JP7016110B2 - Biomarker for differentiating Still's disease from sepsis - Google Patents

Description

The present invention relates to biomarkers for differentiating Still's disease from septic patients. More specifically, the present invention relates to a biomarker for differentiating Still's disease consisting of any of FGF-2, IL-18, G-CSF and GM-CSF from sepsis, and further relates to a test method using the biomarker and the like.

Still's disease can be classified into still's disease (SJIA: systemic type of juvenile idiopathic arthritis) that develops in children and adult Still's disease (ASD, also called adult-onset Still's disease AOSD). Still's disease is an intractable autoinflammatory disease mainly consisting of fever, arthralgia, and rash, and all of these Still's diseases are designated intractable diseases. There is information that there are an estimated 2,000 and 5,000 patients in Japan, respectively, but it is said that there are actually more than 10 times as many potential patients. Not only is Still's disease difficult to diagnose, but the serious complications HPS and disseminated intravascular coagulation (DIC) significantly reduce the prognosis of life and require intensive and rapid treatment. Is. The diagnosis of Still's disease is based on the exclusion diagnosis currently called "Yamaguchi criteria (Non-Patent Document 1)".

Sepsis, on the other hand, is a life-threatening organ disorder caused by an uncontrolled biological response to an infectious disease and is now recognized by the World Health Organization (WHO) as a global health priority, with the American Intensive Care Medicine Society and the European Intensive Care Medicine. The guidelines for the "Surviving Sepsis Campaign," which is a joint research of the medical society, were revised in 2016, and it is a disease that is drawing attention. Sepsis has a survival rate of about 80% when antibiotic treatment is started within 1 hour after diagnosis, but it is said that the survival rate decreases by 7.6% for every 1 hour delay in treatment, and the survival rate when the initial treatment is inappropriate. Is also reported to decrease by 5 times (Non-Patent Document 2). The latest diagnostic criteria for sepsis are left to the physician's findings by infection (including suspicion) and quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) scores.

Therefore, for the treatment of Still's disease and septicemia, rapid diagnosis of these diseases is required, and IL-1β and IL-6 are useful serum biomarkers for the diagnosis of Still's disease and the evaluation of the disease state. Etc. have been reported (Non-Patent Document 3). However, these biomarkers are also known to be elevated in infectious diseases and other autoinflammatory diseases.

Yamaguchi M. et al.,. J. Rheumatol, 19: 424-30, 1992 Kumar A. et al., Crit Care Med, 34: 1589-1596, 2006 Jung, et al., Scand J. Rheumatol, 43 (2): 162-9, 2014

Therefore, it is an object of the present invention to provide a biomarker capable of quickly and accurately distinguishing between Still's disease and sepsis, and a method for distinguishing between Still's disease and sepsis using the biomarker.

In order to solve the above problems, the present inventors used a statistical method combined with deterministic tree analysis based on the results of comprehensive analysis of serum cytokines and chemokines to obtain cytokines with a high accuracy rate. The smallest combination was identified. As a result, it was found that the serum concentrations of FGF-2, IL-18, G-CSF and GM-CSF in adult Still's disease (AOSD) patients were significantly higher than those in septic patients. As a result of further research based on these findings, the present inventors have completed the present invention.

