPO8, HPRT, GAPDH, PSMB4, tubulin, and β-actin.

[0052] As used herein, the term “reference value” refers to an experimental value used as a reference to values / data obtained from a sample taken from a subject. A reference value (or reference level) may be an absolute value, a relative value, a value with upper and / or lower limits, a set of values, a mean, a median, a mean, or a value expressed in reference to a control or reference value. A reference value may be based on a value taken from a sample of an individual, e.g., a value taken from a sample of the subject of study, but obtained at a past point in time. A reference value may be based on a large number of samples, e.g., values ​​taken from a population of subjects of a chronological age group corresponding to that of the subject of study, or on a set of samples that meet inclusion criteria or exclusion criteria. For example, a reference value may be based on the expression level of a marker being analyzed, taken from a subject with cirrhosis or late-onset pre-ACLF. In another embodiment, the reference value for the expression level of one or more genes of interest is the mean level of expression of the one or more genes in a pool of samples of the same type as the sample being analyzed, taken from multiple subjects suffering from cirrhosis or late-onset pre-ACLF.

[0053] Once a reference value is established, the levels of HMGB2, RETN, ZNF608, and PYCARD genes expressed in samples from the subject, as well as optionally IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, or IGKV2-28, are compared to this reference value, and thus the levels can be assigned as "increased," "decreased," or "equal."

[0054] In the context of the present invention, HMGB2, RETN, ZNF608, PYCARD, IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1 are found in samples from the subject. The expression levels of the SAMD3, MS4A4A, and IGKV2-28 genes are considered "increased" or "greater than" the reference value of the marker when the expression levels of these genes in a sample from a subject increase by, for example, 5%, 10%, 25%, 50%, 100%, or more compared to the reference value of the gene, or when they increase by, for example, at least 1.1 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more compared to the reference value of the marker.

[0055] In the context of the present invention, the target marker in a sample from a subject (i.e., HMGB2, RETN, ZNF608, PYCARD, IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASL The expression levels of the G, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes are also considered "decreased" or "below" the reference value of the marker when the expression levels of these genes in a sample from a subject are lower than, for example, 5%, 10%, 25%, 50%, 75%, or even 100% compared to the reference value of the marker.

[0056] In the context of the present invention, the expression levels of target markers (i.e., HMGB2, RETN, ZNF608, PYCARD, IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 genes) in a sample from a subject are also considered "equivalent" to the reference values ​​of the markers if the expression levels of these genes do not substantially change compared to the reference values. For example, HMGB2, RETN, ZNF608, PYCARD, IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISP in samples from subjects under study. The expression levels of the LD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes are considered "equivalent" to the reference value if the level differs by less than or equal to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or the same percentage value as the error associated with the experimental method used for the determination.

[0057] By comparing the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject with reference values ​​of the markers, the first method of the present invention makes it possible to determine whether a subject has ACLF based on whether the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes are increased compared to the reference values.

[0058] In certain embodiments, the first method of the present invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, - Are the expression levels of the following genes increased compared to the reference value: GFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - If the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 gene are lower than the reference value, This indicates that the subject has ACLF.

[0059] The second method of the present invention The scoring system developed by the authors makes it possible to distinguish patients with compensated cirrhosis (phenotype 1), ADC (phenotype 2), or late-onset ACLF (phenotype 3) from patients with early ACLF (phenotype 4) or ACLF 1, 2, and 3 (phenotypes 5, 6, and 7). Since the presence of late-onset ACLF or cirrhosis can lead to the development of ACLF, the method developed by the inventors also makes it possible to predict whether patients with cirrhosis or late-onset pre-ACLF are at high risk of developing ACLF. Accordingly, in another aspect, the present invention relates to an in vitro method for determining the risk of developing ACLF in subjects with cirrhosis or late-onset pre-ACLF, hereafter referred to as the second method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, The fact that the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are higher than the reference values ​​for these genes indicates that the subject is at high risk of developing ACLF.

[0060] In a particular embodiment, the second method of the present invention is a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression levels of the genes with their reference values, The expression level of the gene changes compared to the reference value of the gene, and the change in the expression level is - Increased expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - Decreased expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes. If this is the case, it indicates that the subject has a high risk of developing ACLF.

[0061] Therefore, in certain embodiments, the second method of the present invention is the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes, and at least two, at least three, at least four genes, selected from the group consisting of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes. Each method includes determining the expression levels of 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes, or at least 34 genes.

[0062] As used herein, “determine risk” or “predict risk” or similar expressions are synonymous with “assess risk” or “assess risk,” and mean that the present invention enables the prediction, estimation, or assessment of the risk of developing ACLF in patients with cirrhosis or late-onset pre-ACLF. Risk prediction generally includes either an increase or decrease in risk. As will be understood by those skilled in the art, the prediction (or risk) does not need to be accurate for 100% of cirrhotic or late-onset pre-ACLF patients to be assessed, although it is preferable that it is. However, the term requires that a statistically significant proportion of cirrhotic or late-onset pre-ACLF patients can be identified as likely to have ACLF. Whether a subject is statistically significant can be determined by those skilled in the art without further effort by using various well-known statistical assessment tools, such as determining confidence intervals, p-values, Student's t-tests, Mann-Whitney tests, etc. Further details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%. The p-values ​​are preferably 0.05, 0.025, 0.001, 0.0001, or less.

[0063] The terms “ACLF,” “cirrhosis,” “compensated cirrhosis,” “decompensated cirrhosis,” “pre-ACLF,” “marker,” “subject,” and “sample” are defined or described above, and these definitions are applicable to the second method of the present invention.

[0064] In certain embodiments, the subject suffers from unstable or stable acute decompensated cirrhosis of the liver.

[0065] In another specific embodiment, the sample is a biological fluid. In a more specific embodiment, the biological fluid is blood.

[0066] In the first step, the second method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0067] In certain embodiments, the second method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0068] The term “expression level” is defined or described in the first method of the present invention, and this definition is applicable to the second method of the present invention.

[0069] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0070] A second step of the second method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0071] In certain embodiments, the second method of the present invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0072] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the second method of the present invention.

[0073] By comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject with reference values ​​of the markers, the second method of the present invention makes it possible to determine whether a subject has a high risk of developing ACLF based on whether the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes are elevated compared to the reference values.

[0074] In certain embodiments, the second method of the present invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, - The expression levels of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, and IGKV2-33 genes are increased compared to the reference value, or - If the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 gene are lower than the reference value, This indicates that the subject is at high risk of developing ACLF.

[0075] Third method of the present invention The authors also observed that the score acts as a marker of disease progression and can therefore be used to monitor the clinical course of patients. Thus, in another aspect, the present invention relates to an in vitro method for monitoring the progression of ACLF in a subject, hereafter referred to as a third method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject. b) Comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point after the onset of ACLF, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes are lower than the aforementioned reference values, it indicates ACLF resolution.

[0076] In a particular embodiment, the third method of the present invention is a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time after the onset of ACLF, The change in the expression level of the gene in the sample compared to the reference value of the gene indicates ACLF resolution, and the change in the expression level indicates - Decreased expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - This is an increase in the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes.

[0077] Therefore, in certain embodiments, the third method of the present invention is the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes, as well as at least two, at least three, at least four genes, selected from the group consisting of IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes. Each method includes determining the expression levels of 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes, or at least 34 genes.

[0078] The terms "ACLF," "marker," "subject," and "sample" are defined or described above, and these definitions are applicable to the third method of the present invention.

[0079] In certain embodiments, the sample is a biological fluid. In more specific embodiments, the biological fluid is blood.

[0080] As used herein, the term “resolved ACLF” refers to the situation in which a subject previously diagnosed with ACLF recovers from the disease. Determining whether a subject has recovered from the disease can be performed using different parameters commonly used to measure severity, such as, among other things, changes in the international normalized ratio (INR), total bilirubin (TB), and total creatinine (TC) at different time points. A decrease in INR to less than 2.5 is considered a resolution of the coagulation disorder, resolution of hepatic failure is defined as TB < 12 mg / dl, and resolution of renal failure is defined as TC < 2 mg / dl. Criteria for defining resolution of vascular, respiratory, and cerebral failure include discontinuation of vasoconstrictors, P, etc., respectively. a O2 / F i O2 > 200, and hepatic encephalopathy < 3.

[0081] In the first step, the third method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0082] In certain embodiments, the third method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0083] The term “expression level” is defined or described in the first method of the present invention, and this definition is applicable to the third method of the present invention.

[0084] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0085] The second step of the third method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0086] In certain embodiments, the third method of the present invention further includes comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0087] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the third method of the present invention. In the third method of the present invention, the reference value is the expression level of the same gene obtained from the patient at an earlier point in time after the onset of ACLF. In certain embodiments, the reference value is obtained at the time of diagnosis of the syndrome and daily for the first one to two weeks in patients admitted to the ICU. Patients admitted to general wards require fewer controls and are typically obtained three times a week.

[0088] Therefore, according to the third method of the present invention, if the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are lower than the expression levels of these genes determined in a sample from the patient in the early stages after the onset of ACLF, it indicates ACLF resolution.

[0089] In a particular embodiment, the third method of the present invention further includes determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, - A decrease in the expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, and IGKV2-33, as determined from samples taken from the aforementioned patients in the early stages after the onset of ACLF, indicates the resolution of ACLF, or An increase in the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes relative to the expression levels of these genes determined in samples from the aforementioned patients in the early post-onset period of ACLF indicates ACLF resolution.

[0090] Method 4 of the present invention In another aspect, the present invention relates to an in vitro method for monitoring the effects of a therapy in a subject suffering from ACLF, hereinafter referred to as a fourth method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time, If the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are lower than the reference values, it indicates that the therapy is effective.

[0091] In a particular embodiment, the fourth method of the present invention is a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time, A change in the expression level of the gene in the sample compared to the reference value of the gene indicates that the therapy is effective, and the change in the expression level indicates that - Decreased expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - This is an increase in the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes.

[0092] Therefore, in certain embodiments, the fourth method of the present invention is the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes, as well as at least two, at least three, at least four genes, selected from the group consisting of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes. Each method includes determining the expression levels of 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes, or at least 34 genes.

[0093] As used herein, the term “therapy” typically refers to an attempt to correct a health problem after diagnosis, or to prevent the onset of a health problem. Therefore, it does not necessarily mean a cure, i.e., a complete reversal of the disease. Such therapy may or may not be known to have a positive effect on a particular disease. The term encompasses both therapeutic measures and preventive or prophylactic means, the purpose of which is to prevent or halt (reduce) undesirable physiological changes or impairments, such as ACLF. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, symptom relief, stabilization (especially non-aggravation) of a pathological condition, slowing or halting disease progression, and improvement or mitigation of pathological impairment. In particular, for the purposes of this invention, therapy aims to delay the progression of liver damage and reduce the risk of further complications. It may also include extending survival compared to the survival expected without treatment. Subjects requiring treatment include those already suffering from a condition or impairment, as well as those prone to developing a condition or impairment, or those who must prevent the condition or impairment.

