Xylose as diagnostic marker for non-responsive children with Kawasaki disease and its application in the preparation of kawasaki disease diagnostic products

Abstract

The application provides use of L-xylulose as a diagnostic marker for children with Kawasaki disease of immunoglobulin G non-responsiveness, and application of L-xylulose as a diagnostic marker in preparation of a Kawasaki disease diagnosis product, the diagnostic marker being a marker in a sample of children with Kawasaki disease, and the Kawasaki disease diagnosis product including a product for predicting and / or evaluating treatment effect of Kawasaki disease. The application first proposes a diagnostic marker of children with Kawasaki disease of immunoglobulin G non-responsiveness of L-xylulose, and uses the diagnostic marker for predicting and / or evaluating treatment effect of Kawasaki disease, and has good stability and repeatability.

Description

Technical Field

[0001] This invention belongs to the field of diagnostic product technology, and specifically relates to the application of a metabolomics-based diagnostic biomarker, namely, the use of L-xylulose as a diagnostic biomarker for children with gamma globulin-unresponsive Kawasaki disease, and the application of L-xylulose as a diagnostic biomarker in the preparation of Kawasaki disease diagnostic products. Background Technology

[0002] Kawasaki disease (KD) is the leading cause of acquired heart disease in children in developed countries, primarily affecting infants and young children under five years old, and is characterized by inflammation of small and medium-sized vessels. The most serious complication of this disease is coronary artery damage, including coronary artery dilation and aneurysm formation, which can lead to myocardial ischemia and even sudden death in severe cases. Current treatments for KD are not always effective in all children. For example, intravenous immunoglobulin (IgM, primarily composed of IgG) is currently the main clinical treatment for KD, and most children experience significant symptom improvement after treatment. However, approximately 15%–25% of KD children do not respond to IgM treatment, exhibiting persistent or recurrent fever 36 hours after injection. These children have a significantly increased risk of coronary artery damage and long-term sequelae.

[0003] Currently, the assessment of the therapeutic effect of intravenous immunoglobulin (IVIG) for Kassym-Jomart Toleriane (KD) relies primarily on post-administration response, mainly depending on non-specific clinical characteristics and laboratory indicators. For example, IVIG responsiveness is identified based on younger age, elevated inflammatory markers (such as C-reactive protein and erythrocyte sedimentation rate), hypoalbuminemia, and elevated lactate dehydrogenase. Additionally, some studies suggest that abnormal expression of eosinophils and inflammatory factors also has some predictive value. However, these indicators lack specificity, and their predictive performance varies significantly across different studies, making it difficult to establish a unified standard. Meanwhile, some studies have proposed gene testing and immune cell ratio detection as potential methods for predicting IVIG responsiveness. For instance, in genetics, polymorphisms in immune regulation-related genes (such as Fcγ receptor-related genes and ITPKC genes) are considered associated with treatment response, but their clinical application is limited due to their complexity and high cost. At the immune mechanism level, decreased regulatory T cells and abnormally elevated inflammatory factors are believed to contribute to IVIG non-responsiveness, but related indicators lack stability and operability.

[0004] In recent years, metabolomics technology has provided new research tools for screening disease biomarkers. Previous studies have found that phenylalanine metabolism and tryptophan can serve as metabolic biomarkers for predicting or assessing the efficacy of Kayser-Disease (KD) treatment, particularly for gamma globulin-unresponsive KD. However, metabolic biomarkers with high sensitivity and specificity have not yet been identified. In summary, current methods for identifying the efficacy of KD treatment suffer from drawbacks such as poor specificity, low reproducibility, and a lack of stable and reliable predictive indicators. Summary of the Invention

[0005] The purpose of this invention is to address the problems of poor specificity, low repeatability, and lack of stable and reliable predictive indicators in existing methods for identifying the therapeutic effects of Kawasaki disease.

[0006] To address the aforementioned technical problems, embodiments of the present invention disclose the use of L-xylinose as a diagnostic marker for children with gamma globulin-unresponsive Kawasaki disease.