That is, the present invention comprises the following.
[1] A biomarker for distinguishing Still's disease from sepsis, which comprises any of the following (a) to (d).
(a) FGF-2 protein or FGF-2 transcript,
(b) IL-18 protein or IL-18 transcript,
(c) G-CSF protein or G-CSF transcript, and
(d) GM-CSF protein or GM-CSF transcript [2] The test animal comprises a step of detecting or quantifying one or more of the biomarkers described in [1] in a sample derived from the test animal. A method of assisting in determining whether a person has Still's disease or sepsis.
[3] A method for analyzing the possibility of Still's disease or sepsis, which comprises a step of detecting or quantifying one or more of the biomarkers according to [1] in a sample derived from a test animal.
[4] The method according to [2], which comprises the following steps (i) to (iii).
(I) A step of quantifying one or more of the biomarkers described in [1] in a sample derived from a test animal.
(Ii) When the amount of biomarker quantified in (i) is greater than or equal to the reference value based on the step of comparing the amount of biomarker quantified in (i) with the reference value and (iii) based on the results of (ii). In addition, a step of determining that the test animal has developed Still's disease and determining that the test animal has developed septicemia when the value is less than the reference value [5] The following (i) to The method according to [3], which comprises the step (iii).
(I) A step of quantifying one or more of the biomarkers described in [1] in a sample derived from a test animal.
(Ii) When the amount of biomarker quantified in (i) is greater than or equal to the reference value based on the step of comparing the amount of biomarker quantified in (i) with the reference value and (iii) based on the results of (ii). In addition, a step of determining that the test animal may have developed Still's disease, and if it is less than the reference value, it is determined that the test animal may have developed septicemia [. 6] The method according to any one of [2] to [5], which comprises using two or more kinds of biomarkers according to [1].
[7] The biomarker is
[2]-[containing (1) FGF-2 protein or FGF-2 transcript, and (2) GM-CSF protein or GM-CSF transcript, and / or IL-18 protein or IL-18 transcript. 6] The method according to any one of.
[8] The method according to any one of [2] to [7], which comprises detecting or quantifying the biomarker using any of the following (a) to (f).
(a) An antibody capable of specifically recognizing an FGF-2 protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing an FGF-2 transcript,
(b) A nucleic acid probe or nucleic acid primer capable of specifically recognizing an IL-18 protein or an IL-18 transcript,
(c) An antibody capable of specifically recognizing a G-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a G-CSF transcript,
(d) An antibody capable of specifically recognizing a GM-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a GM-CSF transcript,
(e) The antibody, nucleic acid probe or nucleic acid primer of (a), and the antibody, nucleic acid probe or nucleic acid primer of (b), and
(f) The antibody, nucleic acid probe or nucleic acid primer of (a), and the antibody, nucleic acid probe or nucleic acid primer of (d) [9] The reference value is a value set based on a decision tree analysis or ROC analysis. , [4] to any one of [8].
[10] The method according to any one of [2] to [9], wherein the test animal is a human.
[11] The method according to any one of [2] to [10], wherein the sample is serum.
[12] A test kit for distinguishing Still's disease from sepsis, which comprises one or more of the following (a) to (f).
(a) An antibody capable of specifically recognizing an FGF-2 protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing an FGF-2 transcript,
(b) A nucleic acid probe or nucleic acid primer capable of specifically recognizing an IL-18 protein or an IL-18 transcript,
(c) An antibody capable of specifically recognizing a G-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a G-CSF transcript,
(d) An antibody capable of specifically recognizing a GM-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a GM-CSF transcript,
(e) The antibody, nucleic acid probe or nucleic acid primer of (a), and the antibody, nucleic acid probe or nucleic acid primer of (b), and
(f) The antibody of (a), a nucleic acid probe or a nucleic acid primer, and the antibody of (d), a nucleic acid probe or a nucleic acid primer [13], further comprising one or more of the biomarkers described in [1], [12]. The kit described.
[14] A method for treating Still's disease, which comprises the following steps (i) to (iv).
(I) A step of quantifying one or more of the biomarkers described in [1] in a sample derived from a test animal.
(Ii) A step of comparing the amount of the biomarker quantified in (i) with the reference value,
(Iii) Based on the results of (ii), when the amount of the biomarker quantified in (i) is equal to or greater than the reference value, the step of determining that the subject animal has developed Still disease, and (iv). ) A step of administering a therapeutic agent for still disease to a test animal determined to have developed still disease based on the result of (iii) [15] The therapeutic agent for still disease is a steroid drug, tocilizumab and The method according to [14], which is selected from the group consisting of immunosuppressive agents.
[16] A method for treating sepsis, which comprises the following steps (i) to (iv).
(I) A step of quantifying one or more of the biomarkers described in [1] in a sample derived from a test animal.
(Ii) A step of comparing the amount of the biomarker quantified in (i) with the reference value,
(Iii) Based on the results of (ii), when the amount of the biomarker quantified in (i) is less than the reference value, the step of determining that the subject animal has sepsis, and (iv). Step of administering a therapeutic agent for sepsis to a test animal determined to have developed sepsis based on the result of (iii) [17] The therapeutic agent for sepsis is a group consisting of an antibacterial agent and a circulatory agonist. The method according to [16], which is selected from the above.
[18] The method according to any one of [14] to [17], which comprises using two or more kinds of biomarkers according to [1].
[19] The biomarker is
[14]-[containing (1) FGF-2 protein or FGF-2 transcript, and (2) GM-CSF protein or GM-CSF transcript, and / or IL-18 protein or IL-18 transcript. 18] The method according to any one of.

The FGF-2, IL-18, G-CSF and GM-CSF of the present invention can all be biomarkers for differentiating Still's disease from sepsis. Furthermore, by quantifying one or more of these cytokines as biomarkers, it is possible to determine whether Still's disease or sepsis has developed.

FIG. 1 shows a Random Forest analysis of cytokine levels in AOSD and septic patients. The vertical axis shows each cytokine. The horizontal axis represents the importance of each cytokine. FIG. 2 shows the results of a decision tree analysis that distinguishes AOSD patients from septic patients using GM-CSF and FGF-2.

1. 1. Biomarker of the present invention The present invention provides a biomarker for distinguishing Still's disease (preferably adult Still's disease) from septicemia (hereinafter, may be referred to as "biomarker of the present invention"). Specific examples of the biomarkers of the present invention include (a) FGF-2 protein or FGF-2 transcript, (b) IL-18 protein or IL-18 transcript, and (c) G-CSF protein or G-. CSF transcripts and (d) GM-CSF protein or GM-CSF transcripts. Hereinafter, one or more proteins selected from FGF-2 protein, IL-18 protein, G-CSF protein and GM-CSF protein may be referred to as "biomarker protein of the present invention". Further, one or more transcripts selected from FGF-2 transcript, IL-18 transcript, G-CSF transcript and GM-CSF transcript may be referred to as "biomarker transcript of the present invention". ..

The FGF-2, IL-18, G-CSF and GM-CSF proteins contained in the biomarkers of the present invention are known proteins, and GenPept Accession No .: P09038 (FGF-2) and GenPept Accession No., respectively. : Q14116 (IL-18), GenPept Accession No .: P09919 (G-CSF), and GenPept Accession No .: P04141 (GM-CSF) disclose amino acid sequences. In the present invention, each protein of FGF-2, IL-18, G-CSF and GM-CSF has SEQ ID NO: 2 (FGF-2), SEQ ID NO: 4 (IL-18), and SEQ ID NO: 6 (G). -CSF) and an amino acid sequence represented by SEQ ID NO: 8 (GM-CSF) may be used, and a protein containing substantially the same amino acid sequence as these may be used.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8 is, for example, 60% or more, preferably 70% or more, more preferably 80% or more of these amino acid sequences. , More preferably 90% or more, even more preferably 95% or more, most preferably 98% or more amino acid sequences and the like. Here, "identity" means the optimum alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is the sequence for the optimum alignment). It means the ratio (%) of the same amino acid and similar amino acid residues to all the overlapping amino acid residues in (which may consider the introduction of a gap in one or both). "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. Such substitutions with similar amino acids are expected to result in no change in protein phenotype (ie, conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literatures (see, eg, Bowie et al., Science, 247: 1306-1310 (1990)). The amino acid sequence identity in the present specification is determined by using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gap is allowed; matrix = BLOSUM62; filtering. = OFF) can be calculated.