[0094] The term “treatment,” as used herein, refers to both therapeutic and preventive or prophylactic measures, the objective of which is to prevent or slow (mitigate) undesirable physiological changes or disorders, such as ACLF. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, stabilization (especially non-exacerbation) of the pathological condition, slowing or halting disease progression, and improvement or mitigation of the pathology. In particular, the objective of the present invention is for treatment to delay the progression of liver damage and reduce the risk of further complications.

[0095] The terms "ACLF," "marker," "subject," and "sample" are defined or described above, and these definitions are applicable to the fourth method of the present invention.

[0096] In certain embodiments, the sample is a biological fluid. In more specific embodiments, the biological fluid is blood.

[0097] In the first step, the fourth method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0098] In certain embodiments, the fourth method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0099] The term “expression level” is defined or described in the first method of the present invention, and this definition is applicable to the fourth method of the present invention.

[0100] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0101] The second step of the fourth method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0102] In certain embodiments, the fourth method of the present invention further includes comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0103] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the fourth method of the present invention. In the fourth method of the present invention, the reference value is the expression level of the same gene obtained from the patient at an earlier point in time after the onset of ACLF.

[0104] According to aspects of the present invention, the HMGB2, RETN, ZNF608 and PYCARD genes (and optionally the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZ The expression levels of one or more of the following genes (MH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28) are determined in the first sample obtained from a subject with ACLF (first sample), and the expression levels of these genes are determined in a sample obtained from the same subject during a second period of treatment (second sample). The two values ​​are then compared to enable monitoring of the therapeutic effect in the subject with ACLF.

[0105] The second sample can be taken at any point after the first sample, for example, one day, one week, two weeks, three weeks, one month, two months, three months, or more after the first subject sample. In certain embodiments, the first sample is taken after the subject has begun treatment for the clinical condition. Therapies that can be used to treat patients suffering from ACLF are concentrated to treat the liver failure and peripheral organ damage associated with the condition. Examples of therapies include, but are not limited to, therapies aimed at reducing the immune response, e.g., pentoxifylline; therapies to prevent the migration of aerobic bacteria from the intestines, e.g., norfloxacin or rifaximin; therapies to treat portal hypertension and portal hypertension-related complications (e.g., varicose bleeding), e.g., somatostatin or terlipressin; therapies to treat bacterial infections, including systemic administration of antibiotics; therapies to treat circulatory dysfunction, including administration of vasoconstrictors; therapies to treat hepatic encephalopathy, including lactulose, lactitol, and cleansing enemas; therapies to treat respiratory failure, including oxygen administration or assisted ventilation; therapies to treat renal failure, including hemodilysis or filtration therapy; therapies to treat hepatic failure, including so-called liver support devices such as MARS or Prometheus or plasma exchange; or any other therapies aimed at improving the effects of hepatic failure. The effectiveness of therapies after treatment can be easily tracked in accordance with the teachings of the present invention.

[0106] Therefore, according to the fourth method of the present invention, the therapy is effective if the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject are lower than the expression levels of these genes determined in a sample taken from the patient at an earlier time.

[0107] In a particular embodiment, the fourth method of the present invention further includes determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, - A decrease in the expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33 compared to the expression levels of these genes determined in samples from the patient at an earlier point in the course of therapy indicates that the therapy is effective, or An increase in the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes relative to the expression levels of these genes determined in samples taken from the patient at an earlier time indicates that the therapy is effective.

[0108] Fifth method of the present invention In another aspect, the present invention relates to an in vitro method for selecting a therapy for a patient suffering from ACLF, hereafter referred to as the fifth method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are elevated compared to the reference values, it indicates that the patient should be treated with therapy aimed at treating the effects of liver failure and / or for liver transplantation.

[0109] As used herein, the term “selecting a patient for treatment” relates to identifying a patient for treatment designed to cure a disease or alleviate symptoms associated with one or more diseases or conditions. In the specific case of ACLF therapy, any therapy that neutralizes, delays, or reduces symptoms associated with liver failure is understood. Details of suitable therapies that can be used according to the present invention to treat ACLF include antibiotics, diuretics, albumin, lactulose, β-adrenergic blockers, vasoconstrictors, sclerosants, intrahepatic portosystemic shunts, plasmapheresis, ventilator support, renal hemodialysis, albumin dialysis, liver transplantation, and / or combinations thereof.

[0110] The terms "ACLF," "marker," "subject," and "sample" are defined or described above, and these definitions are applicable to the fourth method of the present invention.

[0111] In certain embodiments, the sample is a biological fluid. In more specific embodiments, the biological fluid is blood.

[0112] In the first step, the fifth method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0113] In certain embodiments, the fifth method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0114] The term “expression level” is defined or described in the first method of the present invention, and this definition is applicable to the fourth method of the present invention.

[0115] In certain embodiments, at least two, at least three, at least four, at least five, or at least six genes selected from the group consisting of at least HMGB2, RETN, ZNF608, PYCARD genes, and IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes. The determination of the expression levels of genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes, or at least 34 genes is performed by RNA-seq.

[0116] The second step of the fifth method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0117] In certain embodiments, the fifth method of the present invention further includes comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0118] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the fourth method of the present invention. In the fourth method of the present invention, the reference value is the expression level of the same gene obtained from the patient at an earlier point in time after the onset of ACLF.

[0119] Therefore, according to the fifth method of the present invention, the therapy is effective if the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject are lower than the expression levels of these genes determined in a sample from the patient at an earlier point in time.

[0120] In another embodiment, the fourth method of the present invention further includes determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, - Increased expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - A decrease in the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes indicates This indicates that the therapy is ineffective and that the patient is a candidate for treatment with therapies aimed at treating the effects of liver failure and / or for liver transplantation.

[0121] As those skilled in the art will understand, it is preferable, but not necessary, that the selection is appropriate for 100% of the subjects selected according to the fourth method of the present invention. However, this term requires that a statistically significant proportion of the subjects have been correctly selected. Whether the selection of patients in the subject population is statistically significant can be determined by those skilled in the art without further effort by using various well-known statistical evaluation tools, such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p-value is preferably 0.2, 0.1, or 0.05.

[0122] The term "therapy aimed at treating the effects of hepatic failure" includes, but is not limited to, therapies aimed at reducing the immune response, e.g., pentoxifylline; therapies to prevent the migration of aerobic bacteria from the intestines, e.g., norfloxacin or rifaximin; therapies to treat portal hypertension and portal hypertension-related complications (e.g., varicose bleeding), e.g., somatostatin or terlipressin; therapies to treat bacterial infections, including systemic administration of antibiotics; therapies to treat circulatory dysfunction, including administration of vasoconstrictors; therapies to treat hepatic encephalopathy, including lactulose, lactitol, and cleansing enemas; therapies to treat respiratory failure, including oxygen administration or assisted ventilation; therapies to treat renal failure, including hemodilysis or filtration therapy; therapies to treat hepatic failure, including so-called liver support devices such as MARS or Prometheus or plasma exchange; or any other therapy aimed at improving the effects of hepatic failure. The effectiveness of therapies after treatment can be easily tracked in accordance with the teachings of this invention.

[0123] As used herein, the term “liver transplant” refers to the replacement of a diseased liver with a part or all of a healthy liver from another person. The most commonly used technique is orthotopic transplantation, in which the original liver is removed and replaced with a liver from an organ donor located in the same anatomical position as the original liver.

[0124] Sixth method of the present invention In another aspect, the present invention relates to an in vitro method for determining the degree of systemic inflammation in a subject, hereinafter referred to as the sixth method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, The fact that the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are higher than the reference values ​​for these genes indicates that the subject has severe systemic inflammation.

[0125] In a particular embodiment, the sixth method of the present invention is a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression levels of the genes with their reference values, The expression level of the gene changes compared to the reference value of the gene, and the change in the expression level is - Increased expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - Decreased expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes. If this is the case, it indicates that the subject has severe systemic inflammation.

[0126] Accordingly, in certain embodiments, the sixth method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes, as well as the expression levels of at least two genes selected from the group consisting of IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes.

[0127] Systemic inflammation is a result of the release of pro-inflammatory cytokines from immune-related cells and chronic activation of the innate immune system. It can contribute to the development or progression of certain conditions, such as cardiovascular disease, cancer, diabetes, chronic kidney disease, non-alcoholic fatty liver disease, autoimmune disorders, and neurodegenerative disorders.

[0128] The terms “marker,” “subject,” and “sample” are defined or described above, and these definitions are applicable to the fifth method of the present invention.

[0129] In certain embodiments, the sample is a biological fluid. In more specific embodiments, the biological fluid is blood.

[0130] In certain embodiments, the subject is suffering from ACLF or an infection. The term "ACLF" is defined in the first method of the present invention, and this definition is applicable to the fifth method of the present invention. In more specific embodiments, ACLF is ACLF1, ACLF2, ACLF3, or early ACLF.

[0131] As used herein, the term “infection” refers to the invasion of tissue by pathogens, their proliferation, and the host tissue’s response to infectious agents and the toxins they produce. Infectious diseases, also called transmissible diseases, are illnesses resulting from infection. Common types of infections in patients with liver disease include spontaneous bacterial peritonitis (SBP), urinary tract infections (UTIs), pneumonia, bacteremia, and soft tissue infections.

[0132] In certain embodiments, the infection is a bacterial infection, a viral infection, a parasitic infection, a fungal infection, or a prion infection.

[0133] In more specific embodiments, the infection is a bacterial infection. In preferred embodiments, the bacterial infection is caused by Escherichia coli, Klebsiella species, Enterobacter species, Pseudomonas aeruginosa, non-enterococcal streptococci, enterococci, or Staphylococcus aureus.

[0134] In the first step, the sixth method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0135] In certain embodiments, the sixth method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0136] The term “expression level” is defined or described in the first method of the present invention, and this definition is applicable to the fifth method of the present invention.

[0137] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0138] The second step of the sixth method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0139] In certain embodiments, the sixth method of the present invention further includes comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0140] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the fifth method of the present invention.

[0141] By comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject with reference values ​​of the markers, the fifth method of the present invention makes it possible to determine whether a subject has severe systemic inflammation based on whether the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes are elevated compared to the reference values.