[0007] The embodiments of the present invention also disclose the application of L-xylinose as a diagnostic marker in the preparation of Kawasaki disease diagnostic products. The diagnostic marker is a marker collected from samples of children with Kawasaki disease, and the Kawasaki disease diagnostic products include products for predicting and / or evaluating the treatment effect of Kawasaki disease.

[0008] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention for predicting and / or evaluating the treatment effect of Kawasaki disease includes predicting and / or evaluating the treatment effect of gamma globulin on children with Kawasaki disease.

[0009] According to another specific embodiment of the present invention, the Kawasaki disease diagnostic product also includes a product for assessing the severity of Kawasaki disease.

[0010] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention includes Kawasaki disease diagnostic products including reagents, kits, systems, models, and chips for detecting diagnostic markers.

[0011] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention, the system for detecting diagnostic biomarkers includes at least one of a liquid chromatography apparatus, a liquid chromatography-mass spectrometry apparatus, an enzyme-linked immunosorbent assay (ELISA) apparatus, and a gas chromatography-mass spectrometry apparatus.

[0012] According to another specific embodiment of the present invention, the application disclosed in this embodiment of the invention, a system for detecting diagnostic biomarkers includes an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) device, wherein the detection conditions of the liquid chromatography include: using a C18 bonded phase column, a column temperature of 40°C; mobile phase A is a 5% acetonitrile aqueous solution, and mobile phase B is 47.5% acetonitrile, 47.5% isopropanol, and 5% water; the gradient elution program in positive ion mode includes: 0-3 min, 0-20% B; 3-4.5 min, 20-35% B; 4.5-5 min, 35-100% B; 5-6.3 min, 100% B. B; 6.3-6.4 min, 100-0% B; 6.4-8 min, 0% B; The gradient elution program in negative ion mode includes: 0-1.5 min, 0-5% B; 1.5-2 min, 5-10% B; 2-4.5 min, 10-30% B; 4.5-5 min, 30-100% B; 5-6.3 min, 100% B; 6.3-6.4 min, 100-0% B; 6.4-8 min, 0% B; The flow rate is 0.4 mL / min.

[0013] According to another specific embodiment of the present invention, the application disclosed in the embodiment of the present invention includes the following detection conditions for mass spectrometry: the ion source is an electrospray ion source, the ion source voltage is 3.5 kV, the capillary temperature is 326°C, the heating temperature is 425°C, the sheath gas flow rate is 50 arb, the auxiliary gas flow rate is 13 arb, the scanning range is 70-1050 Da, the full scan resolution is 60,000, and the secondary mass spectrometry scan resolution is 7500.

[0014] According to another specific embodiment of the present invention, the application disclosed in the embodiments of the present invention uses serum, plasma or urine as samples.

[0015] According to another specific embodiment of the present invention, the diagnostic markers disclosed in the embodiments of the present invention further include at least one of ribulose, phenylalanine and tryptophan.

[0016] Beneficial technical effects of the present invention:

[0017] This invention proposes for the first time a diagnostic biomarker for children with L-xylinus-resistant Kawasaki disease (KD) who are unresponsive to gamma globulin (Gamma globulin), and uses it to predict and / or assess the treatment efficacy of KD. By simply quantitatively analyzing the L-xylinus content in samples from treated KD children, KD can be predicted and / or assessed; the operation is simple and rapid. Furthermore, experimental verification by this invention has shown that L-xylinus, as a diagnostic biomarker for Gamma globulin-resistant KD, has good stability and reproducibility, indicating that detecting the diagnostic biomarker in samples can accurately, stably, and objectively assess the treatment efficacy of KD, and has high diagnostic value. Further, this invention discovers that the diagnostic biomarker can not only be used to determine the reactivity of KD children to Gamma globulin, but also as an indicator of the severity of KD, demonstrating high diagnostic efficacy. Attached Figure Description

[0018] Figure 1 This is a diagram showing the PCA results in the non-targeted metabolomics analysis of IgG-sensitive and IgG-unresponsive KD children in Example 1 of the present invention;