The biomarker protein of the present invention can be produced according to a known protein synthesis method, for example, a solid phase synthesis method, a liquid phase synthesis method, or the like. The obtained protein can be purified and isolated by a known purification method, for example, solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, a combination thereof, or the like. Further, it may be isolated and purified from a biological sample by a method known per se. Alternatively, the biomarker protein of the present invention can also be produced by culturing a transformant containing a nucleic acid encoding the same and separating and purifying the protein from the obtained culture. Such nucleic acid may be DNA, RNA, or DNA / RNA chimera, but is preferably DNA. The nucleic acid may be double-stranded or single-stranded.

Further, as an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8, one or two or more of these amino acid sequences (preferably about 1 to 100). It contains an amino acid sequence in which about 1 to 50, more preferably about 1 to 10, and particularly preferably 1 to several (2, 3, 4 or 5) amino acids are substituted, inserted and / or deleted. Also includes proteins and the like.
When the amino acid sequence is substituted, inserted and / or deleted as described above, the position of the substitution, insertion and / or deletion shall be the position where the activity of the protein (eg, inflammatory activity) is retained. Is preferable. The activity can be measured according to a method known per se.

The FGF-2, IL-18, G-CSF and GM-CSF transcripts contained in the biomarkers of the present invention are known transcripts, and are Genbank Accession No .: NM_002006 (FGF-2) and Genbank Accession, respectively. The base sequences are disclosed as No .: NM_001562 (IL-18), Genbank Accession No .: NM_000759 (G-CSF) and Genbank Accession No .: M11734 (GM-CSF). In the present invention, each transcript of FGF-2, IL-18, G-CSF and GM-CSF has SEQ ID NO: 1 (FGF-2), SEQ ID NO: 3 (IL-18), SEQ ID NO: 5 ( G-CSF) and nucleic acids containing the same or substantially the same base sequence as the base sequence represented by SEQ ID NO: 7 (GM-CSF) can be mentioned.

As a nucleic acid containing a base sequence substantially the same as the base sequence represented by SEQ ID NO: 1, 3, 5 or 7, for example, 60% or more, preferably 70% or more, more preferably 70% or more of these base sequences. It contains a base sequence having 80% or more, more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more identity, and has substantially the same activity as the biomarker protein of the present invention. Examples thereof include a nucleic acid encoding a protein having the above-mentioned biomarker, and a homologue of a gene encoding the above-mentioned biomarker. For the identity of the base sequence in the present specification, the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) is used, and the following conditions (expected value = 10; gap is allowed; filtering = ON; match). It can be calculated by score = 1; mismatch score = -3).

The biomarker transcript of the present invention can be obtained, for example, by isolating and purifying a biological sample containing the transcript by a method known per se.

2. 2. Testing and Analytical Methods of the Invention As described in the Examples below, the inventors have found that the level of the biomarker of the invention in the serum of a patient clinically diagnosed with Still disease is septicemia. It was found to be significantly higher than that of patients who were diagnosed with the disease. Accordingly, the present invention comprises detecting or quantifying the biomarker of the present invention in a sample derived from a test animal (that is, a sample collected from the test animal), wherein the test animal has Still's disease (preferably an adult). A method of performing a test for determining whether or not Still's disease (Still's disease) is occurring or a septicemia (eg, judgment, diagnosis, judgment, differentiation), or a method of assisting the judgment or the test (hereinafter) Then, these may be collectively referred to as "the inspection method of the present invention"). Alternatively, a method for analyzing the possibility of still disease or septicemia (eg, analysis, evaluation), including detecting or quantifying the biomarker of the present invention in a sample derived from a test animal (hereinafter, "analysis of the present invention"). May be referred to as "method"). Hereinafter, the inspection method of the present invention and the analysis method of the present invention may be collectively referred to as "the method of the present invention".

Specifically, the method of the present invention comprises (i) a step of quantifying the biomarker of the present invention in a sample derived from a test animal, and (ii) a step of comparing the amount of the biomarker quantified in (i). It may be a method including. In the test method of the present invention, in addition to the above (i) and (ii), the subject animal has developed Still's disease or sepsis based on the results of (iii) and (ii). It may include a step of determining that. Further, in the analysis method of the present invention, in addition to the above (i) and (ii), the subject animal may have developed Still's disease based on the results of (iii') and (ii). Alternatively, it may include a step of determining that sepsis may have occurred.

