Method for determining mortality risk from infectious inflammatory diseases based on WARS and cytokine concentrations

Abstract

The present invention relates to a method for determining the risk of death from infectious inflammatory diseases based on the concentrations of tryptophanyl-tRNA synthetase (WARS) and cytokines. The method for determining the risk of death of the present invention can effectively select patients at high risk of death from infectious inflammatory diseases in a short period of time, and can greatly facilitate timely treatment.

Description

[Technical field]

[0001] The present invention relates to a method for determining the mortality risk of infectious inflammatory diseases based on WARS and cytokine concentrations. [Background technology]

[0002] Aminoacyl-tRNA synthetases (ARSs) are enzymes that mediate the reaction of specifically binding amino acids to tRNAs and play a central role in protein synthesis. In recent years, it has been discovered that these ARSs are involved in various biological phenomena such as apoptosis, angiogenesis, and inflammatory responses in addition to their essential functions. In particular, the general function of human tryptophanyl-tRNA synthetase 1 (WARS1) is to act as an aminoacyl-tRNA synthetase that links tryptophan-recognizing tRNAs for protein synthesis, but a recent interest is the role of WARS1 in host defense mechanisms against infection. The present inventors previously reported that within minutes of infection, monocytes immediately secrete pre-existing WARS1 into the extracellular space without de novo synthesis (Young Ha Ahn. et al. Secreted tryptophanyl-tRNA synthetase as a primary defense system against infection. Nature Microbiology 2:16191(2016)).

[0003] By functioning as an endogenous ligand for TLR4 and TLR2, secreted WARS1 activates macrophages and induces innate immune activation. WARS1 also initiates the production of proinflammatory cytokines and chemokines, including TNF-α, IL-6, MIP-1α, IL-8, and IFN-γ, and induces neutrophil infiltration. WARS1 gene expression is then activated by IFN-γ, maintaining the continuous secretion of WARS1. Consistent with these findings, we confirmed that WARS1 is highly elevated in the blood of patients with sepsis, but not in patients with sterile chronic inflammatory disorders, and is secreted regardless of the type of pathogen, including gram-positive and negative bacteria, viruses, and fungi.

[0004] Sepsis is a systemic inflammatory response syndrome that appears as a complication of infectious diseases. If the cause cannot be diagnosed quickly and accurately at an early stage, it can progress to severe sepsis, septic shock, multiple organ dysfunction syndrome (MODS), which can lead to dysfunction of the lungs, kidneys, liver, circulatory system, etc., disseminated intravascular coagulation (DIC), acute respiratory distress syndrome (ARDS), or acute kidney injury (AKI), and can be fatal.

[0005] In Korea, the number of patients who were hospitalized or treated for sepsis, an infectious inflammatory disease, increased from 22,430 in 2004 to 34,371 in 2008 (National Health Insurance Service, Sepsis Incidence Statistics, 2009). The mortality rate from severe sepsis has also reached 30% (Jeong, Tae-young et al., Application of the MEDS (Mortality in Emergency Department Sepsis) scoring system as a prognostic tool for patients suspected of sepsis at the emergency treatment stage. Korean Journal of Emergency Medicine 2007;18(2):150-57.). Augus et al. (Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 2001;29:1303-10.) stated that more than half of sepsis patients need to be treated in an intensive care unit. Since it is difficult to predict the occurrence of such serious life-threatening conditions, it is also difficult to detect the conditions early and provide prompt and appropriate treatment within the golden time frame.

[0006] In order to predict the serious condition more accurately and reduce the mortality rate of patients, it is urgent to develop a method for quickly and accurately distinguishing or diagnosing the severity of a patient. Korean Patent Registration No. 10-1771697 discloses a method for diagnosing sepsis or septic shock by comparing the expression level of WARS with that of normal subjects. However, the patent only confirmed that the WARS level in deceased patients was significantly higher than that in surviving patients, and did not disclose a method for more accurately distinguishing a patient group with a high probability of death even if the WARS concentration is low (or a patient group with a high probability of survival even if the WARS concentration is high) by investigating the correlation with other cytokines and / or chemokines.