[0019] Figure 2 This is a heatmap of correlation analysis of differential metabolites in non-targeted metabolomics analysis of IgG-sensitive and IgG-unresponsive KD children in Example 1 of the present invention;

[0020] Figure 3 This is a heatmap of differential metabolites in non-targeted metabolomics analysis of IgG-sensitive and IgG-unresponsive children with KD in Example 1 of the present invention;

[0021] Figure 4 This is a bar chart of KEGG pathway enrichment analysis in non-targeted metabolomics analysis of IgG-sensitive and IgG-unresponsive KD children in Example 1 of the present invention;

[0022] Figure 5 This is a bar chart showing the quantitative analysis results of L-xylulose concentration in the serum of IgG-sensitive and IgG-unresponsive children with KD in Example 2 of the present invention.

[0023] Figure 6 This is a schematic diagram of the process for constructing a KD mouse model and an IgG non-responsive KD mouse model in Embodiment 3 of the present invention;

[0024] Figure 7 These are images of the tongue and abdominal aorta of the KD mice, IgG non-responsive KD mice, IgG sensitive KD mice, and normal mice constructed in Example 3 of this invention.

[0025] Figure 8 This is a diagram showing the staining results of abdominal aortic tissue from KD mice, IgG non-reactive KD mice, IgG sensitive KD mice, and normal mice constructed in Example 3 of this invention.

[0026] Figure 9 This is a bar chart showing the abdominal aortic diameter / body weight ratio analysis of KD mice, IgG non-responsive KD mice, IgG sensitive KD mice, and normal mice constructed in Example 3 of this invention;

[0027] Figure 10 This is a diagram showing the staining results of heart tissue from KD mice, IgG non-reactive KD mice, IgG sensitive KD mice, and normal mice constructed in Example 3 of this invention.

[0028] Figures 11-12 This is a diagram showing the expression of inflammatory factors and vascular damage factors in the heart tissues of KD mice, IgG non-responsive KD mice, IgG sensitive KD mice, and normal mice constructed in Example 3 of this invention;

[0029] Figure 13 This is a bar chart showing the concentration of L-xylulose in the serum of KD mice, IgG non-responsive KD mice, IgG sensitive KD mice, and normal mice in Example 4 of the present invention.

[0030] Figure 14 These are images of the tongue and abdominal aorta of normal mice, KD mice, IgG-sensitive KD mice, KD mice supplemented with L-xylulose, and IgG-sensitive KD mice supplemented with L-xylulose in Example 5 of this invention.

[0031] Figure 15 This is a diagram showing the staining results of abdominal aortic tissue from normal mice, KD mice, IgG-sensitive KD mice, KD mice supplemented with L-xylulose, and IgG-sensitive KD mice supplemented with L-xylulose in Example 5 of this invention.

[0032] Figure 16 This is a bar chart analyzing the abdominal aortic diameter / body weight ratio of normal mice, KD mice, IgG-sensitive KD mice, KD mice supplemented with L-xylulose, and IgG-sensitive KD mice supplemented with L-xylulose in Example 5 of the present invention.

[0033] Figure 17 This is a diagram showing the staining results of heart tissue from normal mice, KD mice, IgG-sensitive KD mice, KD mice supplemented with L-xylulose, and IgG-sensitive KD mice supplemented with L-xylulose in Example 5 of this invention.

[0034] Figures 18-19 This is a diagram illustrating the expression of inflammatory factors and vascular damage factors in the cardiac tissue of normal mice, KD mice, IgG-sensitive KD mice, KD mice supplemented with L-xylulose, and IgG-sensitive KD mice supplemented with L-xylulose in Example 5 of this invention.

[0035] Figure 20 This is a bar chart showing the concentration analysis of L-xylulose in serum samples from IgG-sensitive and IgG-unresponsive KD children in Example 6 of this invention.