[0142] In a particular embodiment, if the sixth method of the present invention further includes determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, then by comparing the expression levels of these genes in a sample from the subject with reference values ​​for the markers, the fifth method of the present invention can determine whether the subject has severe systemic inflammation. - Whether the expression levels of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, and IGKV2-33 genes are elevated relative to the reference value, or - This allows for determination based on whether the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes are reduced compared to a reference value.

[0143] The seventh method of the present invention In another aspect, the present invention relates to an in vitro method for determining the presence of a bacterial infection accompanied by sepsis in a subject, hereafter referred to as the sixth method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, The fact that the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are higher than the reference values ​​for these genes indicates that the subject has a bacterial infection accompanied by sepsis.

[0144] In a particular embodiment, the seventh method of the present invention is c) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and d) further comprising comparing the expression levels of the genes with their reference values, The expression level of the gene changes compared to the reference value of the gene, and the change in the expression level is - Increased expression levels of the genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - Decreased expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes. If this is the case, it indicates that the subject has a bacterial infection accompanied by sepsis.

[0145] Accordingly, in certain embodiments, the seventh method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608 and PYCARD genes, as well as the expression levels of at least two genes selected from the group consisting of IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes.

[0146] The term "bacterial infection" refers to an infection caused by bacteria. Common types of infections in patients with liver disease include spontaneous bacterial peritonitis (SBP), urinary tract infections (UTIs), pneumonia, bacteremia, and soft tissue infections. In certain embodiments, bacterial infections are caused by Escherichia coli, Klebsiella species, Enterobacter species, Pseudomonas aeruginosa, non-enterococcal streptococci, enterococci, or Staphylococcus aureus.

[0147] The terms “marker,” “subject,” and “sample” are defined or described above, and these definitions are applicable to the sixth method of the present invention.

[0148] In certain embodiments, the sample is a biological fluid. In more specific embodiments, the biological fluid is blood.

[0149] The term "sepsis" refers to a syndrome of life-threatening physiological, pathological, and biochemical abnormalities associated with infection. These abnormalities are secondary to a disproportionate immune response to the infection, ultimately damaging the body's own tissues and organs and leading to multi-organ dysfunction. Systemic inflammatory response syndrome (SMS) is associated with sepsis, but it is not the absolute cause of sepsis, as sepsis involves activation of both pro-inflammatory and anti-inflammatory responses. SMS is a serious condition associated with systemic inflammation, organ dysfunction, and organ failure. It is a subset of cytokine storms, in which abnormal regulation of various cytokines is present.

[0150] In certain embodiments, the subject has septic shock. The term "septic shock" refers to a type of sepsis characterized by circulatory, cellular, and metabolic abnormalities that are significant enough to substantially increase mortality.

[0151] In the first step, the seventh method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0152] In certain embodiments, the seventh method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0153] The term “expression level” is defined or described in the first method of the present invention, and these definitions are applicable to the seventh method of the present invention.

[0154] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0155] A second step of the seventh method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0156] In certain embodiments, the seventh method of the present invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0157] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the seventh method of the present invention.

[0158] By comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject with the reference values ​​of the markers, the seventh method of the present invention makes it possible to determine whether a subject has a bacterial infection accompanied by sepsis based on whether the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes are elevated relative to the reference values.

[0159] In a particular embodiment, if the seventh method of the present invention further includes determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, then by comparing the expression levels of these genes in a sample from a subject with reference values ​​for the markers, the sixth method of the present invention can determine whether the subject has a bacterial infection with sepsis. - Whether the expression levels of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, and IGKV2-33 genes are elevated relative to the reference value, or - This allows for determination based on whether the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes are reduced compared to a reference value.

[0160] The present invention kit In another aspect, the present invention relates to a kit or assay device comprising reagents suitable for determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes, hereinafter referred to as the first kit or assay device of the present invention. It will be understood that the reagents suitable for implementation will vary depending on the nature of the method.

[0161] In the context of the present invention, “kit” is understood as a product containing various reagents necessary to carry out the method of the present invention, packaged to allow for transport and storage. Suitable materials for packaging the components of the kit include glass, plastics (such as polyethylene, polypropylene, and polycarbonate), bottles, vials, paper, sachets, and the like. If the kit has two or more components, they may be packaged together if appropriate, or the kit generally includes a second, third, or other additional container in which additional components may be placed separately. However, in some embodiments, a particular combination of components may be packaged together and contained in a single container means. The kit may also include means for sealing and housing any reagent containers for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which desired vials are held. One or more compositions of the kit may be lyophilized. In some embodiments, all compositions of the kit of the present disclosure are lyophilized. In some embodiments, the kit of the present disclosure containing one or more lyophilized agents will be supplied with a reconstituted buffer. The reagents and components of the kit may be contained in one or more suitable container means. The container means may generally include at least one vial, test tube, flask, bottle, syringe or other container means in which components can be arranged, preferably appropriately divided.

[0162] In a particular embodiment, the reagent is for detecting and / or quantifying the mRNA of the HMGB2, RETN, ZNF608, and PYCARD genes. In another embodiment, the reagent is for detecting and / or quantifying the proteins encoded by the HMGB2, RETN, ZNF608, and PYCARD genes.

[0163] In certain embodiments, the reagent for detecting and / or quantifying the mRNA of the HMGB2, RETN, ZNF608, and PYCARD genes comprises a probe that hybridizes with the cDNA of the mRNA of HMGB2, RETN, ZNF608, and PYCARD, or a pair of oligonucleotide primers that hybridize with the mRNA of HMGB2, RETN, ZNF608, and PYCARD or with the cDNA of the mRNA of HMGB2, RETN, ZNF608, and PYCARD.

[0164] As used herein, the term “primer” refers to an oligonucleotide that can specifically hybridize to a target polynucleotide sequence by sequence complementarity of at least a portion of the primer within the sequence of the target polynucleotide sequence. The length of the primer may be at least 8 nucleotides, typically 8–70 nucleotides, and usually 18–26 nucleotides. For proper hybridization to the target sequence, the primer may have sequence complementarity of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% with the portion of the target polynucleotide sequence to be hybridized. Oligonucleotides useful as primers can be chemically synthesized using an automated synthesizer such as that described in Needham-Van Devanter et al, Nucleic Acids Res. (1984) 12:6159–6168, following the solid-phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. (1981) 22:1859–1862. Primers are useful in nucleic acid amplification reactions in which the primer is extended to generate a new chain of polynucleotides. Primers can be readily designed by those skilled in the art using general knowledge known in the art so as to be able to specifically anneal to the nucleotide sequence of the target nucleotide sequence of at least one biomarker provided herein. Typically, the 3' nucleotide of the primer is designed to be complementary to the target sequence at the corresponding nucleotide position to provide optimal primer extension by polymerase.

[0165] As used herein, the term “probe” refers to an oligonucleotide or its analogue that can specifically hybridize to a target polynucleotide sequence by sequence complementarity of at least a portion of the probe within the sequence of the target polynucleotide sequence. Exemplary probes may be, for example, DNA probes, RNA probes, or protein nucleic acid (PNA) probes. The length of a probe may be at least 8 nucleotides, typically 8–70 nucleotides, and usually 18–26 nucleotides. For proper hybridization to the target sequence, the probe may have sequence complementarity of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% with the portion of the target polynucleotide sequence to be hybridized. Probes may also be chemically synthesized according to the solid-phase phosphoramidite triester method described above. Methods for preparing DNA and RNA probes, as well as the conditions for their hybridization to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11.

[0166] In a preferred embodiment, the reagent suitable for determining the expression level of one or more genes comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the total amount of the reagent suitable for determining the expression level of genes that forms the kit.

[0167] In another embodiment, the reagent for detecting and / or quantifying proteins encoded by the HMGB2, RETN, ZNF608, and PYCARD genes is an antibody that recognizes HMGB2, RETN, ZNF608, and PYCARD.

[0168] Therefore, in certain embodiments, the reagents for detecting and / or quantifying the proteins encoded by HMGB2, RETN, ZNF608, and PYCARD are antibodies that recognize HMGB2, RETN, ZNF608, and PYCARD, respectively.

[0169] In another embodiment, the present invention relates to a kit or assay device comprising reagents suitable for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A and IGKV2-28, hereafter referred to as a second kit or assay device of the present invention.

[0170] In certain embodiments, the reagent is for detecting and / or quantifying mRNA of at least the HMGB2, RETN, ZNF608, and PYCARD genes, as well as one or more mRNAs of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28.

[0171] In another specific embodiment, the reagent is for detecting and / or quantifying proteins encoded by the HMGB2, RETN, ZNF608, and PYCARD genes, as well as one or more proteins encoded by the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28.

[0172] In another specific embodiment, the HMGB2, RETN, ZNF608 and PYCARD genes, as well as the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, F The reagent for detecting and / or quantifying one or more mRNAs of ASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 comprises a probe that hybridizes to the cDNA of the mRNA, or a pair of oligonucleotide primers that hybridize to the mRNA or to the cDNA of the mRNA.

[0173] In another specific embodiment, the reagent for detecting and / or quantifying proteins encoded by the HMGB2, RETN, ZNF608, and PYCARD genes is an antibody that recognizes proteins encoded by the HMGB2, RETN, ZNF608, and PYCARD genes, and includes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IG Reagents for detecting and / or quantifying proteins encoded by KV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are antibodies that recognize proteins encoded by one or more of these genes.

[0174] In certain embodiments, the second kit of the present invention comprises one or more reagents for detecting and / or quantifying the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes and / or the proteins encoded by said genes, as well as at least two, at least three, and at least four genes selected from the group consisting of IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 genes. The reagents include for detecting and / or quantifying the expression levels of at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 11 genes, at least 12 genes, at least 13 genes, at least 14 genes, at least 15 genes, at least 16 genes, at least 17 genes, at least 18 genes, at least 19 genes, at least 20 genes, at least 21 genes, at least 22 genes, at least 23 genes, at least 24 genes, at least 25 genes, at least 26 genes, at least 27 genes, at least 28 genes, at least 29 genes, at least 30 genes, at least 31 genes, at least 32 genes, at least 33 genes or at least 34 genes, and / or for detecting and / or quantifying the proteins encoded by said genes.

[0175] In addition to these means, the first and second kits of the present invention may include other components useful for carrying out the invention, such as buffers, material supports, and positive and / or negative control components. In addition to the components described above, the kit may also include instructions for carrying out the objectives of the present invention. These instructions may be present in the aforementioned kits in various forms, and one or more of these forms may be present in the kit. One form in which these instructions may be present is information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, kit packaging, or accompanying documents. Another medium may be a computer-readable medium on which the information is recorded, such as a CD or USB. Another medium that may be present is a website address, which can be used via the Internet to access the information remotely. Any convenient medium may be present in the kit of the present invention.