[0007] The items described above as background art are intended merely to enhance understanding of the background of the present invention, and should not be considered as prior art already known to those having ordinary skill in the art. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have made an intensive effort to find a method for effectively selecting patients with a high risk of death using samples from patients with infectious inflammatory diseases such as sepsis. As a result, they have developed a method for identifying patients with a high risk of death with high accuracy by measuring the concentration of cytokines (e.g., IL-8) together with WARS, and have completed the present invention.

[0009] Therefore, an object of the present invention is to provide a method for providing information necessary for determining the risk of death due to infectious inflammatory diseases, which includes a step of measuring the concentrations of WARS (tryptophanyl-tRNA synthetase) and cytokines in a sample from a subject.

[0010] Another object of the present invention is to provide a kit for determining the risk of death due to an infectious inflammatory disease, comprising: a preparation for measuring the expression level of a WARS protein or a gene encoding the same; and a preparation for measuring the expression level of a cytokine protein or a gene encoding the same.

[0011] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings. [Means for solving the problem]

[0012] According to one aspect of the present invention, there is provided a method for providing information necessary for determining a risk of death due to an infectious inflammatory disease, comprising the step of measuring the concentrations of WARS (tryptophanyl-tRNA synthetase) and cytokines in a sample from a subject.

[0013] The inventors have made extensive efforts to find a method for effectively selecting patients at high risk of death using samples from patients with infectious inflammatory diseases such as sepsis, and as a result, have developed a method for identifying patients at high risk of death with high accuracy by measuring the concentration of cytokines (e.g., IL-8) in conjunction with WARS.

[0014] As used herein, the term "WARS" refers to tryptophanyl-tRNA synthetase, also known as tryptophan-tRNA ligase, TrpRS, WRS, etc. WARS is an enzyme that mediates the aminoacylation reaction between the amino acid tryptophan and tRNA. WARS is encoded by the WARS gene in humans, and the amino acid sequence of the protein and the mRNA nucleotide sequence are publicly known under Genbank accession number NP_004175.2 (protein) and Genbank accession number NM_004184.3 (mRNA nucleotide sequence), etc. WARS has two isoforms: a cytoplasmic form (WARS1 or tryptophanyl-tRNA synthetase 1, cytoplasmic) and a mitochondrial form (WARS2 or tryptophanyl-tRNA synthetase 2, mitochondrial).

[0015] According to a preferred embodiment of the present invention, the WARS of the present invention is WARS1 (first sequence in the sequence listing).

[0016] According to a preferred embodiment, the present invention further comprises the step of providing a sample from a subject.

[0017] As used herein, the term "subject" refers to an individual suffering from, suspected of suffering from, or at risk of suffering from an infectious inflammatory disease (including individuals exposed to a virus, bacteria, fungus, etc. that causes an infectious inflammatory disease), and includes mammals, including humans (dogs, cats, cows, horses, etc.), birds, insects, amphibians, etc. Preferably, the subject is a human.

[0018] As used herein, the term "infectious inflammatory disease" refers to an infectious disease accompanied by inflammation caused by infection with a virus, bacteria, or fungus, and is preferably one or more diseases selected from the group consisting of pneumonia, pulmonary tuberculosis, tuberculosis, sepsis, and septic shock, but is not limited thereto.

[0019] The sepsis includes, but is not limited to, early sepsis, severe sepsis, septic shock, and sepsis-associated multiple organ dysfunction syndrome (MODS), disseminated intravascular coagulation syndrome (DIC), acute respiratory distress syndrome (ARDS), or acute kidney injury (AKI).

[0020] According to a preferred embodiment, the infectious inflammatory disease is sepsis or septic shock.

[0021] As used herein, the term "sample" refers to a biological sample naturally or artificially separated or collected from a subject, preferably, but not limited to, a cell, a tissue, blood, whole blood, plasma, or serum.

[0022] As used herein, the term "cytokine" refers to a protein immunoregulator secreted from immune cells, and is used to include chemokines.

[0023] The cytokines include IL-8, IL-6, MIP-1α, INF-γ, TNF-α, IL-1β, IL-10, and the like.

[0024] The concentration of the WARS and / or cytokines can be confirmed by measuring their expression levels. The expression levels of the WARS and / or cytokines can be the expression levels of these proteins or genes encoding them.

