Method for predicting child non-reactive Kawasaki disease based on T lymphocyte activation related cytokines

Abstract

The invention provides an application of a T lymphatic activation related cytokine as a biomarker in prediction of children immune globulin non-reactive Kawasaki disease. The team finds that in patients of an IVIG sensitive group and an IVIG non-response group, differences of expression levels of IL-6 and IL-17 before and after treatment have statistical significance; meanwhile, ROC curve analysis shows that IL-6 and IL-17 serve as early warning signals, and the prediction efficiency of the IVIG-unreacted Kawasaki disease is remarkably superior to that of existing prediction factors. Particularly, the levels of IL-6 and IL-17 are re-evaluated after initial treatment, so that the early prediction accuracy of the IVIG unresponsive Kawasaki disease can be further improved. By introducing IL-6 and IL-17 as novel biomarkers, early accurate prediction of the IVIG unresponsive Kawasaki disease is realized. Compared with the prior art, the method can provide a quicker and more accurate risk assessment basis for clinicians, so that early recognition of high-risk patients, optimization of subsequent treatment schemes and reduction of serious complications are facilitated, and a new thought and direction are provided for clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis technology, in particular to a method for predicting unresponsive Kawasaki disease in children by using T lymphocyte activation-related factors as biomarkers. Background Art

[0002] Kawasaki disease (KD) is an acute systemic vasculitis that manifests in early childhood. Coronary artery aneurysm (CAA) is its most important complication, which can cause myocardial ischemia or acute coronary syndrome. A single intravenous injection of human immunoglobulin (IVIG) 2g / kg and aspirin is the standard treatment for KD, but some children do not respond to IVIG, with an incidence of about 10-20%, and need to receive additional immunoglobulin (IVIG) treatment and / or other treatments. Such patients are classified as immunoglobulin-unresponsive KD. Immune globulin-unresponsive KD is a high risk factor for CAA. Unresponsiveness to IVIG may reflect the severity of the underlying inflammation. In a highly inflammatory state, more serious complications such as Kawasaki disease shock syndrome (KDSS) or Kawasaki disease combined with macrophage activation syndrome (MAS) may also occur, endangering the child's life. Unresponsive Kawasaki disease (KD, also known as refractory KD) is a difficult problem faced by clinicians, requiring secondary treatment after standard treatment. Early recognition is crucial for initiating IVIG and further additional treatment.

[0003] Predicting resistance to immunoglobulin (IVIG) is the key to strengthening initial treatment and preventing cardiac complications. Existing scoring systems for predicting resistance to immunoglobulin (IVIG) vary from country to country. Since 2006, about 17 prediction models have been published worldwide, but these prediction models are all single-center studies. There is currently no globally unified prediction model. For example, the earliest Kobayashi scoring model (2006) has certain value in predicting treatment failure of unresponsive Kawasaki disease in the Japanese population, but may have limited applicability in the UK, the US, and other regions. Therefore, existing technologies lack objective and reliable experimental markers to predict unresponsive Kawasaki disease in children. Summary of the invention

[0004] To solve the above problems, the present invention provides a new method for predicting unresponsive Kawasaki disease in children based on T lymphocyte activation cytokines. The method and system can improve the accuracy of predicting unresponsive Kawasaki disease, thereby guiding clinicians to develop more effective treatment plans. To achieve the above objectives, the present invention adopts the following technical solutions:

[0005] The first aspect of the present invention provides a method for predicting whether a child suffers from Kawasaki disease by using T lymphocyte activation-related factors as biomarkers.

[0006] Furthermore, the Kawasaki disease is Kawasaki disease complicated by coronary artery damage or immunoglobulin-unresponsive Kawasaki disease;

[0007] Furthermore, the Kawasaki disease is immunoglobulin-unresponsive Kawasaki disease;

[0008] Furthermore, the T lymphocyte activation-related factor is selected from one or more of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17 and tumor necrosis factor-α (TNF-α);

[0009] In some embodiments, the T lymphocyte activation-related factor is IL-6 or IL-17.

[0010] In some embodiments, the T lymphocyte activation-related factors are IL-6 and IL-17.