[0176] Use of the present invention In another aspect, the present invention relates to the use of reagents or a first kit of the present invention that are specific to determining the expression levels of at least HMGB2, RETN, ZNF608, and PYCARD genes, for determining the presence of ACLF in a subject.

[0177] In another aspect, the present invention relates to the use of reagents or a first kit of the present invention that are specific to determining the expression levels of at least HMGB2, RETN, ZNF608, and PYCARD genes, for determining the risk of developing ACLF in a subject.

[0178] In another aspect, the present invention relates to the use of reagents or a first kit of the present invention that are specific to determining the expression levels of at least HMGB2, RETN, ZNF608, and PYCARD genes, for monitoring the progression of ACLF in a subject.

[0179] In another aspect, the present invention relates to the use of reagents or a first kit of the present invention that are specific to determining the expression levels of at least HMGB2, RETN, ZNF608, and PYCARD genes, for monitoring the effect of ACLF therapy in a subject.

[0180] In another aspect, the present invention relates to the use of reagents or a first kit of the present invention that are specific to determining the expression levels of at least HMGB2, RETN, ZNF608, and PYCARD genes, for selecting patients suffering from ACLF for therapeutic purposes.

[0181] In another aspect, the present invention relates to the use of reagents or a first kit of the present invention that are specific to determining the expression levels of at least HMGB2, RETN, ZNF608, and PYCARD genes, for determining the degree of systemic inflammation in a subject.

[0182] In another aspect, the present invention relates to the use of reagents or a first kit of the present invention that are specific to determining the expression levels of at least HMGB2, RETN, ZNF608, and PYCARD genes, for determining the presence of a bacterial infection with sepsis in a subject.

[0183] In another aspect, the present invention relates to the use of reagents or a second kit of the present invention that are specific for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A and IGKV2-28, or for determining the presence of ACLF in a subject.

[0184] In another aspect, the present invention relates to the use of reagents or a second kit of the present invention that are specific for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A and IGKV2-28, or for determining the risk of developing ACLF in a subject.

[0185] In another aspect, the present invention relates to the use of reagents or a second kit of the present invention, which are specific for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A and IGKV2-28, for monitoring the progression of ACLF in a subject.

[0186] In another aspect, the present invention relates to the use of reagents or a second kit of the present invention, which are specific for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A and IGKV2-28, for monitoring the effect of ACLF therapy in a subject.

[0187] In another aspect, the present invention relates to the use of reagents or a second kit of the present invention, which are specific for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A and IGKV2-28, for selecting patients suffering from ACLF for therapy.

[0188] In another aspect, the present invention relates to the use of reagents or a second kit of the present invention that are specific for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A and IGKV2-28, or for determining the degree of systemic inflammation in a subject.

[0189] In another aspect, the present invention relates to the use of reagents or a second kit of the present invention that are specific for determining the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and for determining the expression levels of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A and IGKV2-28, or for determining the presence of a bacterial infection with sepsis in a subject.

[0190] Computer-related aspects In any particular embodiment of the method of the present invention, the method is computer-implemented.

[0191] In another embodiment, the present invention relates to a computer system comprising one or more programs, wherein the one or more programs comprises instructions for performing the method of the present invention.

[0192] In another aspect, the present invention relates to a computer-readable storage medium for storing one or more programs, wherein the one or more programs, when executed by one or more processors of an electronic device, include instructions causing the electronic device to perform a method of the present invention.

[0193] As used herein, the term “computer implementation” refers to the fact that steps of a method are performed by a computer or computer system. The term “computer implementation” also includes the term “partial computer implementation,” which refers to a method in which only certain steps, such as the calculation step, are computer implemented, while other steps of the method are not.

[0194] A further aspect of the present invention relates to a computer system comprising one or more programs, wherein the one or more programs comprises instructions for performing methods I, II, and / or III of the present invention.

[0195] Another aspect of the present invention relates to a computer-readable storage medium for storing one or more programs, wherein the one or more programs, when executed by one or more processors of the electronic device, include instructions causing the electronic device to perform methods I, II, and / or III of the present invention.

[0196] As those skilled in the art will understand, computer-readable instructions cause a computing system to perform the method according to the present invention. The device may take the form of an embodiment of the entire hardware or a combination of software and hardware embodiments. Furthermore, the present invention may include computer program products on a computer-readable storage medium having computer-readable program code means embodied in the medium. Any suitable computer-readable medium may be utilized, including hard disks, CD-ROMs, optical storage devices, or magnetic storage devices. Computer-readable or computer-readable media may be, or include, electronic, magnetic, optical, electromagnetic, infrared, or semiconductor systems, apparatus, devices, or transmission media, for example, but not limited to these. More specific examples of computer-readable media (a non-exhaustive list) include electrical connections having one or more wires, portable computer diskettes, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), optical fibers, and portable compact disk read-only memory (CD-ROM), CD-ROMs, DVDs (digital video discs), or other electronic storage media. It should be noted that computer-readable or computer-readable media may be paper or other materials. A suitable medium on which a program is printed can electronically capture the program, for example, through optical scanning of paper or other media, and then compile, interpret, or otherwise process it in an appropriate manner as needed, and then store it in computer memory.

[0197] The computer program code for performing the operations of the present invention may be written in an object-oriented programming language, such as R (which is the language used in this study), Python, Matlab, Java, Javascript, C#, Smalltalk, or C++. However, the computer program code for performing the operations of the present invention may also be written in a conventional procedural programming language, such as C or FORTRAN or assembly language. The program code can run entirely on the user's computer, partially on the user's computer, as a standalone software package, partially on the user's computer and partially on a remote computer, or entirely on a remote computer. In the latter scenario, the remote computer may be connected to the user's computer via a local area network (LAN) or wide area network (WAN), or it may be connected to an external computer (for example, via the Internet using an Internet service provider).

[0198] Method 8 of the present invention In another aspect, the present invention relates to a method for determining the presence of acute chronic liver failure (ACLF) in subjects suffering from cirrhosis or late-onset pre-ACLF, or in patients suffering from ACLF, and for treating the patient, hereinafter referred to as the eighth method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the patient, and b) Comparing the expression level of the gene with the reference value of each gene, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a patient's sample are higher than their respective reference values, the patient shall be treated with appropriate therapies, including antibiotics, diuretics, albumin, lactulose, β-adrenergic blockers, vasoconstrictors, sclerosants, intrahepatic portosystemic shunts, plasmapheresis, mechanical ventilation, renal hemodialysis, hepatic hemodialysis, liver transplantation, and / or combinations thereof.

[0199] The terms "ACLF," "cirrhosis," "compensated cirrhosis," "decompensated cirrhosis," "marker," "subject," and "sample" are defined or described in the first method of the present invention, and these definitions are applicable to the seventh method of the present invention.

[0200] In certain embodiments, the subject suffers from unstable or stable acute decompensated cirrhosis of the liver.

[0201] In another specific embodiment, the sample is a biological fluid. In a more specific embodiment, the biological fluid is blood.

[0202] In the first step, the eighth method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0203] In certain embodiments, the eighth method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0204] The term “expression level” is defined or described in the first method of the present invention, and this definition is applicable to the eighth method of the present invention.

[0205] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0206] The second step of the eighth method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0207] In certain embodiments, the eighth method of the present invention further includes comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0208] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the seventh method of the present invention.

[0209] When the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are compared with the reference values ​​of the markers, if the expression levels of these genes are greater than their respective reference values, the therapy is administered to the subject who would benefit from the administration of the therapy.

[0210] In certain embodiments, the method of the present invention further includes determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, -When the expression levels of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33 genes are elevated relative to a reference value, the therapy is administered to the subject who would benefit from the administration of the therapy, or -When the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes are reduced relative to a reference value, the therapy is administered to the subject who would benefit from the administration of the therapy.

[0211] The terms “therapy” and “treatment” are defined or described above, and these definitions are applicable to the seventh method of the present invention.

[0212] As is well known, the treatment of ACLF depends on the patient or the severity of ACLF. Therapies that can be used to treat patients with ACLF are concentrated to treat the liver failure and peripheral organ damage associated with the condition. Examples of therapies include, but are not limited to, therapies aimed at attenuating the immune response, e.g., pentoxifylline; therapies to prevent the migration of aerobic bacteria from the intestines, e.g., norfloxacin or rifaximin; therapies to treat portal hypertension and portal hypertension-related complications (e.g., varicose bleeding), e.g., somatostatin or terlipressin; therapies to treat bacterial infections, including systemic administration of antibiotics; therapies to treat circulatory dysfunction, including administration of vasoconstrictors; therapies to treat hepatic encephalopathy, including lactulose, lactitol, and cleansing enemas; therapies to treat respiratory failure, including oxygen administration or assisted ventilation; therapies to treat renal failure, including hemodilysis or filtration therapy; therapies to treat hepatic failure, including so-called liver support devices such as MARS, Prometheus, or plasma separation; or any other therapies aimed at improving the effects of hepatic failure. The effectiveness of therapies after treatment can be easily tracked in accordance with the teachings of the present invention.

[0213] In certain embodiments, the therapy includes antibiotics, diuretics, albumin, lactulose, β-adrenergic blockers, vasoconstrictors, sclerosants, intrahepatic portosystemic shunts, plasma exchange, ventilator support, renal hemodialysis, hepatic hemodialysis, liver transplantation, and / or combinations thereof.

[0214] The term "antibiotic" refers to a type of antimicrobial substance that is active against bacteria, including classic beta-lactams, carbapenems, newer cephalosporins, tetracyclines, quinolones, glycopeptides, and lipopeptides.

[0215] The term “diuretic” refers to any substance that promotes diuresis, an increase in urine production. This includes forced diuresis. In certain embodiments, diuretics are high-sealing / loop diuretics, thiazides, carbonic anhydrase inhibitors, potassium-sparing diuretics, calcium-sparing diuretics, osmotic diuretics, or low-sealing diuretics.

[0216] The term "albumin" refers to a family of globular proteins, the most common of which is serum albumin. Albumin is generally found in plasma and differs from other blood proteins in that it is not glycosylated. Albumin supports circulation and reduces systemic inflammation. In addition to its colloid osmotic function, it also acts as an antioxidant, radical scavenger, and immunomodulator. Long-term albumin therapy in patients with cirrhosis and ascites improves survival rates, prevents complications, simplifies ascites management, and reduces hospitalization rates.

[0217] The term "lactulose" refers to a non-absorbable sugar used to treat constipation and hepatic encephalopathy. Specifically, it is effective as a secondary prevention of hepatic encephalopathy in patients with cirrhosis.

[0218] The term "beta-adrenergic blockers" refers to drugs that lower blood pressure. Beta-blockers work by blocking the action of the hormone epinephrine, also known as adrenaline. Beta-blockers are competitive antagonists that block the receptor sites for the endogenous catecholamines epinephrine (adrenaline) and norepinephrine (noradrenaline) on adrenergic beta receptors in the sympathetic nervous system, which mediate the fight-or-flight response.