[0025] As used herein, the term "expression" refers to the production of a protein or nucleic acid in a cell. Protein is used interchangeably with polypeptide or peptide. Polynucleotide or nucleic acid refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single or double stranded form, and includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, unless otherwise limited. mRNA is RNA that conveys genetic information (gene-specific base sequences) from a particular gene to ribosomes that specify the amino acid sequence during protein synthesis.

[0026] The expression level of the protein can be detected or measured using an antibody or a fragment thereof (preferably an antigen-binding fragment) that specifically binds to a WARS and / or cytokine protein. The expression level of the protein can be measured by any method known in the art, including, but not limited to, western blotting, dot blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation assay, flow cytometry (FACS), or protein chip methods. Preferably, the ELISA method can be used.

[0027] As used herein, the term "antibody" is a term of the art and may be used interchangeably herein to refer to a molecule having an antigen-binding site that specifically binds to an antigen. As used herein, the term includes whole antibodies and any fragments (i.e., "antigen-binding portions") or single chains thereof. According to one embodiment of the present invention, an "antibody" refers to a glycoprotein or antigen-binding portion thereof that comprises at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. According to another embodiment of the present invention, an "antibody" refers to a single-chain antibody that comprises a single variable region (domain), e.g., a VHH domain. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Generally, the heavy chain constant region comprises three domains, CH1, CH2, and CH3, and each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises one domain, i.e., CL.

[0028] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), which are located between the more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino-terminus to the carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0029] As used herein, the term "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of an immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or the first component (C1q) of the classical complement system. Thus, Fc includes the constant region of an antibody excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). In IgG, IgA, and IgD antibody isotypes, the Fc region includes two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the antibody's two heavy chains, while IgM and IgE Fc regions include three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. The Fc provided herein can be a native sequence Fc or an Fc variant (e.g., a non-naturally occurring Fc), including any allotypic variant. The Fc may also be an Fc-containing protein polypeptide, such as a "binding protein comprising an Fc region," also referred to as an "Fc fusion protein" (eg, an antibody or immunoconjugate).

[0030] The antibody can have any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) or any subclass (e.g., IgG1, IgG2, IgG3, and IgG4 in humans, or IgG1, IgG2a, IgG2b, and IgG3 in mice, etc.) of immunoglobulin molecule. Immunoglobulins, e.g., IgG1, exist in several allotypes that differ from each other in some amino acids. The antibodies disclosed herein may be derived from any one of the commonly known isotypes, types, subclasses, and allotypes. In certain embodiments, the antibodies presented herein belong to the IgG1, IgG2, IgG3, or IgG4 subclass, or any hybrid thereof. In certain embodiments, the antibodies belong to the IgG2, IgG4, or IgG2 / IgG4 subclass.

[0031] Examples of antibodies include naturally occurring or non-naturally occurring antibodies; monoclonal or polyclonal antibodies; chimeric or humanized antibodies; human or non-human antibodies; fully synthetic antibodies; single chain antibodies; monospecific antibodies; and multispecific antibodies (including bi- or trispecific antibodies).

[0032] As used herein, the term "neutralizing antibody" refers to an antibody that neutralizes the biological effects of pathogens or infectious particles when they penetrate the body and protects cells. Neutralizing antibodies are part of the immune response of the acquired immune system against viruses, bacteria, fungi, and microbial toxins. Neutralizing antibodies are produced in a specialized form and bind to the surface structures of infectious particles, achieving immunity by preventing infectious antigens from interacting with host cells.

[0033] In the present specification, the term "fragment" is interpreted to include one or more fragments of an antibody that retains the ability to specifically bind to an antigen, or all forms in which the "one or more fragments of an antibody that retains the ability to specifically bind to an antigen" are linked to other molecules (including a portion of the same or another antibody (such as Fc)). Examples of fragments include a monovalent fragment (Fab fragment) composed of the VL, VH, CL, and CH1 domains; a bivalent fragment (F(ab')2 fragment) containing two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment composed of the VH and CH1 domains; an Fv fragment composed of the VL and VH domains of one arm of an antibody and a disulfide-linked Fv (sdFv); a dAb fragment composed of the VH domain; and a combination of an isolated complementarity determining region (CDR) or two or more isolated CDRs that can be selectively linked by a linker. The VL and VH regions may also be linked by a linker to form a single protein chain that pairs to form a monovalent molecule (single-chain Fv (scFv)). Such single-chain antibodies are also included in antibody fragments. The antibodies or antibody fragments also include tetrameric antibodies comprising two heavy chain molecules and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers; intrabodies; monovalent antibodies; camelid antibodies; and single-domain antibodies (sdAbs).