[0036] Figure 21 This is a correlation analysis graph of L-xylulose concentration and KD children's responsiveness to IgG in Example 6 of the present invention;

[0037] Figure 22 This is the ROC curve of L-xylulose concentration and IgG reactivity in the clinical sample of Example 7 of this invention;

[0038] Figure 23 This is a bar chart analyzing the coronary artery injury grade and L-xylulose concentration of clinical samples in Example 8 of this invention;

[0039] Figure 24 This is a correlation analysis graph of L-xylulose concentration and coronary artery injury grade in Example 8 of the present invention;

[0040] Figure 25 This is the ROC curve of L-xylulose concentration and coronary artery injury grade in clinical samples in Example 9 of this invention. Detailed Implementation

[0041] This invention provides the use of L-xylidene ketone as a diagnostic marker for children with gamma globulin-unresponsive Kawasaki disease.

[0042] Furthermore, the present invention also provides the application of L-xylulose as a diagnostic biomarker in the preparation of Kawasaki disease diagnostic products, wherein the diagnostic biomarker is a biomarker collected from samples of children with Kawasaki disease, and the Kawasaki disease diagnostic products include products for predicting and / or evaluating the treatment effect of Kawasaki disease.

[0043] Specifically, L-xylulose is an important intermediate product in the glucuronic acid metabolism pathway and the pentose phosphate pathway, participating in energy metabolism and redox reactions in vivo. Previous studies have found that it is abnormally elevated in the serum of children with metabolic diseases, diabetes, and liver diseases. The inventors of this invention, through non-targeted metabolomics screening, determined that L-xylulose is associated with the treatment efficacy of Kawasaki disease (KD), and for the first time confirmed that L-xylulose can serve as a diagnostic biomarker for children with gamma globulin-unresponsive KD. Further, a Kawasaki disease diagnostic product was prepared to predict and / or assess the treatment efficacy of Kawasaki disease by detecting L-xylulose in samples collected from children with Kawasaki disease. In addition, the inventors of this invention, through animal experiments and clinical validation, determined that L-xylulose is a key metabolic biomarker for recognizing gamma globulin-unresponsive KD. It should be noted that in this invention, L-xylulose can be used alone as a diagnostic biomarker to predict and / or assess the treatment efficacy of Kawasaki disease; alternatively, other metabolic biomarkers can be used in combination with L-xylulose. In one specific embodiment of this invention, the diagnostic biomarker also includes at least one of ribulose, phenylalanine, and tryptophan.

[0044] Furthermore, the outcome of Kawasaki disease treatment specifically includes treatment effectiveness or ineffectiveness, i.e., sensitivity to the treatment method or no response. Further, treatment effectiveness can be categorized into different levels based on symptom severity, such as complete effectiveness (complete disappearance of symptoms) and partial effectiveness (partial disappearance or improvement of some symptoms). Predicting or assessing the treatment effect of Kawasaki disease, depending on the treatment method, can specifically include: predicting or assessing the treatment effect of intravenous immunoglobulin, and / or predicting or assessing the treatment effect of oral anti-inflammatory drugs (e.g., aspirin), or predicting or assessing the treatment effect of one of the above treatment methods combined with other methods.

[0045] The samples for testing can be collected blood, serum, plasma, urine, saliva, sputum, lymph, cerebrospinal fluid, pleural fluid, or other body fluids. In one specific embodiment of the present invention, the sample is serum, plasma, or urine; preferably, the sample is serum.

[0046] The present invention preferably includes predicting and / or evaluating the therapeutic effect of gamma immunoglobulin (GI) on children with Kawasaki disease. Specifically, this can be for predicting and / or evaluating the therapeutic effect of GI injection, or for predicting and / or evaluating the effect of GI injection combined with other methods. When the concentration of L-xylulose in the sample is determined to be higher than a predetermined threshold, the child is judged to be unresponsive to GI, and the ineffectiveness or poor efficacy of GI injection is predicted and / or evaluated. The predetermined threshold can be specifically determined based on clinical data and can vary depending on the child's age, weight, etc.