[0219] The term "vasoconstrictor" refers to drugs that cause vasoconstriction, which is the narrowing of blood vessels resulting from the constriction of the muscular walls of blood vessels, particularly the aorta and arterioles. In certain embodiments, the vasoconstrictor is selected from the group consisting of, among others, norepinephrine, somatostatin, octreotide, terlipressin, and methylene blue.

[0220] The term "sclerosing agent" refers to the drugs used in sclerotherapy, typically polidocanol and ethanolamine.

[0221] The term "intrahepatic portosystemic shunt" refers to an artificial channel within the liver that establishes communication between the inflowing portal vein and the outflowing hepatic vein. This is often used to treat portal hypertension (often caused by cirrhosis), which can lead to intestinal bleeding, life-threatening esophageal bleeding (esophageal varices), and fluid accumulation in the abdominal cavity (ascites). Interventional radiologists create the shunt using an image-guided endovascular (vascular) approach, with the jugular vein as the usual entry site.

[0222] The term "plasma exchange" refers to a therapeutic procedure used to treat a wide variety of diseases by removing large amounts of plasma. Therapeutic plasma exchange (TPE) has been shown to reduce levels of inflammatory cytokines, modulate adaptive immunity which may reduce susceptibility to infection, and lower levels of albumin-binding toxins and water-binding toxins in liver failure.

[0223] The term "ventilator support" is a medical term referring to providing complete or partial mechanical ventilation using a machine called a ventilator.

[0224] The term "hemodialysis" refers to a renal replacement therapy aimed at partially replacing kidney function. In hepatic failure, hemodialysis (HD) is highly effective in removing small and medium-sized water-soluble unbound molecules (e.g., ammonia, urea) from circulation across a semipermeable membrane into a hypotonic dialysate fluid along their concentration gradient.

[0225] Method 9 of the present invention In another aspect, the present invention relates to a method for determining the risk of developing ACLF in a subject suffering from cirrhosis or late-onset pre-ACLF and for treating the patient, hereinafter referred to as Method 9 of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are greater than the reference values ​​for the said genes, the patient is treated with a therapy appropriate for the prevention of ACLF, including antibiotics, diuretics, albumin, lactulose, β-adrenergic blockers, terlipressin, sclerosing agents, intrahepatic portosystemic shunts, and / or combinations thereof.

[0226] The terms "ACLF," "cirrhosis," "compensated cirrhosis," "decompensated cirrhosis," "marker," "subject," "sample," and "determine risk" are defined or described in the first and second methods of the present invention, and these definitions are applicable to the eighth method of the present invention.

[0227] In certain embodiments, the subject suffers from unstable or stable acute decompensated cirrhosis of the liver.

[0228] In another specific embodiment, the sample is a biological fluid. In a more specific embodiment, the biological fluid is blood.

[0229] In the first step, the ninth method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0230] In certain embodiments, the ninth method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0231] The term "expression level" is defined or explained in the first method of the present invention, and this definition is applicable to the ninth method of the present invention.

[0232] In certain embodiments, the determination of the expression level of at least the HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 is performed by RNA-seq. [[ID=⑨]]

[0233] The second step of the eighth method of the present invention comprises comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with a reference value.

[0234] In certain embodiments, the ninth method of the present invention further comprises comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 with a reference value.

[0235] The terms "reference value", "increased", "decreased", and "equivalent" are defined or explained in the first method of the present invention, and these definitions are applicable to the eighth method of the present invention.

[0236] When the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are compared with the reference values of the markers, if the expression levels of these genes are greater than their respective reference values, the therapy will be administered to the subject who will benefit from the administration of the therapy.

[0237] In certain embodiments, when the method of the present invention further comprises determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 -When the expression levels of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33 genes are elevated relative to a reference value, the therapy is administered to the subject who would benefit from the administration of the therapy, or -When the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes are reduced relative to a reference value, the therapy is administered to the subject who would benefit from the administration of the therapy.

[0238] The terms “therapy” and “treatment” are defined or described above, and these definitions are applicable to the eighth method of the present invention.

[0239] Appropriate treatments for the prevention of ACLF include, but are not limited to, antibiotics, diuretics, albumin, lactulose, β-adrenergic blockers, terlipressin, sclerosants, intrahepatic portosystemic shunts, and / or combinations thereof.

[0240] The tenth method of the present invention In another aspect, the present invention relates to a method for determining the degree of systemic inflammation in a subject and treating the subject, hereinafter referred to as the 10th method of the present invention, which is: a) Determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are greater than the reference values ​​for those genes, the patient is treated with a therapy appropriate for severe systemic inflammation, including antibiotics, corticosteroids, albumin, plasmapheresis, and / or a combination thereof.

[0241] The terms “systemic inflammation,” “marker,” “subject,” and “sample” are defined or explained above, and these definitions are applicable to the ninth method of the present invention.

[0242] In certain embodiments, the sample is a biological fluid. In more specific embodiments, the biological fluid is blood.

[0243] In the first step, the tenth method of the present invention includes determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject under study.

[0244] In certain embodiments, the tenth method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0245] The term “expression level” is defined or described in the first method of the present invention, and this definition is applicable to the tenth method of the present invention.

[0246] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0247] A second step of the tenth method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0248] In certain embodiments, the tenth method of the present invention further includes comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0249] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the ninth method of the present invention.

[0250] When the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are compared with the reference values ​​of the markers, if the expression levels of these genes are greater than their respective reference values, the therapy is administered to the subject who would benefit from the administration of the therapy.

[0251] In certain embodiments, the method of the present invention further includes determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, -When the expression levels of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33 genes are elevated relative to a reference value, the therapy is administered to the subject who would benefit from the administration of the therapy, or -When the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes are reduced relative to a reference value, the therapy is administered to the subject who would benefit from the administration of the therapy.

[0252] The terms “therapy” and “treatment” are defined or described above, and these definitions are applicable to the tenth method of the present invention.

[0253] Appropriate therapies for the treatment of severe systemic inflammation include, but are not limited to, antibiotics, corticosteroids, albumin, plasmapheresis, and / or combinations thereof.

[0254] Method 11 of the present invention In another aspect, a method of determining the presence of a bacterial infection associated with sepsis in a subject and treating the subject, hereinafter referred to as the 11th method of the present invention, the method comprising: a) determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject, and b) comparing the expression levels of the genes in the sample to their reference values, wherein when the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject are greater than the reference values of the genes, the patient is treated with a therapy suitable for bacterial infection and / or sepsis, including organ support including antibiotics, vasoconstrictors, renal hemodialysis, and / or ventilator support.

[0255] The terms "bacterial infection", "sepsis", "marker", "subject", and "sample" are defined or described in the 1st method of the present invention, and these definitions are applicable to the 10th method of the present invention.

[0256] In certain embodiments, the sample is a biological fluid. In more specific embodiments, the biological fluid is blood.

[0257] In certain embodiments, the bacterial infection is caused by Escherichia coli, Klebsiella species, Enterobacter species, Pseudomonas aeruginosa, non-enterococcal streptococci, enterococci, and Staphylococcus aureus.

[0258] In the first step, the 11th method of the present invention comprises determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject under study.

[0259] In certain embodiments, the eleventh method of the present invention further comprises determining the expression level of one or more of the following genes in a sample from a subject under study: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28.

[0260] The term “expression level” is defined or described in the first method of the present invention, and this definition is applicable to the eleventh method of the present invention.

[0261] In certain embodiments, the expression levels of at least HMGB2, RETN, ZNF608, PYCARD genes, as well as one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 are determined by RNA-seq.

[0262] A second step of the eleventh method of the present invention includes comparing the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes obtained in the first step of the method with reference values.

[0263] In certain embodiments, the eleventh method of the present invention further includes comparing the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28 to a reference value.

[0264] The terms “reference value,” “increased,” “decreased,” and “equivalent” are defined or described in the first method of the present invention, and these definitions are applicable to the tenth method of the present invention.

[0265] When the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from a subject are compared with the reference values ​​of the markers, if the expression levels of these genes are greater than their respective reference values, the therapy is administered to the subject who would benefit from the administration of the therapy.

[0266] In certain embodiments, the method of the present invention further includes determining the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, -When the expression levels of the IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33 genes are elevated relative to a reference value, the therapy is administered to the subject who would benefit from the administration of the therapy, or -When the expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes are reduced relative to a reference value, the therapy is administered to the subject who would benefit from the administration of the therapy.

[0267] As is well known, the treatment of bacterial infections and / or sepsis depends on the patient or the severity of the infection. Appropriate therapies for treating bacterial infections and / or sepsis include, but are not limited to, antibiotics, vasoconstrictors, and organ support including renal hemodialysis and / or ventilator support.

[0268] The present invention is illustrated below by the following embodiments, which should be interpreted as merely illustrative and not as limiting the scope of the invention. [Examples]

[0269] material and method patient. This study was conducted using data and biobanking materials collected during two multicenter prospective observational studies, the Predict and Aclara studies, involving 1273 and 1274 hospitalized patients with ADC, respectively. These studies aimed to characterize ACLF syndrome and other phenotypes of ADC clinically and through genomic, transcriptome, and metabolomics assessments. Informed consent was obtained from patients or their representatives, and both studies were approved by the corresponding research and ethics committees at each institution. Authorization was also granted to use data and samples for supplementary studies.

[0270] This study involved 700 patients (Cohort 1) (383 from Predict and 317 from the Aclara study) who were hospitalized with ADC regardless of the presence of ACLF and were investigated by whole blood RNA sequencing (RNA-seq) within 2 days of admission (Time 1, T1). In 375 patients (Subcohort 1a), a second RNA-seq was performed at T2 (median duration of T1-T2 period: 7 days; 75%). The study was performed with a CI of 5-8 days. Finally, 59 patients from subcohort 1a were investigated by one or two additional RNA-seqs after T2 to form subcohort 1b. The CLF-SIG score was designed in cohort 1. The initial course of the CLIF-SIG score was estimated in subcohort 1a and estimated in more detail in subcohort 1b. Cohort 1 and subcohort 1a included patients with and without ACLF at T1 (Tables 1 and 2). Subcohort 1b included patients without ACLF at T1.

[0271] Based on disease severity at T1, patients were classified into a "low-severity group" (including 403 patients with "late-onset" pre-ACLF and unstable or stable decompensated cirrhosis) and a "high-severity group" (including 297 patients with "early-onset" pre-ACLF (development of ACLF within 21 days after T1) or ACLF).