[0034] According to a preferred embodiment of the present invention, the antibody fragment of the present invention is a fragment selected from the group consisting of Fab, F(ab')2, Fd, sdFv, Fv, dAb, scFv, sdAb and tetramer, or a combined form of said fragment and Fc.

[0035] The expression level of the gene encoding the WARS and / or cytokine can be measured by measuring the mRNA level of the protein, which can be measured by amplifying mRNA or cDNA from a sample of the subject using a primer set or a probe that specifically binds to the mRNA of the WARS and / or cytokine, or by measuring the presence and expression amount of the mRNA in the sample of the subject using hybridization with the probe. When measuring the mRNA expression level, any method commonly used in the art for confirming the expression level may be used without limitation, including, but not limited to, reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA sequencing, hybridization using nanostring, and in situ hybridization of tissue sections.

[0036] As used herein, the term "primer" refers to a short single strand oligonucleotide that acts as a starting point for DNA synthesis. A primer specifically binds to a template polynucleotide under suitable buffer and temperature conditions, and DNA is synthesized by DNA polymerase adding a nucleotide triphosphate having a base complementary to the template DNA to the primer. A primer generally consists of a 15-30 base sequence, and the temperature at which it binds to the template strand (melting temperature, Tm) varies depending on the base composition and length. A person skilled in the art can easily design primers by referring to the mRNA or cDNA base sequence of WARS and / or cytokines, and use them to determine the risk of death due to infectious inflammatory diseases.

[0037] In this specification, the term "probe" refers to a polynucleotide fragment such as RNA or DNA having a length of several to several hundred base pairs that can specifically bind to the mRNA or cDNA (complementary DNA) of a specific gene, and is labeled, so that the presence or absence and expression level of the target mRNA or cDNA to which it binds can be confirmed. For the purpose of the present invention, a hybridization reaction between a probe complementary to WRS mRNA and a test sample is performed, and the expression level of WARS and / or mRNA is measured, thereby enabling the determination of the risk of death due to infectious inflammatory diseases. The selection of the probe and the hybridization conditions can be appropriately selected by techniques known in the art.

[0038] According to a preferred embodiment, the present invention includes measuring the concentrations of WARS (tryptophanyl-tRNA synthetase) and IL-8.

[0039] According to a preferred embodiment, the present invention includes determining whether the concentration of WARS is 100 ng / ml or more and the concentration of IL-8 is 100 pg / ml or more.

[0040] According to a preferred embodiment, the present invention includes determining whether the concentration of WARS is 100 ng / ml or more and less than 200 ng / ml, and the concentration of IL-8 is 400 pg / ml or more.

[0041] When the WARS concentration is 100 ng / ml or more and less than 200 ng / ml, and the IL-8 concentration is 400 pg / ml or more, preferably 430 pg / ml or more, more preferably 440 pg / ml or more, even more preferably 450 pg / ml or more, even more preferably 460 pg / ml or more, and most preferably 466.5 pg / ml or more, information useful for distinguishing patients from a patient group at high risk of death can be provided.

[0042] According to a preferred embodiment of the present invention, the present invention includes identifying a patient group with a high risk of death when the WARS concentration is 100 ng / ml or more but less than 200 ng / ml and the IL-8 concentration is 466.5 pg / ml or more.

[0043] According to one embodiment of the present invention, when the WARS concentration was greater than 100 ng / ml but less than 200 ng / ml and the IL-8 concentration was greater than 466.5 pg / ml, all of the sepsis patients died (6 / 6) (Figure 3a).

[0044] According to a preferred embodiment, the present invention includes determining whether the concentration of WARS is 200 ng / ml or more and the concentration of IL-8 is 100 pg / ml or more.

[0045] When the WARS concentration is 200 ng / ml or higher, and the IL-8 concentration is 100 pg / ml or higher, preferably 105 pg / ml or higher, more preferably 110 pg / ml or higher, even more preferably 115 pg / ml or higher, and most preferably 115.65 pg / ml or higher, information useful for distinguishing patients from those at high risk of death can be provided.