[0011] Furthermore, the method comprises the following steps:

[0012] (1) Peripheral venous blood samples were collected from children with KD before and after a single intravenous immunoglobulin (IVIG) treatment;

[0013] (2) detecting the levels of T lymphocyte activation-related cytokines in blood samples;

[0014] (3) To compare the differences in cytokine levels between the immunoglobulin-unresponsive Kawasaki disease (hereafter referred to as “unresponsive KD”) group and the sensitive Kawasaki disease group (hereafter referred to as “sensitive KD”);

[0015] (4) ROC curve analysis was used to evaluate the predictive ability of T lymphocyte activation-related cytokine levels for unresponsive KD;

[0016] The results showed that cytokines IL-2, IL-7, IL-12, IL-15, IL-6, IL-17 and TNF-α were related to lymphocyte activation in unresponsive KD, especially IL-6 and IL-17, which had significant advantages in the comparison of each group. ROC curve analysis showed that the area under the curve (AUC) of IL-6 increased from 0.859 to 0.920 after IVIG treatment; the AUC of IL-17 increased from 0.699 to 0.884 after IVIG treatment.

[0017] Furthermore, in step (2), the T lymphocyte activation-related cytokines are selected from one or more of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17 and tumor necrosis factor α (TNF-α);

[0018] Furthermore, in step (2), the T lymphocyte activation-related cytokines are IL-6 and / or IL-17.

[0019] In some embodiments, the T lymphocyte activation-related cytokines are IL-6 and IL-17;

[0020] In some embodiments, the T lymphocyte activation-related cytokine is IL-6;

[0021] In some embodiments, the T lymphocyte activation-related cytokine is IL-17;

[0022] Further, in step (2), the level of cytokines is detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry;

[0023] Furthermore, in step (3), the cytokine levels detected in step (2) are compared with the cytokine levels of a known non-responsive Kawasaki disease group or a sensitive Kawasaki disease group.

[0024] Furthermore, the method also includes, if the child to be tested is diagnosed with immunoglobulin-unresponsive Kawasaki disease, administering immunoglobulin injection combined with hormone treatment to the child.

[0025] Furthermore, the immunoglobulin injection is IVIG injection, and the injection dose is 2 g / kg body weight each time, once a day.

[0026] Furthermore, the hormone is a glucocorticoid, specifically methylprednisolone, which is administered orally or intravenously, at a dosage of 1-2 mg / kg body weight, twice a day, for 3-5 days, such as 3 days, 4 days or 5 days, and can be reduced or stopped within 15 days of administration according to the C-reactive protein and body temperature conditions.

[0027] A second aspect of the present invention provides a kit for predicting non-responsive Kawasaki disease in children;

[0028] Furthermore, the kit comprises the T lymphocyte activation-related factor described above;

[0029] Furthermore, the T lymphocyte activation-related factor is selected from one or more of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17 and tumor necrosis factor α (TNF-α);

[0030] Furthermore, the T lymphocyte activation-related cytokines are IL-6 and / or IL-17;

[0031] Further, the reagent is an enzyme-linked immunosorbent assay (ELISA) reagent or a flow cytometry reagent;

[0032] Furthermore, the kit also includes software or instructions for drawing a ROC curve and determining a cutoff value.

[0033] In some embodiments, the ROC curve drawn shows that the sensitivity of IL-6 before IVIG treatment is 87.50%, and the specificity is 82.81%; after IVIG treatment, the sensitivity is 90.91%, and the specificity is 88.00%.

[0034] In some embodiments, the ROC curve drawn shows that the sensitivity of IL-17 before IVIG treatment is 72.73%, and the specificity is 68.75%; after IVIG treatment, the sensitivity is 100%, and the specificity is 76.00%.

[0035] In some embodiments, the ROC curve plotted shows that the area under the curve (AUC) of IL-6 for predicting immunoglobulin-unresponsive Kawasaki disease ranges from 0.80 to 0.90 before immunoglobulin treatment and ranges from 0.90 to 0.95 after treatment;

[0036] In some embodiments, the ROC curve plotted shows that the area under the curve (AUC) of IL-17 for predicting immunoglobulin-unresponsive Kawasaki disease ranges from 0.65-0.75 before immunoglobulin treatment and ranges from 0.85-0.95 after immunoglobulin treatment.