In the method of the present invention, the biomarker may be used alone or in combination of two or more. When two types are used in combination, for example, two types of biomarkers are detected or quantified, and based on the results, it is determined that the test animal has Still's disease or sepsis (). Alternatively, it may be determined that these diseases may have developed). Alternatively, first, the first type of biomarker is detected or quantified, and based on the result, it is determined whether the test animal has Still's disease (or sepsis), and Still's disease (or sepsis) is developed. If it is not determined, a second type of biomarker can be further detected or quantified, and the result can be used to determine whether Still's disease or sepsis has occurred. When three or more types are used, the same determination can be made. When two or more kinds are used in combination, (1) FGF-2 protein or FGF-2 transcript, and (2) GM-CSF protein or GM-CSF transcript, and / or IL-18 protein or Combinations of IL-18 transcripts include, but are not limited to.

The animal that can be the subject of the method of the present invention (that is, the animal to be tested) is not particularly limited, but is preferably an animal suspected of developing Still's disease or sepsis, or clinically known to have developed. Examples include animals that have been diagnosed with. Examples of animal types include mammals (eg, humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, mice, rats, etc.), birds (eg, chickens, etc.). And so on. It is preferably a mammal, more preferably a human.

The sample derived from the test animal is not particularly limited as long as it can contain the biomarker of the present invention, and for example, blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal juice, semen, breast water, gastrointestinal secretion. Examples thereof include fluid, cerebrospinal fluid, interstitial fluid, and lymph fluid, preferably serum or plasma, and more preferably serum. These samples can be obtained by a method known per se, for example, serum or plasma can be prepared by collecting blood from a test animal according to a conventional method and separating humoral components, and cerebrospinal fluid can be prepared. , Can be collected by known means such as spinal puncture.

For the detection or quantification of the biomarker of the present invention in a sample derived from a test animal, an RNA (eg, total RNA, mRNA) fraction is prepared from the sample, and the biomarker transcript of the present invention contained in the fraction is prepared. Can be investigated by detecting or quantifying. Accordingly, in one embodiment, the method of the invention comprises detecting or quantifying using a nucleic acid probe or nucleic acid primer capable of specifically recognizing the biomarker transcript of the invention, respectively.

The RNA fraction can be prepared using a known method such as guanidine-CsCl ultracentrifugation method or AGPC method, and a small amount can be prepared using a commercially available RNA extraction kit (eg, RNeasy Mini Kit; manufactured by QIAGEN, etc.). It is also possible to quickly and easily prepare high-purity total RNA from a sample. As a means for detecting or quantifying the biomarker transcript of the present invention in the RNA fraction, for example, a method using hybridization (Northern blot, dot blot, etc.), PCR (RT-PCR, competitive PCR, real-time PCR, etc.), etc. ) Etc. can be mentioned. Quantitative PCR methods such as competitive PCR and real-time PCR are preferable in that expression fluctuations of each gene encoding the biomarker of the present invention can be detected quickly and easily and quantitatively from a small amount of sample.

In the case of Northern blot or dot blot hybridization, the detection or quantification of the gene of the biomarker of the present invention can be performed, for example, using a nucleic acid probe capable of specifically recognizing each transcript of the biomarker of the present invention. .. As such a nucleic acid probe, among the above-mentioned known polynucleotides which are transcripts of FGF-2, IL-18, G-CSF or GM-CSF, 15 bases or more, preferably 18 to 500 bases, more preferably. Examples thereof include polynucleotides containing a continuous nucleotide sequence of 18 to 200 bases, more preferably 18 to 50 bases, or a complementary sequence thereof. The nucleic acid may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA. Further, the nucleic acid used as a probe may be double-stranded or single-stranded. In the case of double strand, it may be double-stranded DNA, double-stranded RNA or a hybrid of DNA: RNA. In the case of a single strand, one containing an antisense strand sequence can be used.

The above-mentioned nucleic acid probe is a polynucleotide containing a nucleotide sequence that hybridizes to the polynucleotide that is the biomarker transcript of the present invention under stringent conditions. Hybridization can be performed according to a method known per se, specifically, the method described in Molecular Cloning 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). .. Stringent conditions include a hybridization reaction at 45 ° C. in 6 × SSC (sodium chloride / sodium citrate) followed by one or more washes at 65 ° C. in 0.2 × SSC / 0.1% SDS.

The nucleic acid probe uses a primer set capable of amplifying a part or all of the transcript of the biomarker gene of the present invention, and uses cDNA or genomic DNA derived from the cell of the test animal as a template to have a desired length by the PCR method. Amplify the nucleic acid of the above, or clone the above gene or cDNA from the cDNA or genomic DNA library by colony or plaque hybridization, etc., and if necessary, use a restricting enzyme or the like to form a fragment of an appropriate length. It can be obtained by. Alternatively, it can also be obtained by chemically synthesizing using a commercially available automatic DNA / RNA synthesizer or the like.

The nucleic acid probe is preferably labeled with a labeling agent in order to enable detection and quantification of the target nucleic acid. As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ³² P], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably stable and has a large specific activity, and for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescamine isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. In addition, biotin- (streptavidin) avidin can be used to bind the probe to the labeling agent.

In the case of Northern hybridization, the RNA fraction prepared as described above is separated by gel electrophoresis and then transferred to a membrane such as nitrocellulose, nylon, or polyvinylidene difluoride, and prepared as described above. After specific hybridization in a hybridization buffer containing the labeled probe, the transfer amount of the biomarker of the present invention is measured by measuring the labeled amount bound to the membrane for each band by an appropriate method. can do. Also in the case of dot blotting, the transfer amount of the biomarker of the present invention can be measured by subjecting the membrane spotted with the RNA fraction to the hybridization reaction in the same manner and measuring the labeled amount of the spot.