[0046] According to a preferred embodiment of the present invention, the present invention includes identifying a patient group with a high risk of death when the WARS concentration is 200 ng / ml or more and the IL-8 concentration is 115.65 pg / ml or more.

[0047] According to one embodiment of the present invention, when the WARS concentration was 200 ng / ml or more and the IL-8 concentration was 115.65 pg / ml or more, more than 94% (17 / 18) of sepsis patients died (Figure 3a).

[0048] According to a preferred embodiment, the present invention includes determining whether the concentration of WARS is less than 200 ng / ml and the concentration of IL-6 is more than 1,000 pg / ml.

[0049] When the WARS concentration is less than 200 ng / ml, and the IL-6 concentration is 1000 pg / ml or more, preferably 1200 pg / ml or more, more preferably 1300 pg / ml or more, even more preferably 1500 pg / ml or more, even more preferably 1600 pg / ml or more, and most preferably 1654.5 pg / ml or more, information useful for distinguishing patients from a patient group at high risk of death can be provided.

[0050] According to a preferred embodiment of the present invention, the present invention includes identifying a patient group with a high risk of death when the WARS concentration is less than 200 ng / ml and the IL-6 concentration is 1654.5 pg / ml or more.

[0051] According to one embodiment of the present invention, when the WARS concentration was less than 200 ng / ml and the IL-6 concentration was more than 1654.5 pg / ml, 79.2% (19 / 24) of sepsis patients died (Figure 2d).

[0052] According to a preferred embodiment of the present invention, if the WARS concentration is less than 200 ng / ml and the IL-6 concentration is less than 1654.5 pg / ml, the method further includes a step of confirming the concentration of one or more cytokines selected from the group consisting of IFN-γ, IL-6, MIP-1α, IL-8 and TNF-α.

[0053] The concentration of IFN-γ can be further confirmed when the concentration of WARS is less than 200 ng / ml while the concentration of IL-6 is less than 1654.5 pg / ml.

[0054] According to a preferred embodiment of the present invention, the present invention includes identifying a patient group having a low risk of death when the IFN-γ concentration is 2.99 pg / ml or more and the IL-6 concentration is less than 54.96 pg / ml.

[0055] According to one embodiment of the present invention, when the cases where the WARS concentration was less than 200 ng / ml and the IL-6 concentration was less than 1654.5 pg / ml and the IFN-γ concentration was 2.99 pg / ml or more were classified, and then the cases where the IL-6 concentration was less than 54.96 pg / ml were classified again, it was confirmed that 100% of the sepsis patients survived (35 / 35) (Figure 2d).

[0056] According to a preferred embodiment of the present invention, the present invention includes identifying a patient group having a low risk of death when the IFN-γ concentration is less than 2.99 pg / ml and the IL-8 concentration is less than 22.91 pg / ml.

[0057] According to one embodiment of the present invention, when the WARS concentration was less than 200 ng / ml, the IL-6 concentration was less than 1654.5 pg / ml, the IFN-γ concentration was less than 2.99 pg / ml, and the IL-8 concentration was less than 22.91 pg / ml, it was confirmed that 78.6% of sepsis patients survived (11 / 14) (Figure 2d).

[0058] According to a preferred embodiment of the present invention, the present invention includes identifying a patient group with a low risk of death when the IFN-γ concentration is less than 2.99 pg / ml, the IL-8 concentration is 22.91 pg / ml or more, and the TNF-α concentration is 37.21 pg / ml or more.

[0059] According to one embodiment of the present invention, when the WARS concentration was 200 ng / ml, the IL-6 concentration was less than 1654.5 pg / ml, the IFN-γ concentration was less than 2.99 pg / ml, the IL-8 concentration was 22.91 pg / ml or more, and the TNF-α concentration was 37.21 pg / ml, it was confirmed that 69.2% of sepsis patients survived (9 / 13) (Figure 2d).

[0060] According to another aspect of the present invention, there is provided a kit for determining risk of death due to an infectious inflammatory disease, comprising: a preparation for measuring the expression level of a WARS protein or a gene encoding the same; and a preparation for measuring the expression level of a cytokine protein or a gene encoding the same.

[0061] According to a preferred embodiment, the cytokine of the present invention is IL-8 or IL-6.

[0062] The expression level of the protein can be detected or measured using an antibody or a fragment thereof (preferably an antigen-binding fragment) that specifically binds to the WARS and / or cytokine protein.