[0272] Clinical features of systemic inflammation, biomarkers (CRP, WBC, neutrophil-to-lymphocyte ratio, and cytokines), and exacerbation events (acute alcoholic hepatitis or bacterial infection) were recorded at T1 in patients of Cohort 1, T1 and T2 in Subcohort 1a, and T1, T2, T3, and possibly T4 in Subcohort 1b. Acute alcoholic hepatitis was diagnosed according to NIAAA criteria.

[0273] New score. CLIF-SIG score. This study was designed in 700 patients in Cohort 1 based on top genes that distinguish patients into high-severity and low-severity groups, and aimed to obtain a single continuous variable to estimate the individual magnitude of systemic inflammation at a given time point. The change in CLIF-SIG score between T2 and T1 (CLIF-SIG delta score) estimates the course of systemic inflammation during the study period.

[0274] The strategy for designing the CLIF-SIG score relied on two evidence-based concepts and one assumption. The first concept was based on research supporting the idea that chronic systemic inflammation, a characteristic of patients with decompensated cirrhosis [patients with a history of acute decompensation (AD)], is a result of the continuous migration of bacterial and / or pathogen-associated molecular patterns (PAMPs) due to leaks from the intestinal mucosa. ADCs occur within the context of transient bursts of systemic inflammation, beyond the background of moderate chronic systemic inflammation likely associated with transiently enhanced bacterial migration or exogenous pro-inflammatory precipitants, primarily acute alcoholic hepatitis, or bacterial infections. Therefore, systemic inflammation in cirrhosis is a result of an innate immune response involving neutrophils, basophils, monocytes, macrophages, NK cells and dendritic cells, and B lymphocytes, responding to PAMPs by the rapid release of low-specific polyclonal antibodies. The second concept relies on unpublished data from the Predict study, particularly data obtained for the design of this study, which show that systemic inflammation is moderate in patients with “late-onset” pre-ACLF or unstable decompensated cirrhosis and stable decompensated cirrhosis, but severe in patients with “early” pre-ACLF or ACLF.

[0275] As a result of these two concepts, the inventors hypothesized that the top genes associated with innate immune cells and B cells that distinguish patients from high-severity and low-severity groups by T1 would include genes with high sensitivity for estimating the scale of systemic inflammation in ADC patients.

[0276] Chronic liver failure (CLIF)-systemic inflammation composite (SIC) score (CLIF-SIC score). This study was designed in 654 patients with T1 who had a complete set of well-recognized markers of systemic inflammation, including cytokines (IL-6, IL-8, IL-1RA, IP-10, MCP1, MIP1β, and TNFα) and proteins such as CRP, as well as cells such as WBCs and the neutrophil-to-lymphocyte ratio, which are known to be significantly elevated in patients with ACLF compared to patients without ACLF.

[0277] Calculating the score. The inventors randomly divided Cohort 1 into a training set (n=463) and a test set (n=237). The inventors then used the training set to compare whole blood transcriptomes from the high-severity group with those from the low-severity group using GSEA analysis of 1 MSigDB collection (Gene Ontology) and Blood Transcription Module (BTM) space. In doing so, the inventors identified four notable gene sets that were significantly richer in the high-severity group. These gene sets included BTM(M37.0) associated with immune activation generic clusters, BTM(S4) associated with monocyte surface signatures, BTM(M7.2) and immunoglobulin complex (GOCC 0019814) which were rich in NK cells (I). Across the four gene sets used to calculate the CLIF-SIG score, there were 220 leading edge genes (Supplementary Table 1). This involved using the glmnet R package to compute the Lasso solution and then finding the optimal λ in a 10-fold CV to select the gene that optimizes misclassification error when comparing patients in the above groups. The resulting score was the risk probability.

[0278] Patients with a complete set of inflammatory markers were randomly selected for the training set (n=430) and the test set (n=224). CLIF-SIC scores were calculated using a stepwise selection method comparing patients in the high-severity and low-severity groups using logistic regression. However, during the calculation, neither cytokines nor neutrophil-to-lymphocyte ratios remained in the model. Therefore, CLIF-SIC scores were based solely on CRP and WBC.

[0279] Characterization of the CLIF-SIG score and comparison with other markers of systemic inflammation. Novel scores, CRP, WBC count, and neutrophil-to-lymphocyte ratio characterizations to distinguish between high-severity and low-severity groups were performed at T1 in patients from the Predict and Aclara studies. However, characterization of the CLF-SIG score in each of the seven phenotypes of ADC is based only on patients from the Predict study, as follow-up clinical and laboratory data for the period 28–90 days after T1 were not available in the Aclara study. Indeed, stable and unstable decompensated cirrhosis, as well as the "late-onset" pre-ACLF phenotype, require the availability of these data for identification. The CLIF-SIG score was also characterized in patients with varying types and numbers of major complications and exacerbating events.

[0280] The CLIF-SIG score will be tested as a marker of systemic inflammation. To test whether a score based on gene transcription, such as the CLIF-SIG score, can estimate the scale of systemic inflammation, the inventors correlated this score with the CLIF-SIC score at T1.

[0281] Furthermore, ACLF is known to exhibit an extremely dynamic clinical course, closely related to changes in systemic inflammation, with recovery, improvement, stabilization, and worsening occurring within a few days of hospitalization. Therefore, the inventors investigated whether the CLIF-SIG score could detect these follow-up changes in systemic inflammation in the high-severity group.

[0282] Limitation of subcohort 1a for estimating the progression of CLIF-SIG scores. The course of CLIF-SIG scores in subcohort 1a was based on only two time points and a median study period of 7 days. To limit the possibility of misinterpretation in the characteristics of the acute inflammatory response, the inventors expanded the study to subcohort 1b, where patients had one or two additional RNA-seqs and a longer observation period. Subcohort 1b included 15 patients with “early” pre-ACLF, 4 patients with “late” pre-ACLF (ACLF onset 21 days after T1), and 38 patients with unstable or stable decompensated cirrhosis.

[0283] Characterization of CLIF-SIG score genes in publicly available RNA-seq datasets from unselected patients with bacterial infections. To evaluate whether the genes selected for the CLIF-SIG score also estimate the magnitude of systemic inflammation in other diseases associated with systemic inflammation, the inventors investigated a recently published RNA-seq dataset reported by Herwanto et al, BMC Res Notes (2021) 14:76, which included 40 healthy subjects, 32 patients with infections (20 including sepsis), and 19 patients with septic shock.

[0284] RNA-seq and other methods for statistical and bioinformatics evaluation. Results were presented as median and IQR for non-normally distributed data, mean and SD for normally distributed data, and count and relative percentage for categorical data. Statistical analyses were performed accordingly. Monaco database signatures were used to identify cell populations associated with the CLIF-SIG score gene.

[0285] RNA preparation from whole blood RNA was isolated from blood stored in Tempus tubes using the Tempus® Spin RNA Isolation Kit (reference 4380204, Applied Biosystems). RNA quality was assessed using Agilent RNA 6000 Nano and Pico Chips (Agilent Technologies, catalog numbers #5067-1511 and #5067-151, respectively), and concentration was assessed using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific, catalog number #Q32855). Sequencing libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Globin kit (Illumina Inc., catalog number 20020612) and TruSeq RNA CD Index Plate (Illumina Inc., catalog number 20019792) according to the TruSeq Stranded Total RNA Sample Prep-guide (part number 15031048 Rev.E). In short, we started with 500 ng of total RNA, depleted rRNA and globin mRNA, purified the remaining RNA, fragmented it, and primed it for cDNA synthesis. The first strand of cDNA was synthesized using SuperScript-II Reverse Transcriptase (Thermo Fisher Scientific, catalog number #18064-014) at 25°C for 10 minutes, 42°C for 15 minutes, 70°C for 15 minutes, and paused at 4°C. The second strand of cDNA was synthesized using Illumina reagent at 16°C for 1 hour. A-tailing and adapter ligation were then performed. Finally, library enrichment was achieved by PCR (30 seconds at 98°C; 15 cycles of 10 seconds at 98°C, 30 seconds at 60°C, and 30 seconds at 72°C; 5 minutes at 72°C and paused at 4°C).Subsequently, the library was visualized using the Agilent High Sensitivity DNA Kit (Agilent Technologies, catalog number #5067-4626) on an Agilent 2100 Bioanalyzer, quantified using the Qubit dsDNA HS DNA Kit (Thermo Fisher Scientific, catalog number #Q32854), and sequenced with at least 100 million paired-end 100nt reads on a NovaSeq-6000 (Illumina Inc.). Reads were aligned to the hg38 genome assembly and transcriptome using STAR v2.5.3a and GENCODE 26 annotation.

[0286] Gene expression was quantified using STAR (-quantMode GeneCounts) and VSN conversion (variance-stabilized normalization). Non-biological variability was then normalized using quantile normalization. Using the HUGO Genome Nomenclature Committee (HGNC), a resource for approved human gene nomenclature, the inventors included protein-coding genes, particularly locus types, immunoglobulin genes, and T cell receptor genes. Genes mediated by sex chromosomes and mitochondrial DNA were not included in the analysis.

[0287] Determination of gene expression of the 28 genes that constitute the CLIF-SIG score and their correlation with that gene expression. The expression of 28 genes constituting the CLIF-SIG score was determined using real-time PCR with a low-density microfluidic array (Fluidigm, Standard BioTools Inc., South San Francisco, CA) in whole blood samples collected in Tempus tubes from 40 patients with acute decompensated cirrhosis (in which the expression of the above genes had been previously measured by RNA-seq). The expression of the 28 genes constituting the CLIF-SIG score in the same patient group was also determined by digital quantification of nucleic acids using nCounter technology (Nanostring, Seattle, WA).

[0288] result Patient characteristics. Table 1 shows unpaired comparisons of clinical characteristics, laboratory data and scores at T1, and 28-day and 90-day mortality between patients in the low-severity and high-severity groups of Cohort 1. There were no differences in demographic data or the etiology of cirrhosis. Among the major complications at admission, ascites and hepatic encephalopathy were more frequent, while gastrointestinal bleeding was less frequent in patients in the high-severity group. The prevalence of acute alcoholic hepatitis and bacterial infections was higher, liver and kidney function was impaired, and biomarkers and scores indicating systemic inflammation were increased in the high-severity group. Mortality at 28 and 90 days post-admission was lower in the low-severity group and significantly higher in the high-severity group.