According to another preferred embodiment, a quantitative PCR method is used as a method for measuring the transcription amount of the biomarker of the present invention. Examples of the set of oligonucleotides used as primers in PCR include nucleic acid primers capable of specifically recognizing the transcript of the biomarker of the present invention. In one preferred embodiment, as the nucleic acid primer used in the method of the present invention, for example, among known polynucleotides which are transcripts of the biomarkers of the present invention, 15 or more bases, preferably 15 to 50 bases, more preferably. Has a length of contiguous nucleotide sequences of 15-30 bases, more preferably 15-25 bases, and is complementary to a polynucleotide (sense strand) sequence designed to amplify a DNA fragment of 100 bp to several kbp. And a set of oligonucleotides of a polynucleotide capable of hybridizing to a polynucleotide (antisense chain) having a nucleotide sequence complementary to the above-mentioned polynucleotide sequence. Such nucleic acid primers can also be prepared, synthesized, or the like by the same method as the nucleic acid probe.

Alternatively, for detection or quantification of the biomarker of the present invention in a sample derived from a test animal, a protein fraction is prepared from the sample and a translation product of the gene contained in the fraction (that is, the biomarker of the present invention). It can be investigated by detecting or quantifying the protein). Detection or quantification of these proteins can be performed by immunoassay (eg, ELISA, FIA, RIA, Western blot, etc.) using an antibody that specifically recognizes each protein. Thus, in one embodiment, the method of the invention comprises detecting or quantifying the biomarker using an antibody capable of specifically recognizing each of the biomarker proteins of the invention.

An antibody capable of specifically recognizing each of the biomarker proteins of the present invention can be produced by an existing general production method using a partial peptide having these proteins or epitopes as an immunogen. In the present specification, the antibody includes a polyclonal antibody, a natural antibody such as a monoclonal antibody (mAb), a chimeric antibody that can be produced using a gene recombination technique, a humanized antibody or a single-stranded antibody, and a binding property thereof. Fragments and the like are included, but not limited to these. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof. The binding fragment means a region of a part of the above-mentioned antibody having specific binding activity, and specific examples thereof include F (ab') ₂ , Fab', Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996). The class of antibody is not particularly limited and includes an antibody having any isotype such as IgG, IgM, IgA, IgD or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of easiness of purification and the like. Further, in the present invention, it is also preferable to use a commercially available antibody or a kit or array containing the antibody as an antibody capable of specifically recognizing the biomarker protein of the present invention.

No special conditions, operations, etc. are required to apply the individual immunoassays to the method of the present invention. The biomarker measurement system of the present invention may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, review articles, books, etc. can be referred to. For example, Hiroshi Irie ed. "Radioimmunoassay" (Kodansha, published in 1974), Hiroshi Irie ed. "Radioimmunoassay" (Kodansha, published in 1979), Eiji Ishikawa et al. (Published in 1993), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical Shoin, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. Published in 1987), "Methods in ENZY MOLOGY" Vol.70 (Immunochemical Techniques (Part A)), the same book Vol.73 (Immunochemical Techniques (Part B)), the same book Vol.74 (Immunochemical Techniques (Part C)), the same book Vol. .84 (Immunochemical Techniques (Part D: Selected Immunoassays)), the same book Vol.92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), the same book Vol.121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) )) (The above is published by Academic Press).

Further, the biomarker of the present invention can be detected or quantified by proteome analysis using a combination of iTRAQ ^TM reagent (ABI) capable of high-throughput protein detection or quantitative analysis and a mass spectrometer. good.

The examples described below show that the concentration of the biomarker of the present invention in serum in adult Still's disease patients is significantly higher than that in septic patients. Therefore, the comparison of the amount of biomarkers is made, for example, a sample derived from a control animal that has developed or is likely to develop septicemia (hereinafter, may be referred to as a “control sample”), and a target. This can be done by quantifying the concentration of the biomarker of the present invention in a sample derived from a test animal and comparing the concentrations of both.

Alternatively, this may be done by comparing the amount of biomarker with the reference value.
As the "reference value" used in the present invention, the amount of the biomarker of the present invention in the control sample may be used. Alternatively, a preset value from the quantitative value of the biomarker in the control sample may be used. In this case, as a reference value, for example, an average value or a mode value of the measured values of a plurality of individuals may be adopted with a plurality of individuals as a control group.

The reference value may be a reference value of a branch point set by the decision tree analysis. The turning point is determined by determining the amount of the biomarker of the present invention in a sample (eg, serum) collected from, for example, an individual or population developing Still's disease and an individual or population developing septicemia, and performing a decision tree analysis. The results are used to create variables (eg, the concentration of the biomarker of the invention in the sample) and their reference values using the impureness of the data represented by criteria such as gini coefficients, entropy, chi-square statistics, etc. , Can be decided. The decision tree analysis can be performed by a method known per se, for example, a method using software for decision tree analysis (statistical analysis software R and rpart package).

In a preferred embodiment, for the bifurcation, the amount of the biomarker of the invention in serum collected from a still disease-developed population and a septicemia-developed population is quantified, a decision tree analysis is performed, and a Gini coefficient is used. The impurities of the represented data are used to create and determine variables and their reference values. As a reference value of a suitable branching point of the biomarker of the present invention in the collected serum, for example, 35.3 (pg / ml) for the FGF-2 protein and 3.09 (pg / ml) for the GM-CSF can be mentioned. However, these values can vary depending on the sample size and other conditions, and are not limited to these values.