[0063] The expression level of the genes encoding the WARS and / or cytokines can be measured by measuring the mRNA level of the proteins, which can be measured by amplifying mRNA or cDNA from a sample of the subject using a primer set or a probe that specifically binds to the mRNA of the WARS and / or cytokine, or by measuring the presence and expression amount of the mRNA in the sample of the subject using a hybridization reaction with the probe.

[0064] In this specification, the contents of the kit for determining the risk of death from an infectious inflammatory disease that are the same as those described in the method for providing information necessary for determining the risk of death from an infectious inflammatory disease are omitted to avoid excessive duplication in the specification.

[0065] According to a preferred embodiment of the present invention, the kit of the present invention comprises: a preparation for measuring the expression level of a WARS protein or a gene encoding the same; and a preparation for measuring the expression level of an IL-8 protein or a gene encoding the same.

[0066] According to a preferred embodiment of the present invention, the kit may be for determining whether the WARS concentration in a subject's sample is 100 ng / ml or more and the IL-8 concentration is 100 pg / ml or more, and determining the risk of death from infectious inflammatory diseases.

[0067] According to a preferred embodiment of the present invention, the kit may further include an instruction manual for determining the risk of death based on the correlation between the concentration of WARS and the concentration of IL-8.

[0068] For example, the instructions may include content for determining whether the WARS concentration is 200 ng / ml or more and the IL-8 concentration is 100 pg / ml or more, preferably, whether the WARS concentration is 200 ng / ml or more and the IL-8 concentration is 115.65 pg / ml or more, or for providing information for identifying a patient group as being at high risk of death.

[0069] The instructions may also include content for confirming whether the WARS concentration is 100 ng / ml or more and less than 200 ng / ml and the IL-8 concentration is 400 pg / ml or more, or preferably, for confirming whether the WARS concentration is 100 ng / ml or more and less than 200 ng / ml and the IL-8 concentration is 466.5 pg / ml or more, or for providing information for identifying a patient group as being at high risk of death.

[0070] According to a preferred embodiment of the present invention, the kit may further comprise an agent for measuring the expression level of IL-6 protein or a gene encoding the same. Effect of the Invention

[0071] The features and advantages of the present invention can be summarized as follows: (i) The present invention provides a method for determining the mortality risk of an infectious inflammatory disease based on the concentrations of tryptophanyl-tRNA synthetase (WARS) and cytokines. (ii) The method of the present invention for determining the risk of death can effectively select patients at high risk of death from infectious inflammatory diseases in a short period of time, greatly facilitating timely treatment. [Brief description of the drawings]

[0072] [Figure 1] FIG. 1 shows the composition of the cohort for this study. [Figure 2a]FIG. 1 shows the results of measuring WARS1 and cytokine concentrations in survivor and non-survivor samples. FIG. 2a shows a comparison of WARS1 concentrations between survivors and non-survivors, FIG. 2b shows a comparison of concentrations between the sepsis group and the septic shock group, FIG. 2c shows the results of the AUC analysis between survivors and non-survivors, FIG. 2d shows a decision tree (DT) analyzed using R2 software using WARS1 and pro-inflammatory cytokine and chemokine values, FIG. 2e shows a distribution diagram of IL-8 and IL-6 concentrations based on WARS1, FIG. 2f shows the analysis results of the AUC curves specified for IL-8 and WARS1, FIG. 2g shows a comparison of survival days according to concentrations of WARS1 200 ng / ml or more and IL-8 115.65 pg / ml, and FIGS. 2h to 2k show the results of closeness and significance between WARS1 200 ng / ml and IL-8 115.65 pg / ml or more and pro-inflammatory cytokines and chemokines in non-survivors. [Figure 2b] See column Figure 2a. [Figure 2c] See column Figure 2a. [Figure 2d] See column Figure 2a. [Figure 2e] See column Figure 2a. [Figure 2f] See column Figure 2a. [Figure 2g] See column Figure 2a. [Figure 2h] See column Figure 2a. [Figure 2i] See column Figure 2a. [Figure 2j] See column Figure 2a. [Figure 2k] See column Figure 2a. [Figure 3a] Fig. 3 shows the results of an analysis of the correlation between WARS1 and IL-8 in survivors and non-survivors, in which Fig. 3a shows a decision tree (DT) analyzed only for WARS1 and IL-8, Fig. 3b shows a distribution diagram of IL-8 concentration based on WARS1, and Fig. 3c shows the results of an analysis of the AUC curves determined by IL-8 and WARS1. [Figure 3b] See column Figure 3a. [Figure 3c] See column Figure 3a. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention in more detail, and that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. EXAMPLES