[0037] Beneficial Effects

[0038] 1. The present invention detects the changes in the levels of T lymphocyte activation-related cytokines before and after immunoglobulin treatment, as well as the differences in the levels of each factor between the sensitive Kawasaki disease group and the immunoglobulin-unresponsive Kawasaki disease group, and finds that the related cytokines of T cell activation are involved in the disease progression of immunoglobulin-unresponsive Kawasaki disease, especially IL-6 and IL-17. Retesting after initial treatment can further improve the predictiveness of immunoglobulin-unresponsive Kawasaki disease, thereby determining independent biomarkers: that is, IL-6 and IL-17 have significant advantages in the comparison of each group, which can be used as new and objective experimental markers for early prediction of immunoglobulin-unresponsive Kawasaki disease in children, thereby improving the accuracy and reliability of the prediction of immunoglobulin-unresponsive Kawasaki disease in children.

[0039] 2. The present invention compares the T lymphocyte activation-related cytokines IL-2, IL-6, IL-7, IL-12, IL-15, and IL-17 in each group, and IL-6 and IL-17 perform outstandingly. In addition to the sensitive KD group having statistical significance before and after treatment for the unresponsive group, the IL-6 sensitive KD group and the unresponsive group have statistical significance before and after treatment, and the IL-17 sensitive KD group has statistical significance before and after treatment for the unresponsive group, and the sensitive KD group within the group has statistical significance after treatment. Therefore, IL-6 and IL-17 can be used as independent biomarkers for early prediction of IVIG unresponsive Kawasaki disease, and by monitoring the changes in their content, they can provide a basis for early diagnosis of Kawasaki disease.

[0040] 3. The method provided by the present invention for using T lymphocyte activation-related factors as biomarkers to predict unresponsive Kawasaki disease in children has the advantages of high sensitivity and high accuracy, and the method is simple and easy to implement, suitable for clinical diagnosis and formulation of treatment plans, and helps to reduce the incidence of complications in patients with unresponsive Kawasaki disease and improve the treatment effect; it fills the gaps in the existing IVIG resistance prediction scoring system and provides new ideas and methods for the individualized treatment of Kawasaki disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the overall process operation.

[0042] Figure 2 This is a comparison chart of the changes in T lymphocyte activation cytokines IL-2, 6, 7, 12, 15 and 17 and TNF-α before and after treatment between the sensitive KD group and the unresponsive group.

[0043] Among them, A: The IL-2 sensitive KD group was statistically significant compared with the non-responsive group before and after treatment (P<0.05).

[0044] B: There was statistical significance between the IL-6 sensitive KD group and the non-responsive group before and after treatment, there was statistical significance within the sensitive KD group before and after treatment, and there was statistical significance within the non-responsive group before and after treatment (P<0.05).

[0045] C: The IL-7 sensitive KD group was statistically significant before treatment compared with the unresponsive group, and there was significance within the unresponsive group before and after treatment (P<0.05).

[0046] D: The IL-12 sensitive KD group was statistically significant compared with the non-responsive group before treatment (P<0.05).

[0047] E: There was statistical significance between the IL-15 sensitive KD group and the non-responsive group before and after treatment (P<0.05).

[0048] F: There was statistical significance between the IL-17 sensitive KD group and the non-responsive group before and after treatment, and there was statistical significance within the sensitive KD group before and after treatment (P<0.05).

[0049] G: Statistical significance of TNF-α sensitive KD group compared with non-responsive group before treatment (P<0.05).

[0050] Figure 3 ROC curve analysis of the predictive efficacy of IL-6 and IL-17 for unresponsive KD.