Further, the reference value may be a cutoff value. The "cut-off value" is a value that can satisfy both high diagnostic sensitivity (presence-related correct diagnosis rate) and high diagnostic specificity (disease-free correct diagnosis rate) when the disease is determined based on the value. For example, a value showing a high positive rate in a population developing Still's disease and a high negative rate in a population developing sepsis can be set as a cutoff value.

Methods for calculating cutoff values are well known in the art. For example, the amount of the biomarker of the present invention in serum collected from an individual who developed Still's disease and an individual who developed septicemia was quantified, and the diagnostic sensitivity and specificity at the quantified values were determined, and based on these values. , Create a ROC (Receiver Operating Characteristic) curve using commercially available analysis software. Then, a value when the diagnostic sensitivity and the diagnostic specificity are as close to 100% as possible can be obtained, and the value can be used as the cutoff value.

Suitable cutoff values for the biomarkers of the invention in the collected serum include, for example, 35.5 (pg / ml) for the FGF-2 protein, 543 (pg / ml) for the IL-18 protein, and G-. 47.97 (pg / ml) for CSF and 4.44 (pg / ml) for GM-CSF can be mentioned, but these values can vary depending on the sample size and other conditions, and these values are included in this value. Not limited. Although not shown in the examples described later, when G-CSF is used as a biomarker, the sensitivity is 0.837, the specificity is 0.706, the correct diagnosis rate is 0.800, the AUC (Area Under Curve) is 0.721, and the AIC (Akaike's Information). Criterion) was 75.30 (66 sera from patients diagnosed with AOSD, 17 sera from patients diagnosed with septicemia).

As a result of the comparison of the amounts of the biomarkers of the present invention, for example, in the sample derived from the test animal, when the biomarker of the present invention is detected or measured at a higher value than that of the control sample, or when the value is equal to or higher than the above reference value. In some cases, it can be determined that the test animal has developed Still's disease, and when the biomarker of the present invention is detected or measured at the same level or lower than that of the control sample, Alternatively, if it is less than the above reference value, it can be determined that the test animal has developed septicemia.

Alternatively, as a result of comparison of the amounts of biomarkers, for example, in a sample derived from a test animal, when the biomarker of the present invention is detected or measured at a higher value than that of a control sample, or when it is equal to or higher than the above reference value. In the case where it can be determined that the test animal may have developed Still's disease, and the value is detected or measured at the same level or lower than that of the control sample, or less than the above reference value. If this is the case, it can be determined that the test animal may have developed septicemia.

3. 3. Treatment or prevention method of the present invention 2. As a result of the method of the present invention described in the above, when it is determined that the test animal has developed Still disease or septicemia, or when it is determined that the test animal may have developed it. Based on the results of the determination and the like, a therapeutic or prophylactic drug for still disease or septicemia to be administered to the test animal (hereinafter collectively referred to as "therapeutic drug") is selected (or determined), and the test animal is selected. Still disease or septicemia can be treated or prevented by administering a therapeutically or prophylactically effective amount of the therapeutic agent. As used herein, the term "therapeutic agent" includes not only drugs intended for the curative treatment of Still's disease or sepsis, but also drugs aimed at suppressing the progression of these diseases or drugs aimed at alleviating symptoms. It shall be included.

Examples of the therapeutic agent for still disease include, but are not limited to, steroid drugs (eg, prednisolone, methylprednisolone, etc.), tocilizumab, immunosuppressants (cyclosporine, tacrolimus, methotrexate, etc.). Further, examples of the therapeutic agent for sepsis include, but are not limited to, antibacterial agents (eg, meropenem, etc.), circulatory agonists (noradrenaline, etc.), and the like. The above-mentioned therapeutic agents may be used in combination as appropriate according to the patient's symptoms.

The above-mentioned therapeutic agent is administered orally or parenterally as a pharmaceutical composition of an appropriate dosage form by using the active ingredient as it is or by mixing it with a pharmaceutically acceptable carrier, excipient, diluent and the like. You may. Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. Agents, emulsions, suspending agents and the like can be mentioned. On the other hand, as the composition for parenteral administration, for example, injections, suppositories and the like are used, and the injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections and the like. The dosage form may be included. In addition, the dose of the therapeutic agent can be appropriately set depending on various conditions such as the type of the compound, the symptom of the administration target, the age, the body weight, and the drug acceptability.

4. Test Kit of the Present Invention Further, the present invention provides a test kit for distinguishing Still's disease from sepsis (hereinafter referred to as the test kit of the present invention). The test kit of the present invention preferably has
(a) An antibody capable of specifically recognizing an FGF-2 protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing an FGF-2 transcript,
(b) A nucleic acid probe or nucleic acid primer capable of specifically recognizing an IL-18 protein or an IL-18 transcript,
(c) An antibody capable of specifically recognizing a G-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a G-CSF transcript,
(d) An antibody capable of specifically recognizing a GM-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a GM-CSF transcript,
(e) The antibody, nucleic acid probe or nucleic acid primer of (a), and the antibody, nucleic acid probe or nucleic acid primer of (b), and
(f) The antibody, nucleic acid probe or nucleic acid primer of (a), and any of the antibody, nucleic acid probe or nucleic acid primer of (d) are included. Only one type of these may be contained, or a plurality of types may be contained.