[0074] Example 1. Cohort composition To investigate whether high levels of WARS1 are associated with death from sepsis, target patients were selected based on the concentration of WARS1. Plasma samples were provided by Asan Hospital. The samples were collected from patients within 24 hours of admission. The cohort consisted of a total of 243 cases (Figure 1), including 54 cases in the HC group (normal group), 100 cases in the sepsis group, and 89 cases in the septic shock group.

[0075] Example 2. Comparison of WARS1 and cytokine and chemokine concentrations in survivors and non-survivors The differences between survivors and non-survivors were measured in plasma WARS1 and proinflammatory cytokines and chemokines, including IL-1β, IL-6, IL-8, IFN-γ, MIP-1α, and TNF-α. IL-1β, IL-6, IL-8, IFN-γ, MIP-1α, and TNF-α were measured by multiplex assay (Millipore), and WARS1 was measured by ELISA using an antibody against WARS1 (Abiotech). The measurement methods were performed according to the methodology of each manufacturer.

[0076] WARS1 was significantly increased in non-survivors compared to survivors (Fig. 2a), and was significantly increased in the septic shock group compared to the sepsis group (Fig. 2b). Analysis of the AUC with the current biomarkers including WARS1 concentration, procalcitonin (PCT) and C-reactive protein (CRP) showed that PCT (p=0.7112) and CRP (p=0.2380) levels were not significantly different between non-survivors and survivors (Fig. 2c). Only WARS1 showed significant discriminatory ability (p=0.006) to predict mortality.

[0077] Decision trees (DTs) were analyzed and classified through the concentrations of cytokines and chemokines according to the concentration of WARS1 in the plasma of the cohort patients by the “rpart” package analysis in R software version 3.5.1. In the first classification tree root, WARS1 was set at a concentration of 200 ng / ml. In the first group, 17 of 18 sepsis patients with IL-8 ≥ 115.65 pg / ml died (94.4%), and IL-6 was selected as the second root in patients with WARS1 values ​​≤ 200 ng / ml, but 19 of 24 patients with IL-6 ≥ 1654.5 pg / ml died (79.2%) (Fig. 2d). This DT showed a sensitivity of 77.5% and a specificity of 86.2% (Fig. 2e). The AUC curves using WARS1 ≥ 200 ng / ml and IL-8 ≥ 115.65 pg / ml significantly distinguished non-survivors in sepsis patients (P = 0.0003) (Fig. 2f).

[0078] The mean time to death was about 4.3 days in group 1, which was significantly shorter than that of non-survivors with WARS1 <200ng / ml and IL-8 <115.65pg / ml, meaning that they died at an early stage (Fig. 2g).WARS1 in group 1 showed quantitative correlations with MIP-1α (p=0.006), IFN-γ (p=0.011), TNF-α (p=0.003) and IL-8 (p=0.01) (Fig. 2h-k). The MIP-1α, IFN-γ, TNF-α, IL-6, IL-1β, IL-8 and IL-10 were compared between normal subjects, sepsis and septic shock patients. In the septic shock patients, MIP-1α (P<0.0001), TNF-α (P<0.0001), IL-6 (P<0.0001), IL-8 (P<0.0001) and IL-10 (P<0.0001) were significantly increased. In addition, IFN-γ, TNF-α, IL-6, IL-1β, IL-6 and IL-10, except for MIP-1α, were relatively increased in the non-survivors compared to the survivors, and IL-6 (P<0.0001), IL-8 (P<0.0001) and IL-10 (P<0.0001) were significantly increased.