[0051] The results of ROC curve analysis showed that the sensitivity of IL-6 before IVIG treatment was 87.50%, and the specificity was 82.81%, and the sensitivity and specificity after IVIG treatment were 90.91% and 88.00% respectively; the sensitivity of IL-17 before IVIG treatment was 72.73%, and the specificity was 68.75%, and the sensitivity and specificity was 100% and 76.00% respectively after IVIG treatment; before and after treatment, the areas under the curve (AUC) of IL-6 and IL-17 for predicting KD were 0.859, 0.920, 0.699 and 0.884 respectively. DETAILED DESCRIPTION

[0052] In order to find an objective and reliable experimental marker to predict immunoglobulin-unresponsive Kawasaki disease in children, the present invention compares the differences in interleukin-2 (IL-2), IL-6 (IL6), IL-7 (IL7), IL-12 (IL-12), IL-15 (IL-15), IL-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) in patients with Kawasaki disease sensitive KD group (hereinafter referred to as "sensitive KD group") and immunoglobulin-unresponsive Kawasaki disease group (hereinafter referred to as "unresponsive KD group"), to find out whether cytokines can provide effective clues for unresponsive Kawasaki disease and contribute to the existing scoring mechanism. The present invention aims to provide a new biomarker detection method to improve the prediction accuracy of unresponsive Kawasaki disease.

[0053] The present invention is further described below in conjunction with specific embodiments, but the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0054] 1 Research process

[0055] In order to explore the predictive value of T lymphocyte activation-related cytokines for unresponsive Kawasaki disease, the present invention collected 82 children aged 1-14 years who were hospitalized in a specialized children's hospital in Zhejiang Province, China from June 2022 to December 2023. They were divided into a sensitive Kawasaki disease group (n=71) and an unresponsive Kawasaki disease group (n=11) according to the treatment effect. Then, the levels of T lymphocyte activation-related cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-17 (IL-17) and tumor necrosis factor α (TNF-α) in the serum of the two groups were detected before and after IVIG treatment. The differences in the levels of T lymphocyte activation-related cytokines before and after treatment between the two groups were compared. The ROC curve was used to explore the discrimination ability of cytokines for unresponsive Kawasaki disease, and the cutoff value was determined.

[0056] The results showed that before treatment, the interleukin-2, 6, 7, 12, 15, 17, and tumor necrosis factor-α values ​​in the non-responsive Kawasaki disease group were significantly increased compared with the sensitive Kawasaki disease group. After initial treatment, IL-6 and IL-17 in the sensitive Kawasaki disease group decreased significantly, and IL-6 and IL-7 in the non-responsive Kawasaki disease group decreased significantly, while IL-6 and IL-17 in the sensitive KD group increased significantly compared with the non-responsive group.

[0057] The ROC curve showed that the area under the curve (AUC) of IL-6 for predicting non-responsive Kawasaki disease before and after treatment was 0.859 and 0.920, respectively, while the AUC of IL-17 was 0.699 and 0.884, respectively.

[0058] Therefore, the present invention shows that T cell activation-related cytokines, especially IL-6 and IL-17, are involved in the pathogenesis of immunoglobulin unresponsive Kawasaki disease (hereinafter referred to as "unresponsive KD") and can improve the predictability of unresponsive KD within a certain range.

[0059] 2 Inclusion and Exclusion Criteria

[0060] Inclusion criteria:

[0061] 1) Meet the 2017 American Heart Association (AHA) KD diagnostic guidelines;

[0062] 2) The age range is 1-14 years old, regardless of gender;

[0063] 3) Complete hospitalization records;

[0064] Exclusion criteria:

[0065] 1) Combined with other immune diseases or immunodeficiency diseases, such as systemic juvenile idiopathic arthritis and leukemia;

[0066] 2) recently received immunoglobulin therapy;

[0067] 3) incomplete hospitalization data;

[0068] Grouping criteria:

[0069] 1) All patients were given intravenous immunoglobulin (IVIG) at a dose of 2 g / kg and aspirin at a dose of 30-50 mg / kg within 10 days of diagnosis;

[0070] 2) The definition of unresponsive Kawasaki disease is that the fever does not subside (T>38℃) after 36 hours of IVIG treatment or the fever reappears 2 to 7 days after the fever subsides and is accompanied by at least one of the main clinical features of Kawasaki disease.

[0071] Coronary artery abnormality (CAL) criteria:

[0072] 1) Non-invasive: Z value <2;

[0073] 2) Abnormal: Z value ≥ 2 or initial Z value < 2, and Z value decreased by ≥ 1 during follow-up.