[0047] In one specific embodiment of the present invention, the Kawasaki disease diagnostic product also includes a product for assessing the severity of Kawasaki disease. The inventors of this invention have discovered that L-xylulose, as a diagnostic biomarker, can effectively reflect the severity of vascular damage in children and has extremely high accuracy in predicting coronary artery damage. This indicates that the diagnostic biomarker of the present invention can not only be used to predict and / or assess the treatment effect of Kawasaki disease, but also serve as an indicator for assessing the severity of Kawasaki disease, with high assessment efficacy. Specifically, the higher the concentration of L-xylulose detected in the sample, the more severe the assessed Kawasaki disease.

[0048] In one specific embodiment of the present invention, the Kawasaki disease diagnostic product includes reagents, kits, systems, models, and chips for detecting diagnostic biomarkers. Specifically, reagents may include standards for diagnostic biomarkers, detection reagents, etc. Kits may include standards for diagnostic biomarkers, reagents, and detection components, etc., for clinical testing, and the kits may be specific enzyme-linked immunosorbent assay (ELISA) kits for rapid detection. The system includes a device for detecting diagnostic biomarkers, an operating program (including detection conditions) stored in the system, and reagents used, etc. The model may be a diagnostic model obtained by training and learning based on a dataset of clinical characteristics and indicators collected from multiple children, and may specifically include a data acquisition module, a calculation module, a training module, etc.

[0049] Furthermore, in one specific embodiment of the present invention, the system for detecting diagnostic biomarkers includes at least one of a liquid chromatography apparatus, a liquid chromatography-mass spectrometry apparatus, an enzyme-linked immunosorbent assay (ELISA) apparatus, and a gas chromatography-mass spectrometry (GC-MS) apparatus. In one specific embodiment of the present invention, the system for detecting diagnostic biomarkers includes an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-Q Exactive HF-X) device. The detection conditions for liquid chromatography include: using a C18 bonded-phase column, such as an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm id, 1.8 μm; Waters, Milford, USA), with a column temperature of 40°C and a sample manager temperature of 10°C; mobile phase A is 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B is 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). It should be noted that all the above contents are volume percentages; the flow rate is 0.4 mL / min, and the sample loading volume is 3 μL. The gradient elution procedure varies depending on the ion mode selected for mass spectrometry, as shown in Table 1.

[0050] Table 1 Gradient elution program

[0051]

[0052] Furthermore, in one specific embodiment of the present invention, the detection conditions of mass spectrometry in the liquid chromatography-mass spectrometry (LC-MS) device include: the ion source is an electrospray ionization (ESI) source, using positive or negative ion mode, the ion source voltage is 3.5 kV, the capillary temperature is 326 °C, the heating temperature is 425 °C, the sheath gas flow rate is 50 arb, the auxiliary gas flow rate is 13 arb, the scan range is 70-1050 Da, the full scan (FulI MS) resolution is 60,000, and the secondary mass spectrometry scan (MS2) resolution is 7500.

[0053] Furthermore, in the case of serum samples, the following pretreatment steps were performed using liquid chromatography-mass spectrometry (LC-MS) for detection: 100 μL of sample was transferred to a 1.5 mL centrifuge tube, and 400 μL of extraction buffer (methanol:acetonitrile = 1:1 (v:v)) containing 0.02 mg / mL internal standard (L-2-chlorophenylalanine) was added. The sample was extracted at 10 °C with shaking at 1500 rpm for 15 min. The sample was then incubated at -20 °C for 20 min, followed by centrifugation at 18000 g for 20 min at 4 °C. The supernatant was collected, dried under nitrogen, and then reconstituted with 100 μL of reconstitution solution (acetonitrile:water = 1:1 (v:v)). The sample was mixed at 10 °C with shaking at 1500 rpm for 5 min, centrifuged for 10 min, and then the supernatant was transferred to a sample vial for injection and analysis.

[0054] To make the objectives, technical solutions, and effects of the present invention clearer, the embodiments of the present invention will be further described in detail below by way of specific examples.

[0055] Example 1

[0056] This embodiment performs non-targeted metabolomics screening and identifies key metabolites.