[0079] Example 3. Correlation analysis of WARS1 and IL-8 in survivors and non-survivors We drew a decision tree (DT) using only WARS1 and IL-8 through the "rpart" package analysis in R software version 3.5.1, and the classification tree root was set as WARS1 100ng / ml ≤ WARS1 < 200ng / ml at a concentration of 200ng / ml, respectively. In the first group, 17 of 18 sepsis patients with WARS1 ≥ 200ng / ml and IL-8 ≥ 115.65pg / ml died. All 6 of 6 sepsis patients with WARS1 ≥ 100ng / ml ≤ WARS1 < 200ng / ml and IL-8 ≥ 466.5ng / ml died (Figure 3a). This DT was able to target 24 of the 80 overall deaths, and the AUC using WARS1 and IL-8 selective interval values ​​was 0.80, significantly distinguishing between non-survivors in sepsis patients (P = 0.007) (Figures 3b and 3c).

[0080] Although each embodiment of the present invention has been described above, a person having ordinary knowledge in the relevant technical field can modify and change the present invention in various ways by adding, changing, deleting or adding components without departing from the concept of the present invention as described in the claims, and this can also be said to be included in the scope of the present invention.

Claims

1. A preparation for measuring the expression level of a WARS protein or the expression level of a gene encoding the same; and A preparation for measuring the expression level of IL-8 protein or the expression level of the gene encoding same; A kit for determining the risk of death due to sepsis or septic shock, comprising: The kit is a kit for determining risk of death, characterized in that it is used to confirm whether the WARS concentration in a subject sample is 100 ng / ml or more and the IL-8 concentration is 100 pg / ml or more, and to determine the risk of death due to sepsis or septic shock.

2. The death risk assessment kit according to claim 1, further comprising a preparation for measuring the expression level of IL-6 protein or a gene encoding the same.

3. The kit for determining the risk of death as described in claim 1, further comprising instructions for determining the risk of death based on the correlation between the WARS concentration and the IL-8 concentration in a sample.

4. The death risk determination kit according to claim 3, characterized in that the instructions include the following content (1) or (2): (1) determining whether the concentration of WARS is greater than or equal to 100 ng / ml and less than 200 ng / ml, and the concentration of IL-8 is greater than or equal to 400 pg / ml; (2) Confirm that the WARS concentration is 100 ng / ml or more but less than 200 ng / ml, and that the IL-8 concentration is 466.5 pg / ml or more.

5. A method for providing information necessary to determine the risk of death from sepsis or septic shock, comprising the steps of: (a) measuring the concentrations of WARS (tryptophanyl-tRNA synthetase) and IL-8 or IL-6 in a plasma sample taken from a subject; and (b) confirming that: (1) Whether the concentration of the WARS is 100 ng / ml or more and the concentration of IL-8 is 100 pg / ml or more. (2) Whether the concentration of WARS is less than 200 ng / ml and the concentration of IL-6 is 1000 pg / ml or more.

6. The method for providing information according to claim 5, wherein the sepsis or septic shock is caused by infection with one or more selected from the group consisting of viruses, bacteria and fungi.

7. 6. The method of claim 5, wherein step (b) determines whether the WARS concentration is 100 ng / ml or more and less than 200 ng / ml, and the IL-8 concentration is 400 pg / ml or more.

8. 6. The method of claim 5, wherein step (b) comprises determining whether the WARS concentration is 200 ng / ml or more and the IL-8 concentration is 100 pg / ml or more.

9. 6. The method of claim 5, wherein step (b) comprises determining whether the WARS concentration is less than 200 ng / ml and the IL-6 concentration is 1600 pg / ml or more.

10. The method for providing information according to claim 5, further comprising a step (c) of confirming the concentration of one or more cytokines selected from the group consisting of IFN-γ, IL-6, MIP-1α, IL-8 and TNF-α when the concentration of WARS is less than 200 ng / ml and the concentration of IL-6 is less than 1654.5 pg / ml.

11. The method for providing information according to claim 10, characterized in that a patient group having an IFN-γ concentration of 2.99 pg / ml or more and an IL-6 concentration of less than 54.96 pg / ml is identified as a patient group with a low risk of death.

12. The method for providing information according to claim 10, characterized in that when the IFN-γ concentration is less than 2.99 pg / ml and the IL-8 concentration is less than 22.91 pg / ml, the patient group is identified as having a low risk of death.

13. The method for providing information described in claim 10, characterized in that a patient group having an IFN-γ concentration of less than 2.99 pg / ml, an IL-8 concentration of 22.91 pg / ml or more, and a TNF-α concentration of 37.21 pg / ml or more is identified as a patient group with a low risk of death.

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