[0074] KD comorbidity diagnostic criteria:

[0075] 1) Liver dysfunction: mainly characterized by elevated ALT, serum ALT>50U / L;

[0076] 2) Myocardial damage: CK-MB>50U / L or high-sensitivity troponin (hs-cTn)>0.05ng / ml;

[0077] 3) Hypoproteinemia: total protein <60g / L or albumin <25g / L;

[0078] 4) Cholestasis: direct bilirubin>17μmol / L.

[0079] 2 Methods:

[0080] T lymphocyte activation-related cytokine detection uses yellow procoagulant blood collection tubes to collect 2 mL of blood samples from the peripheral vein of the child, place them at room temperature for 15-30 minutes, and then cool them at 4°C after the blood coagulates.

[0081] Centrifuge at 2000g*10min, collect the supernatant, and store at -20℃ or lower for later use. Serum samples will be sent to Hangzhou Dacheng Biotechnology Co., Ltd. for testing. According to the instructions of the ELISA kit (ELK Biotechnology), the levels of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17 and TNF-α were measured.

[0082] 3. Data Collection

[0083] The following clinical data were collected: gender, age, clinical characteristics, and clinical treatment received by the subjects. Clinical characteristics mainly refer to complications that meet the diagnostic criteria for KD. Laboratory data were collected 24 hours before IVIG treatment and within 3 days after the temperature returned to normal after IVIG treatment.

[0084] 4 Statistical analysis methods

[0085] SPSS25.0 statistical software was used for statistical analysis of the data. First, the original data were tested for normality. If they conformed to the normal distribution, the two-independent sample T test was used; if they did not conform to the normal distribution, the two-sample rank sum test was used. Numerical data were expressed as numbers and percentages (%) and compared using the chi-square test. One-way analysis of variance (ANOVA) was performed using Graphpad Prism 9, and graphs were drawn to detect the homogeneity of variance. If the variances were equal, ordinary analysis of variance should be used; if the variances were unequal, the Bute-Forsyth and Welch analysis of variance method should be used. Med Calc statistical software was selected to draw the ROC curve. In all statistical analyses, P<0.05 was considered statistically significant.

[0086] Example 1

[0087] This embodiment uses T lymphocyte activation-related factors as biomarkers to predict Kawasaki disease in children, especially immunoglobulin-unresponsive Kawasaki disease. The T lymphocyte activation-related factors include one or more of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17 and tumor necrosis factor-α (TNF-α), among which IL-6 and / or IL-17 are particularly prominent.

[0088] The overall operation process of this embodiment is as shown in the attached Figure 1 As shown, the specific steps are as follows:

[0089] (1) Peripheral venous blood samples were collected from children with KD before and after a single intravenous injection of human immunoglobulin (IVIG).

[0090] (2) Enzyme-linked immunosorbent assay (ELISA) or flow cytometry was used to detect the levels of T lymphocyte activation-related cytokines in blood samples.

[0091] (3) Compare the differences in cytokine levels between the immunoglobulin-unresponsive Kawasaki disease group and the sensitive Kawasaki disease group. (4) Use ROC curve analysis to evaluate the predictive ability of cytokines for immunoglobulin-unresponsive Kawasaki disease. (5) If the child to be tested is diagnosed with immunoglobulin-unresponsive Kawasaki disease, immunoglobulin injection combined with

[0092] Hormone therapy. Immunoglobulin injection is IVIG injection, the dose is 2g / kg body weight each time, once a day.

[0093] At the same time, glucocorticoids are used for treatment, and the glucocorticoid is methylprednisolone. Methylprednisolone is taken orally or intravenously, with a dose of 1-2 mg / kg body weight each time, twice a day, and continued for 3-5 days, such as 3 days, 4 days or 5 days. The dosage can be reduced or stopped within 15 days according to the C-reactive protein and body temperature.