[0289] Comparing the changes in both severity groups, significant differences were observed only in several parameters of systemic inflammation, including a marked decrease in CRP and a moderate decrease in IL-6 in the low-severity group. CLIF-SIC scores decreased significantly in these patients in the low-severity group but not in patients in the high-severity group, consistent with the decrease in CRP observed in the former group. A significant decrease in CLIF-SIG scores was observed in the high-severity group, not the low-severity group. The improvement in CLIF-SIG scores in the high-severity group was consistent with a significant decrease in the prevalence of ACLF at T2, mainly attributable to the resolution of ACLF-1 in many patients and the resolution of ACLF-2 in some patients. The improvement in CLIF-SIG scores occurred in the high-severity group despite some of the 52 patients with “early” pre-ACLF developing ACLF during the T1-T2 period. There was no change in MELD-sodium scores in either group. The proportion of patients infected at T1 or who acquired an infection during the study period was 22% and 7% in the low-severity group, and 38% and 16% in the high-severity group, respectively.

[0290] In the 44 patients with low severity and the 15 patients with “early” pre-ACLF, differences were observed only in serum bilirubin and MELD scores, and not in any clinical features or biomarkers of systemic inflammation, including cytokines and CLIF-SIC scores. However, CLIF-SIG scores were significantly increased in “early” pre-ACLF patients.

[0291] [Table 1]

[0292] CLIF-SIG score as a marker of systemic inflammation The authors identified 28 genes (4 related to B cells and 24 related to innate immune cells) as independent variables to distinguish patients with high severity from those with low severity, and used them to calculate the CLIF-SIG score. The relative effect of each of these genes on the CLF-SIG score is shown in Figure 1A. For simplification, they are represented uniformly, but 24 genes were upregulated and 4 genes related to NK cells (XCL1, GZMH, GNLY, and FASLG) were downregulated. The CLIF-SIG score at T1 was significantly increased in the high-severity group (showing “early” pre-ACLF and ACLF separately) compared to the low-severity group and healthy subjects (Figure 1B), as well as in patients with ACLF compared to patients with “early” pre-ACLF. The CLIF-SIC score also distinguished patients with low severity from those with high severity and ACLF from “early” pre-ACLF. However, there was significant overlap between the two groups (Figure 1C). To enable comparative estimation, the median CLIF-SIG and CLIF-SIC scores are also given as multiplier increases for each comparison in parentheses at the top of the figure. Figure 1D shows that the area under the ROC curve distinguishing the high-severity group from the low-severity group in the training and test cohorts at T1 is significantly higher when using the CLIF-SIG score than when using the CLIF-SIC score, indicating the higher accuracy of the CLIF-SIG score. Figure 1E shows that the CLIF-SIG score and the CLIF-SIC score were directly and significantly correlated.

[0293] Figures 2A–2D illustrate the characterization of CLIF-SIG and CLF-SIC scores, CRP, and WBC within different phenotypes of ADCs in the Predict study. None of these variables distinguished between stable cirrhosis, unstable cirrhosis, and “late-onset” pre-ACLF, suggesting that these different phenotypes likely develop within a context of moderate systemic inflammation of similar magnitude. The CLIF-SIG score was the only variable that distinguished “early” pre-ACLF from ACLF, and WBC was the only variable that distinguished ACLF-1 from ACLF-2 and ACLF. Finally, all variables except WBC distinguished between low-severity and high-severity groups, but there was strong overlap in all except the CLIF-SIG score.

[0294] The progression of the CLIF-SIG score correlates with the patient's clinical course. definition The authors were the first to identify the magnitude of the delta CLIF-SIG score that better stratified the three inflammatory courses detected in subcohort 1a: upregulation, minimal or no change, or downregulation (Figure 3A).

[0295] The course of patients in the low-severity group during the study period was uneventful, except for a small percentage of patients who developed infections. In contrast, the course of patients in the high-severity group was highly variable and stratified as "good" if ACLF resolved within the first 21 days after enrollment (no ACLF at T2, 60 patients), presented with stable ACLF-1 (ACLF-1 at T1 and T2, 20 patients), or improved from ACLF-3 or ACLF-2 to ACLF-1 (5 patients); "poor" if they had "early" pre-ACLF (all progressed from no ACLF to any grade of ACLF within 21 days after T1) (42 patients); presented with ACLF-1 at T1 which progressed to ACLF-2 or ACLF-3, or presented with ACLF-2 which progressed to ACLF-3 (9 patients); or presented with ACLF-3 at T1 which improved to only ACLF-2, stable ACLF-2, or ACLF-3 (27 patients).

[0296] Finally, outcomes were defined according to whether the patient died or survived within the first 28 days of hospitalization.

[0297] The course of systemic inflammation in subcohorts 1a and 1b shows a significant discrepancy between systemic inflammatory responses and hospitalization.

[0298] The acute systemic inflammatory response in sepsis follows an explosive course, involving an initial elevated-onset (AD) phase, a short-intermediate top-steady (ITS) inflammatory phase, and a final declining-recovery (DR) phase, generally lasting about one week in patients who respond to treatment (Mantovani A, Garlanda C. Humoral innate immunity and acute phase protein. N Engl J Med 2023;388:439-452). In contrast, the authors have previously reported that systemic inflammation in decompensated cirrhosis also manifests as a burst, but with a longer duration (Fernandez J, Claria J, Amoros A, et al. Effects of albumin treatment on systemic and portal hemodynamics and systemic inflammation in patients with decompensated cirrhosis. Gastroenterology 2019;157:149-162). Perhaps due to this, using two study points and a median study period of 7 days, the authors were able to detect only a monophase of the inflammatory process (Figure 3B). Of the 375 patients from subcohort 1a, 76 patients (20.3%) showed upregulation of CLIF-SIG scores, 167 patients (44.5%) showed slight or no change, and 132 patients (35.2%) showed downregulation.

[0299] The discrepancy between the onset of systemic inflammation and hospitalization was a predictable finding because ascites or encephalopathy can take several days to become clinically apparent. However, the authors were surprised by the high frequency of this period in most patients, and especially by its long duration. In fact, the initial AD phase was detected in only 76 patients, the DR phase in 132 patients, and both the initial AD phase and the AD phase + TSI phase occurred before hospitalization in 167 and 132 patients, respectively. Therefore, phase AD or A-D+TSI occurred 1-2 weeks before hospitalization in most patients. While it is difficult to estimate the duration of the systemic inflammatory response from our data, the one-week duration during the observation period of each phase suggests that it may average more than 3 weeks.

[0300] The authors identified a group of only 10 patients (2.6%) with undetectable systemic inflammation (CLIF-SIG scores at T1 and T2 that were lower than the median CLIF-SIG score in healthy subjects) (Figure 3B).

[0301] Figure 3C shows ROC curves estimating the accuracy of the CLIF-SIG score in differentiating all patients in Cohort 1 into low-severity and high-severity groups, and a score value of 0.386 (detected by the best Youden index) as a better cutoff level for differentiating these groups at T1. Subsequently, this critical CLIF-SIG score level (CSL) was applied to T2 to stratify the magnitude of systemic inflammation into moderate (<0.386) or severe (≧0.386).

[0302] Figure 3D shows a complex network of lines connecting 3-4 consecutive CLIF-SIG scores measured in patients of subcohort 1b. However, it is noteworthy that, at first glance, there is a high frequency of patients with severe score downregulation from day 0 or between days 7 and 14, which reflects patients who had an AD + TSI or AD phase of inflammatory response prior to admission, thus supporting the observations in subcohort 1a.

[0303] Figures 3E–3H show estimated graphical representations of the most relevant clinical courses identified in Figure 3D. Only four patients had all or part of the three phases of inflammatory response detected within the first three weeks after admission (Figure 3E). Figures 3F and 3G show representative examples of systemic inflammation curves lacking either the TSI phase + AD phase or only the AD phase. Finally, Figure 3H shows the presence of a small number of patients in subcohort 1b who developed ADC in the absence of a detectable systemic inflammatory response, and patients who developed recurrent systemic inflammatory responses, as indicated by the decline-recovery phase of the previous systemic inflammation burst, followed by an upward phase (3 cases), or the complete course of the second inflammatory response.

[0304] Scale of systemic inflammation and the clinical course of patients To improve the graphical representation of the relationship between the magnitude of systemic inflammation and the clinical course, patients from subcohort 1a were assigned two colors, red or green, according to the course of their CLIF-SIG scores (Figure 4A). Patients whose T2 score was upregulated to a level of 0.386 or higher during the study period, or who were stable but followed a highly upregulated course as also defined by a T2 score of 0.386 or higher, or who were downregulated but did not reach a T2 score of less than 0.386, were assigned red to indicate severe systemic inflammation. The remaining patients were assigned green to indicate moderate systemic inflammation.

[0305] In subcohort 1a, CLIF-SIG scores progressed in 195 patients in the low-severity group and 180 patients in the high-severity group, respectively, with upregulation in 41 and 35 patients (21% and 19.4%), slight changes in 96 and 71 patients (49.2% and 39.4%), and downregulation in 58 and 74 patients (29.7% and 41.1%) (Figure 4A). Severe systemic inflammation was very frequent in patients in the high-severity group (77.4%) and rare in the low-severity group (22.6%). The length of hospital stay was 14 (9-22) days and 7 (5-10) days, respectively.

[0306] Two pieces of evidence from subcohort 1a support the correlation between the severity of systemic inflammation and the patient's clinical course. First, the prevalence of a poor clinical course and the 28-day mortality were 53% and 32%, respectively, in patients with severe systemic inflammation, and 32% and 23%, respectively, in patients with moderate systemic inflammation (p<0.001 and p<0.05, respectively; data below Figure 5B). Second, the frequency of patients developing a poor clinical course was 60.6% (20 out of 33) and 63.2% (43 out of 68) during the AD phase and TSI phase of the inflammatory response, respectively, compared to only 24.3% (17 out of 70) during the DR phase, indicating a significant decrease in the risk of developing a poor clinical course during the DR phase.

[0307] However, the clearest evidence that systemic inflammation correlated with the clinical course was observed in subcohort 1b (Figure 4B). Most patients whose clinical course was within the hypothetical high-inflammatory range (CSL ≥ 0.386) developed ACLF, which was extremely rare in patients whose clinical course was within the low-inflammatory range.

[0308] CLIF-SIG score gene in unselected patients with bacterial infections. Because RNA prepared by Herwanto V, Tang B, Wan Ya, et al. Blood trancriptome analysis of patients with uncomplicated bacterial infection and sepsis. BMC Res Notes (2021) 14:76 is depleted from β-globin RNA in patients with bacterial infection, the authors were able to examine 26 genes for the score rather than IGKV2D-30 and IGKV2D-28. Figure 5A shows the expression of CLIF-SIG score genes across three groups (healthy subjects and non-cirrhotic patients with bacterial infection (with sepsis or septic shock)) as a heatmap. Figures 5B and 5C show that the expression of these genes, as shown in the heatmap and PCA plot, was significantly increased in patients with bacterial infection compared to healthy subjects, and that this distinguished patients with and without septic shock as a surrogate for the severity of systemic inflammation. Figure 5B shows that the downregulated genes (XCL1, GZNH, GNLY, and FASLG) were as accurate as the upregulated genes in identifying subsets of these targets.