When the test kit of the present invention contains the above-mentioned nucleic acid probe or nucleic acid primer (also simply referred to as “nucleic acid”) as a constituent, these nucleic acids may be referred to as the above 2. Examples thereof include nucleic acid for a probe and oligonucleotide for a primer exemplified by the method of the present invention. These nucleic acids can be provided as a solid in a dry state or in an alcohol-precipitated state, or can be provided in a state of being dissolved in water or a suitable buffer solution (eg, TE buffer solution, etc.). When used as a labeling probe, the nucleic acid can be provided in a state of being labeled with any of the above-mentioned labeling substances in advance, or can be provided separately from the labeling substance and labeled at the time of use. Alternatively, the nucleic acid can be provided in a state of being immobilized on a suitable solid phase. Examples of the solid phase include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluorolide, and the like. Further, as an immobilization means, a functional group such as an amino group, an aldehyde group, an SH group or a biotin is introduced into the nucleic acid in advance, and on the other hand, a functional group capable of reacting with the nucleic acid on the solid phase (eg, aldehyde). Groups, amino groups, SH groups, streptavidin, etc.) are introduced to cross-link the solid phase and nucleic acid by covalent bonds between both functional groups, or polyanionic nucleic acid is coated with polycation on the solid phase and statically. Methods such as immobilizing the nucleic acid using an electric bond can be mentioned, but the method is not limited thereto.

When a plurality of types of the above nucleic acids contained in the test kit of the present invention are contained, the expression of the biomarker of the present invention is detected by the same method (eg, Northern blot, dot blot, DNA array technique, quantitative RT-PCR, etc.). Alternatively, it is preferably constructed so that it can be quantified.

When the test kit of the present invention contains the above-mentioned antibodies as a constituent, the above-mentioned 2. Examples of the antibody exemplified by the method of the present invention.

In addition to the nucleic acids and antibodies described above, the test kit of the present invention may contain other substances necessary for the reaction for detecting or quantifying the expression of the biomarker of the present invention. These other substances may be provided in coexistence with nucleic acids, antibodies, etc., or may be provided with a separate reagent, as long as they do not adversely affect the reaction. For example, when the reaction for detecting or quantifying the expression of the biomarker of the present invention is PCR, examples of the other substances include reaction buffers, dNTPs, thermostable DNA polymerase and the like. When competitive PCR or real-time PCR is used, it can further contain a competitor nucleic acid, a fluorescent reagent (the above-mentioned intercalator, a fluorescent probe, etc.) and the like. When the reaction for detecting or quantifying the expression of the biomarker of the present invention is an antigen-antibody reaction, the other substances include, for example, a reaction buffer, a competitor antibody, and a labeled secondary antibody (for example, primary). When the antibody is a rabbit antibody, examples thereof include mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc.), a blocking solution, and a plate for ELISA. In addition, the test kit of the present invention may include an instruction manual that describes how to use the kit and reagents, criteria for determining a disease, and the like. Further, the above-mentioned test kit may contain one or more biomarkers of the present invention for use as a positive control, for example.

Hereinafter, the present invention will be described in more detail with reference to examples, but it is clear that the present invention is not limited thereto.

Patients and controls The study population of this example met the classification criteria of Yamaguchi et al. (1992) at our hospital and related facilities (Kanazawa University Hospital, Nagasaki Medical Center, Sasebo General Medical Center), and other diseases were excluded. Pretreatment serum of 66 patients diagnosed by AOSD and rheumatologist (41 women, mean age 48.1 years, standard deviation 21.2) and sepsis diagnosed at our hospital for circulatory agents or DIC Pretreatment serum in 17 patients with septic shock who required thrombomodulin preparation (9 women, mean age 61.4 years, standard deviation 18.3).
The resulting blood sample was centrifuged at 2,000 g for 5 minutes, the supernatant was collected and stored at -80 ° C until prior to the assay. All patients were informed of their studies based on a protocol (approval number 14092946) approved by the Institutional Review Board of Nagasaki University and related centers, and signed informed consent with consent and consent.

Complex Cytokine Assay The inventors used the obtained blood sample in accordance with the instructions for Bio-Plex Pro Human Cytokine (Bio-Rad, Hercules, CA) and Milliplex MAP Human Cytokine / Chemokine Panel 1 (Millipore, Billerica, MA). ) Was used to perform a complex cytokine bead assay. Interferon-1β (IL-1β), IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL -12 (p70), IL-17, IL-18, tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), IFN-γ, granulocyte macrophage colony stimulator (GM-CSF), Granulocyte colony stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), ICAM-1, VCAM-1, fibroblast growth factor 2 (FGF2), CCL2 [monosphere migration factor-1 (MCP-1) / MCAF], CCL3 [Macrophage Inflammatory Protein-1a (MIP-1a)], CCL4 [Macrophage Inflammatory Protein-1b (MIP-1b)], CCL22 [Human Macrophage Derived Chemokine (MDC)], CXCL1 [Proliferation Control Protein] Of the 45 cytokines containing α precursor (GRO)], CXCL10 [interferon γ-inducible protein 10 (IP-10)] and CX3CL1 (Fractalkine), 30 cytokines with large fluctuations in numerical values were selected for further analysis. I tried it.

Statistical analysis First, using logistic regression analysis, we derived cytokines or combinations thereof that are effective in discriminating between AOSD and sepsis. Table 1 shows the results by ROC analysis. This results in sensitivity, specificity and when using the combination of FGF-2, IL-18, G-CSF, GM-CSF, FGF-2 and IL-18, and the combination of FGF-2 and GM-CSF. High AUC values indicate that one or more of these cytokines are suitable as biomarkers for differentiating still disease from septicemia.