[0094] Example 2

[0095] In this example, based on Example 1, a total of 82 children with Kawasaki disease who were finally included in the study were treated according to the above inclusion, exclusion and grouping criteria. Among the 82 children with Kawasaki disease, 48 were males and 34 were females, with a male-to-female ratio of 1.44:1. Among them, there were 19 children aged 1-12 months, 24 children aged 13-24 months, 21 children aged 25-60 months, and 18 children aged >60 months. According to the persistent fever (T>38°C) after 36 hours of initial IVIG (2g / kg / time) treatment or the recurrence of fever 2-7 days after the fever subsided and accompanied by at least one of the main clinical features of Kawasaki disease, the patients were finally divided into a sensitive Kawasaki disease group of 71 cases and an unresponsive Kawasaki disease group of 11 cases. For the group of children with Kawasaki disease who were resistant to the first intravenous human immunoglobulin (IVIG), samples were drawn after the second IVIG treatment and before steroid treatment. After the body temperature returned to normal on the 2nd-3rd day after steroid treatment, the total lymphocyte subsets or lymphocyte activation-related factors were retested. If there was a third time, the same was true; cardiac ultrasound could be performed before and after IVIG administration, and 4 and 8 weeks after administration; patients who had received treatment or had other diseases that might interfere with this study were excluded. Afterwards, total lymphocyte subsets and flow cytokine samples were drawn before and after each step of treatment to understand the changes in the levels of each item and to observe whether there was an increase or decrease. In particular, there were differences in the level of T cell activation.

[0096] All 11 children in the unresponsive KD group received a second full dose of IVIG (2 g / kg / time, total dose of 4 g / kg) combined with intravenous hormone (1-2 mg / kg each time, 2 times / day, 3-5 days, gradually reduced and stopped within 15 days after the body temperature stabilized and the C-reactive protein level decreased). One of them had fever again after the second round of IVIG shock combined with hormone therapy after the hormone was reduced. Infliximab (IFX) (5 mg / kg) was used once, and the body temperature did not recur. The proportion of patients with liver dysfunction in the unresponsive Kawasaki disease group increased significantly (p = 0.004), and there was no statistical difference in other complications, see Table 1.

[0097] 2 mL of blood samples were collected from the peripheral veins of the children using yellow procoagulant blood collection tubes, the supernatant was collected after centrifugation, and stored at -20°C or lower for later use. Serum samples were sent to a professional biotechnology company for testing to measure the levels of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17, and TNF-α.

[0098] The detected cytokine levels were compared with those of known non-responder Kawasaki disease groups and sensitive Kawasaki disease groups, and the ROC curve was used to analyze the discriminatory ability of cytokines for non-responder Kawasaki disease and determine the cutoff value.

[0099] The results showed that the IL-2, 6, 7, 12, 15, 17 and TNF-α values ​​of the unresponsive Kawasaki disease group before treatment were significantly higher than those of the sensitive Kawasaki disease group; after treatment, the IL-6 and IL-17 levels of the sensitive KD group and the unresponsive KD group decreased significantly, but the levels of IL-6 and IL-17 after treatment were significantly higher than those of the sensitive KD group. Therefore, cytokines related to T cell activation are involved in the pathogenesis of unresponsive KD and can improve the predictability of unresponsive KD within a certain range. Among them, IL-6 has a high accuracy in predicting immunoglobulin unresponsive Kawasaki disease.

[0100] Table 1 Basic clinical data [cases (%), X 2 ,M(P25,P75)]

[0101]

[0102]

[0103] Example 3

[0104] This example detects the levels of T lymphocyte activation-related cytokines, and studies the role of T lymphocyte activation-related cytokines IL-2, IL-6, IL-7, IL-12, IL-15, IL-17 and TNF-α as biomarkers in predicting immunoglobulin-unresponsive Kawasaki disease.

[0105] Specifically, this example compares the changes in T lymphocyte activation cytokines IL-2, 6, 7, 12, 15 and 17 and TNF-α before and after treatment between the sensitive KD group and the unresponsive KD group. Figure 2 .