[0309] CLIF-SIG score, composed of 4 genes The results in Figure 6 show that a model consisting of four genes (HMGB2, RETN, ZNF608, and PYCARD) was able to find significant differences in the distribution of healthy subjects and patients with low and high severity (Figure 6A). As shown in Figure 6B, even with only four genes, this model performed better than the CLIF-SIC score, which consists of 28 genes, in both the training and test sets when distinguishing patients into low and high severity groups. This score also showed a positively significant association with the CLIF-SIC score and an association with the score and systemic inflammation (Figure 6C).

[0310] CLIF-SIG score, composed of 28 genes The expression of the 28 genes constituting the CLIF-SIG score was measured using two additional platforms that provide precise and accurate quantification of individual RNA molecules, similar to that of the RNA sequencing platform. These two platforms were Fluidigm, a low-density microfluidic array system, and Nanostring, a digital molecular barcoding technology, both of which were commonly available in tertiary hospitals. The results of these additional measurements showed a high and significant correlation between the expression data from Fluidigm and Nanostring and the expression results of the 28 genes obtained by RNA sequencing. These findings support the feasibility of measuring the CLIF-SIG score in a clinical setting closer to the patient's bedside (Figure 7).

Claims

1. An in vitro method for determining the presence of acute chronic liver failure (ACLF) in subjects suffering from cirrhosis or late-onset pre-ACLF, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, An in vitro method in which the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are greater than the reference values ​​of the genes, thereby indicating that the subject has ACLF.

2. a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression levels of the genes with their reference values, The expression level of the gene changes compared to the reference value of the gene, and the change in the expression level is - Increased expression levels of the aforementioned genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - Decreased expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes. In that case, the subject has ACLF, The in vitro method according to claim 1.

3. An in vitro method for determining the risk of developing ACLF in subjects suffering from cirrhosis or late-onset pre-ACLF, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, An in vitro method for indicating that the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in a sample from the subject are greater than the reference values ​​for the genes, thereby indicating that the subject is at high risk of developing ACLF.

4. a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression levels of the genes with their reference values, The expression level of the gene changes compared to the reference value of the gene, and the change in the expression level is - Increased expression levels of the aforementioned genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - Decreased expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes. If so, it indicates that the subject has a high risk of developing ACLF. The in vitro method according to claim 3.

5. The in vitro method according to any one of claims 1 to 4, wherein the subject is suffering from unstable or stable acute decompensated cirrhosis of the liver.

6. An in vitro method for monitoring the progression of ACLF in a subject, a) To determine the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, b) Comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time after the onset of ACLF, An in vitro method in which, if the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes are reduced compared to the reference value, this indicates ACLF resolution.

7. a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time after the onset of ACLF, The change in the expression level of the gene in the sample compared to the reference value of the gene indicates ACLF elimination, and the change in the expression level indicates - A decrease in the expression levels of the aforementioned genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - An increase in the expression level of the XCL1, GZMH, GNLY, FASLG, or SAMD3 gene. The in vitro method according to claim 6.

8. An in vitro method for monitoring the effectiveness of therapy in subjects suffering from ACLF, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time, An in vitro method that indicates the effectiveness of the therapy if the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are lower than the reference value.

9. a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression level of the gene with a reference value, which is the expression level of the same gene obtained from the patient at an earlier point in time, A change in the expression level of the gene in the sample compared to a reference value of the gene indicates that the therapy is effective, and the change in the expression level indicates that - A decrease in the expression levels of the aforementioned genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - An increase in the expression level of the XCL1, GZMH, GNLY, FASLG, or SAMD3 gene. The in vitro method according to claim 8.

10. An in vitro method for selecting a therapy for patients suffering from ACLF, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression level of the gene with a reference value which is the expression level of the same gene obtained from the patient at an earlier point in time, An in vitro method for indicating that if the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are elevated relative to the reference value, the patient should be treated with therapy aimed at treating the effects of hepatic failure and / or for liver transplantation.

11. a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression level of the gene with a reference sample, wherein the reference value is the expression level of the same gene obtained from the patient at an earlier point in time. When compared to the aforementioned reference sample, the expression level of the gene changes, and the change in the expression level is - Increased expression levels of the aforementioned genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - A decrease in the expression level of the XCL1, GZMH, GNLY, FASLG, or SAMD3 gene is indicated. This indicates that the patient is a candidate for therapy aimed at treating the effects of liver failure and / or for liver transplantation. The in vitro method according to claim 10.

12. An in vitro method for determining the degree of systemic inflammation in a subject, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, An in vitro method in which the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are greater than the reference values ​​for the genes, indicating that the subject has severe systemic inflammation.

13. a) Determine the expression level of one or more of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, IGKV2-28, and b) further comprising comparing the expression levels of the genes with their reference values, The expression level of the gene changes compared to the reference value of the gene, and the change in the expression level is - Increased expression levels of the aforementioned genes IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, RNASE2, MS4A4A, FPR1, CRISPLD2, C3AR1, STX11, OSCAR, IGKV2-28, IGKV2-33, or - Decreased expression levels of the XCL1, GZMH, GNLY, FASLG, or SAMD3 genes. In that case, it indicates that the subject has severe systemic inflammation. The in vitro method according to claim 12.

14. The in vitro method according to any one of claims 12 or 13, wherein the subject is suffering from ACLF or an infectious disease.

15. The method according to claim 14, wherein the infectious disease is a bacterial infection.

16. The method according to any one of claims 1 to 14, wherein the ACLF is ACLF1, ACLF2, ACLF3, or early ACLF.

17. An in vitro method for determining the presence of a bacterial infection accompanied by sepsis in a subject, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, An in vitro method in which the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are greater than the reference values ​​of the genes, thereby indicating that the subject has a bacterial infection accompanied by sepsis.

18. The method according to claim 17, wherein the subject has septic shock.

19. Expression levels of HMGB2, RETN, ZNF608 and PYCARD genes, as well as IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBO The method according to any one of claims 2, 4, 7, 9, 11, and 13, comprising determining the expression levels of at least two genes selected from the group consisting of the genes AT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28.

20. At least the HMGB2, RETN, ZNF608, and PYCARD genes, as well as the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTBR, ​​PTPRE, and TLR2. The method according to any one of claims 1 to 19, wherein determining the expression level of one or more of MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 comprises determining the mRNA level of the gene or fragment thereof.

21. At least the HMGB2, RETN, ZNF608, and PYCARD genes, as well as the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LILRB2, LTB The method according to claim 20, wherein the determination of the expression level of one or more of R, PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28 is performed by RNA-seq.

22. The method according to any one of claims 1 to 21, wherein the sample is a biological fluid.

23. The method according to claim 22, wherein the biological fluid is blood.

24. A kit or assay device comprising reagents suitable for determining the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes.

25. Determine the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, and also determine the expression levels of the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, ABCA1, LIL A kit or assay device comprising reagents suitable for determining the expression levels of one or more of the following: RB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28.

26. A reagent specific to determining the expression levels of at least HMGB2, RETN, ZNF608, and PYCARD genes, or The kit or assay device according to claim 24 To determine the presence of ACLF in a subject, to determine the risk of developing ACLF, to monitor the progression of ACLF, to monitor the effectiveness of therapy, to select patients with ACLF for therapy, to determine the degree of systemic inflammation, or to determine the presence of a bacterial infection with sepsis. use.

27. Determination of the expression levels of at least HMGB2, RETN, ZNF608 and PYCARD genes, as well as the following genes: IGFBP7, ORM1, ACSL1, BCL6, OLFM4, SERPINB10, STK32B, CYP4F2, CDK14, PCBP3, MCTP1, IGKV2D-28, IGKV2D-30, CD163, A A reagent specific for determining the expression level of one or more of BCA1, LILRB2, LTBR, ​​PTPRE, TLR2, MBOAT7, GZMH, GNLY, FASLG, XCL1, RNASE2, STX11, CRISPLD2, OSCAR, C3AR1, IGKV1D-33, FPR1, SAMD3, MS4A4A, and IGKV2-28, or The kit or assay device according to claim 25 To determine the presence of ACLF in a subject, to determine the risk of developing ACLF, to monitor the progression of ACLF, to monitor the effectiveness of therapy, to select patients with ACLF for therapy, to determine the degree of systemic inflammation, or to determine the presence of a bacterial infection with sepsis. use.

28. A computer system comprising one or more programs, wherein the one or more programs comprises instructions for performing the method according to any one of claims 1 to 23.

29. A computer-readable storage medium for storing one or more programs, wherein when the one or more programs are executed by one or more processors of an electronic device, the computer-readable storage medium includes instructions that cause the electronic device to execute the method according to any one of claims 1 to 23.

30. A method for determining the presence of acute chronic liver failure (ACLF) in a subject suffering from cirrhosis or late-onset pre-ACLF, and for treating the said patient, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the patient, and b) Comparing the expression level of the gene with the reference value of each gene, If the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the patient are greater than their respective reference values, the patient is treated with appropriate therapies including antibiotics, diuretics, albumin, lactulose, β-adrenergic blockers, vasoconstrictors, sclerosants, intrahepatic portosystemic shunts, plasmapheresis, mechanical ventilation, renal hemodialysis, albumin dialysis, liver transplantation, and / or combinations thereof.

31. A method for determining the risk of developing ACLF in subjects suffering from cirrhosis or late-onset pre-ACLF, and for treating patients accordingly, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, A method wherein, if the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are greater than the reference values ​​of the genes, the patient is treated with a therapy suitable for the prevention of ACLF, comprising antibiotics, diuretics, albumin, lactulose, β-adrenergic blockers, terlipressin, sclerosing agents, intrahepatic portosystemic shunts, and / or combinations thereof.

32. A method for determining the degree of systemic inflammation in a subject and treating the subject, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, A method wherein, if the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are greater than the reference values ​​of the genes, the patient is treated with a therapy appropriate for severe systemic inflammation, including antibiotics, corticosteroids, albumin, plasmapheresis, and / or a combination thereof.

33. A method for determining the presence of a bacterial infection accompanied by sepsis in a subject and for treating the subject, a) Determining the expression levels of HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject, and b) Comparing the expression levels of the genes in the sample with their reference values, A method wherein, if the expression levels of the HMGB2, RETN, ZNF608, and PYCARD genes in the sample from the subject are greater than the reference values ​​for the genes, the patient is treated with a therapy appropriate for bacterial infection and / or sepsis, including antibiotics, vasoconstrictors, organ support including renal hemodialysis and / or ventilator support.

Images