In the table, AUC stands for Area Under Curve, AIC stands for Akaike's Information Criterion, TP stands for True Positive, and TN stands for True Negative. FP is an abbreviation for False Positive, and FN is an abbreviation for False Negative. The unit of the cutoff value is pg / ml.

In addition, the inventors of R package random Forest Version 4.5-34,22 (Kokkonen H, Soderstrom I, Rocklov J, et al. Up-regulation of cytokines and chemokines predates the onset of rheumatoid arthritis. Arthritis Rheum. 2010; 62. Cytokine levels were ranked by a multivariate classification algorithm named Random Forest analysis (RFA) using (2): 383). The results are shown in FIG.

Next, a decision tree analysis was performed as an algorithm for discriminating between AOSD patients and septic patients using the above cytokine levels, and a discriminant model was constructed. Decision tree analysis is an analysis method that branches cytokine values and combines them to build a discriminant model.Since simple rules such as binary branching can be obtained regardless of the distribution of cytokines, it is suitable for clinical application. Are better. The appropriate tree size was determined by cross-validation (especially Min-1SE rule), and statistical analysis software R and rpart package were used for analysis. As a result, it was shown that by combining the measurement of GM-CSF protein and FGF-2 protein, AOSD patients and septic patients can be accurately differentiated (Fig. 2).

The FGF-2, IL-18, G-CSF or G-CSF of the present invention is useful as a biomarker for differentiating Still's disease from sepsis.

Claims (13)

A biomarker for distinguishing Still's disease from sepsis, which comprises any of the following (a) to (c).
(a) FGF-2 protein or FGF-2 transcript ,
(b ) G-CSF protein or G-CSF transcript, and
( c ) GM-CSF protein or GM-CSF transcript A method for assisting determination of whether a test animal has still disease or sepsis, which comprises a step of detecting or quantifying one or more of the biomarkers according to claim 1 in a sample derived from a test animal. A method for analyzing the possibility of still disease or sepsis, which comprises a step of detecting or quantifying one or more of the biomarkers according to claim 1 in a sample derived from a test animal. The method according to claim 2, which comprises the following steps (i) to (iii).
(I) A step of quantifying one or more of the biomarkers according to claim 1 in a sample derived from a test animal.
(Ii) When the amount of biomarker quantified in (i) is greater than or equal to the reference value based on the step of comparing the amount of biomarker quantified in (i) with the reference value and (iii) based on the results of (ii). In addition, a step of determining that the test animal has developed Still's disease and determining that the test animal has developed septicemia when the value is less than the reference value. The method according to claim 3, which comprises the following steps (i) to (iii).
(I) A step of quantifying one or more of the biomarkers according to claim 1 in a sample derived from a test animal.
(Ii) When the amount of biomarker quantified in (i) is greater than or equal to the reference value based on the step of comparing the amount of biomarker quantified in (i) with the reference value and (iii) based on the results of (ii). In addition, a step of determining that the test animal may have developed Still's disease, and if it is less than the reference value, it is determined that the test animal may have developed septicemia. It is characterized in that two or more kinds of biomarkers according to claim 1 are used, or one or more kinds of biomarkers according to claim 1 and a biomarker composed of IL-18 protein or IL-18 transcript are used . , The method according to any one of claims 2 to 5. The biomarker
Claims 2-6 comprising (1) FGF-2 protein or FGF-2 transcript, and (2) GM-CSF protein or GM-CSF transcript, and / or IL-18 protein or IL-18 transcript. The method according to any one of the above. The method according to any one of claims 2 to 7, wherein the biomarker is detected or quantified using any one of the following (a) to ( e ).
(a) An antibody capable of specifically recognizing an FGF-2 protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing an FGF-2 transcript ,
(b ) An antibody capable of specifically recognizing a G-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a G-CSF transcript,
( c ) An antibody capable of specifically recognizing a GM-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a GM-CSF transcript,
( d ) An antibody, nucleic acid probe or nucleic acid primer of (a), and a nucleic acid probe or nucleic acid primer capable of specifically recognizing an antibody or IL-18 transcript capable of specifically recognizing an IL-18 protein, and a nucleic acid probe or nucleic acid primer .
( e ) The antibody, nucleic acid probe or nucleic acid primer of (a), and the antibody, nucleic acid probe or nucleic acid primer of ( c ). The method according to any one of claims 4 to 8, wherein the reference value is a value set based on a decision tree analysis or a ROC analysis. The method according to any one of claims 2 to 9, wherein the test animal is a human. The method according to any one of claims 2 to 10, wherein the sample is serum. A test kit for distinguishing Still's disease from sepsis, which comprises one or more of the following (a) to ( e ).
(a) An antibody capable of specifically recognizing an FGF-2 protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing an FGF-2 transcript ,
(b ) An antibody capable of specifically recognizing a G-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a G-CSF transcript,
( c ) An antibody capable of specifically recognizing a GM-CSF protein or a nucleic acid probe or nucleic acid primer capable of specifically recognizing a GM-CSF transcript,
( d ) An antibody, nucleic acid probe or nucleic acid primer of (a), and a nucleic acid probe or nucleic acid primer capable of specifically recognizing an antibody or IL-18 transcript capable of specifically recognizing an IL-18 protein, and a nucleic acid probe or nucleic acid primer .
( e ) The antibody, nucleic acid probe or nucleic acid primer of (a), and the antibody, nucleic acid probe or nucleic acid primer of ( c ). 12. The kit of claim 12, further comprising one or more of the biomarkers of claim 1.

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