[0106] from Figure 2 It can be seen that before immunoglobulin treatment, the IL-2, IL-6, IL-7, IL-12, IL-15, IL-17, and tumor necrosis factor α values ​​in the unresponsive Kawasaki disease group were significantly increased compared with those in the sensitive Kawasaki disease group; after the initial immunoglobulin (IVIG) treatment, IL-6 and IL-17 in the sensitive Kawasaki disease group decreased significantly, and IL-6 and IL-7 in the immunoglobulin unresponsive Kawasaki disease group decreased significantly, while IL-6 and IL-17 in the sensitive Kawasaki disease group unresponsive to immunoglobulin increased significantly.

[0107] ROC curve analysis showed that the sensitivity of IL-6 before IVIG treatment was 87.50 and the specificity was 82.81; the sensitivity after IVIG treatment was 90.91% and the specificity was 88.00%; the sensitivity of IL-17 before IVIG treatment was 72.73% and the specificity was 68.75%; the sensitivity after IVIG treatment was 100% and the specificity was 76.00%; the area under the curve (AUC) of IL-6 for predicting non-responsive Kawasaki disease before and after treatment was 0.859 and 0.920 respectively, while the area under the curve (AUC) of IL-17 for predicting non-responsive Kawasaki disease before and after treatment was 0.699 and 0.884 respectively, indicating that by measuring the content of IL-6 and IL-17 factors before and after reaction, it can provide effective clues for predicting non-responsive Kawasaki disease.

[0108] Example 4

[0109] In this example, the predictive efficacy of IL-6 and IL-17 for Kawasaki disease without immunoglobulin response was verified. The ROC curve analysis results showed that the area under the curve (AUC) for predicting KD before IL-6 treatment, after IL-6 treatment, before IL-17 treatment, and after IL-17 treatment was 0.859, 0.920, 0.699, and 0.884 (see Table 2 and Figure 3 )

[0110] Table 2 Predictive efficacy of IL-6 and IL-17 before and after

[0111]

[0112] The root cause of Kawasaki disease is unknown. It may be caused by the activation of the immune system after genetically susceptible children are infected with unknown pathogens (such as viruses), leading to an inflammatory cascade. No response to intravenous human immunoglobulin (IVIG) There is less information on the pathogenesis of Kawasaki disease, which may involve the interaction of multiple factors, including insufficient IVIG dose, high metabolism, excessive activation of neutrophils, significantly increased cytokine levels, and immature expression of Fc receptors on effector cells.

[0113] The present invention suggests that the levels of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17, and TNF-α in the unresponsive group are significantly increased. In the unresponsive KD, there may be an imbalance of T cells, in which the number of inhibitory regulatory T cells (Treg cells) decreases, while the number of inflammatory T cells (Th17 cells) increases.

[0114] In addition, the present invention has compared the above factors in each group, and the differences in IL-6 and IL-17 levels are particularly prominent. In addition to the differences in IL-6 and IL-17 levels before and after treatment between the sensitive KD group and the unresponsive group, the differences in IL-6 and IL-17 levels before and after treatment within the IL-6 sensitive KD group and the unresponsive group are statistically significant, while the differences in IL-17 sensitive KD group and the unresponsive group are statistically significant before and after treatment, and the differences in the sensitive KD group within the group are statistically significant after treatment.

[0115] The above results show that IL-6 and L-17 increased significantly in the acute phase of unresponsive KD, and after the initial IVIG treatment, there were still differences and high levels were maintained. In particular, IL-6 was statistically significant between the two groups and before and after treatment within the two groups. It was confirmed that IL-6 can predict IVIG unresponsive KD. IL-6 can promote lymphocyte proliferation and macrophage activation and enhance the interaction between immune cells. Other immune cells and inflammatory cells can be further activated. Therefore, IL-6 plays an important regulatory role in the process of lymphocyte activation and plays an important role in immune response, inflammatory response and antibody production.

[0116] IL-6 is a multi-source proinflammatory factor that activates different signaling mechanisms to perform various biochemical functions. It can activate various pathological pathways across signaling mechanisms and cross-regulate CD4+T cells and vascular growth factor levels, and participate in the pathophysiology of various complex diseases. Therefore, IL-6 is extremely sensitive to unresponsive KD. IL-17 is also a proinflammatory cytokine, which is mainly produced by CD4+T cells after activation of the Th17 subset and plays an important role in the activation of lymphocytes. Th17 cells produce IL-17 and are associated with tissue damage and autoimmunity. The proinflammatory cytokine IL-17 acts on multiple types of cells, including neutrophils, monocytes, and fibroblasts, to induce the expression of additional proinflammatory cytokines, such as IL-6, TNF-α, and IL-8. The IL-17 family includes 6 structurally related cytokines that signal to non-hematopoietic cells to induce innate immune defense mechanisms. Most studies on IL-17 focus on IL-17A, while less is known about other members of the family. High IL-17A levels in the blood may be closely related to the severity of KD. IL-17A is present at higher plasma levels in IVIG-resistant KD patients, and the plasma IL-17A concentration in the CAL-positive sensitive KD group is higher than that in the CAL-negative sensitive KD group. Intravenous immunoglobulin (IVIG) can inhibit the differentiation of Th17-type T cells and inhibit the production of IL-17A and IL-17F by T lymphocytes, indicating that the IL-17 cytokine family can be used as a diagnostic marker for Kawasaki disease and a potential risk factor for the occurrence of coronary artery aneurysms, while IVIG unresponsiveness may be related to IL-17 overexpression, that is, the production of a large amount of IL-17A inhibits the anti-inflammatory effect of IVIG.

[0117] In view of the fact that IL-6 and IL-17 are mainly derived from CD4+T, CD4+T is also involved in the process of unresponsive KD. Combined with the ROC curve analysis results of the present invention, the area under the curve (AUC) of IL-6 for predicting unresponsive Kawasaki disease before and after treatment is 0.859 and 0.920, respectively, while the AUC of IL-17 is 0.699 and 0.884, respectively, that is, after the initial treatment with immunoglobulin, the predictive value of IL-6 and IL-17 is higher than that before treatment. Therefore, clinically, IL-6 and IL-17 need to be retested within 36 hours after the initial IVIG shock treatment. If IL-6 does not decrease significantly or / and IL-17 continues to increase, it strongly indicates the possibility that the patient is unresponsive KD. Combined with the clinical manifestations after the initial treatment, the predictability of unresponsive KD can be greatly improved, providing an important supplement to the existing scoring system.

Claims

1. A method for predicting whether a child has Kawasaki disease by using T lymphocyte activation-related factors as biomarkers.

2. The method according to claim 1, characterized in that The Kawasaki disease includes Kawasaki disease complicated with coronary artery damage and immunoglobulin unresponsive Kawasaki disease, and the T lymphocyte activation-related factors are selected from one or more of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17 and tumor necrosis factor α.

3. The method according to claim 1, characterized in that: The method comprises the following steps: (1) Peripheral venous blood samples were collected from children with KD before and after a single intravenous immunoglobulin (IVIG) treatment; (2) detecting the levels of T lymphocyte activation-related cytokines in blood samples; (3) To compare the levels of T lymphocyte activation-related cytokines between the immunoglobulin-unresponsive Kawasaki disease group and the sensitive Kawasaki disease group; (4) ROC curve analysis was used to evaluate the predictive ability of cytokines for immunoglobulin-unresponsive Kawasaki disease.

4. The method according to claim 3, characterized in that In step (2), the T lymphocyte activation-related cytokines are selected from one or more of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17 and tumor necrosis factor α (TNF-α).

5. The method according to claim 3, characterized in that: In step (2), the T lymphocyte activation-related cytokines are IL-6 and / or IL-17.

6. The method according to claim 3, characterized in that In step (2), the cytokine level is detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry; in step (3), the cytokine level detected in step (2) is compared with the cytokine level of a known non-responsive Kawasaki disease group or a sensitive Kawasaki disease group.

7. A kit for predicting unresponsive Kawasaki disease in children, characterized in that: The kit comprises the T lymphocyte activation-related factor as claimed in claim 1.

8. The kit according to claim 7, characterized in that The T lymphocyte activation-related factors are IL-6 and / or IL-17.

9. The kit according to claim 7, characterized in that The reagent is an enzyme-linked immunosorbent assay (ELISA) reagent or a flow cytometry reagent.

10. The kit according to claim 7, characterized in that The kit also includes software or instructions for plotting a ROC curve and determining a cutoff value.

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