[0057] Serum samples were collected from 14 children with KD who were sensitive to gamma globulin (IgG) and 3 children with KD who were unresponsive to gamma globulin (IgG). Each sample was processed as follows: 100 μL of sample was transferred to a 1.5 mL centrifuge tube, 400 μL of extraction buffer (methanol:acetonitrile = 1:1 (v:v)) containing 0.02 mg / mL internal standard (L-2-chlorophenylalanine) was added, and the sample was extracted by shaking at 1500 rpm at 10 °C for 15 min; the sample was then incubated at -20 °C for 20 min, followed by centrifugation at 18000 g at 4 °C for 20 min; the supernatant was collected, dried under nitrogen, and then reconstituted with 100 μL of reconstitution solution (acetonitrile:water = 1:1 (v:v)); the sample was mixed by shaking at 1500 rpm at 10 °C for 5 min, centrifuged for 10 min, and the supernatant was transferred to a sample vial.

[0058] The concentration of metabolites was detected using an ultra-high performance liquid chromatography-tandem Fourier transform mass spectrometry (UHPLC-Q Exactive HF-X) system. The liquid chromatography detection conditions included: an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm id, 1.8 μm; Waters, Milford, The column temperature was 40℃, and the sample manager temperature was 10℃. Mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). The flow rate was 0.4 mL / min, and the sample loading volume was 3 μL. The gradient elution method is shown in Table 1. The mass spectrometry detection conditions included: ESI ion source, ion source voltage of 3.5 kV, capillary temperature of 326℃, heating temperature of 425℃, sheath gas flow rate of 50 arb, auxiliary gas flow rate of 13 arb, scan range of 70-1050 Da, Ful1 MS resolution of 60,000, and MS2 resolution of 7,500.

[0059] After the above tests, non-targeted metabolomics analysis was performed, in which... Figure 1 The results of principal component analysis (PCA) show that there is a clear separation trend between the two groups of patients with KD who are IgG sensitive and those who are IgG unresponsive, indicating that there are systemic differences in the overall metabolic levels of the two groups of patients. Figure 2 A heatmap of correlation analysis of differential metabolites is shown, which reveals that the IgG correlation network structure between metabolites in the two groups of samples is significantly different. Figure 3 A heatmap of differential metabolites is shown, from Figure 3 As can be seen from the data, compared with the IgG-sensitive KD group (IgGsensitive1-14 in the figure), the levels of L-xylulose and ribulose were significantly increased in the IgG-unresponsive KD group (refractory1-3 in the figure), indicating that these two metabolites are associated with non-response to gamma globulin. Figure 4The bar chart showing KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis reveals enrichment in the pentose and glucuronate interconversion pathway, which ranks first in significance. This indicates that differentially metabolites are concentrated in the oxidative stress and pentose phosphate pathways. L-xylulose is a key metabolite in the pentose phosphate pathway bypass, and ribulose is a key intermediate in the pentose phosphate pathway. Therefore, L-xylulose and ribulose are inferred to be important metabolites in gamma globulin-unresponsive KD. Furthermore, L-xylulose is closely related to oxidative stress, thus further confirming its importance as a key metabolite in gamma globulin-unresponsive KD. Further validation of its efficacy as a diagnostic biomarker is needed.

[0060] The results of this embodiment indicate that L-xylinose is a key metabolic regulatory node in gamma globulin-unresponsive KD.

[0061] Example 2

[0062] This embodiment quantitatively analyzes the concentration of L-xylulose in the serum of children with KD who are unresponsive to gamma globulin.

[0063] The concentration of L-xylulose in the serum samples of the two groups of children with koilomah, detected by ultra-high performance liquid chromatography-mass spectrometry in Example 1, was quantitatively analyzed. The results are as follows: Figure 5 As shown, the average concentration of L-xylulose in the serum of children with KD who are sensitive to gamma globulin (IgG) is about 4 µmol / L, while the average concentration in the serum of children with KD who are unresponsive to gamma globulin (IgG) is about 13 µmol / L. This indicates that the concentration of L-xylulose is indeed significantly increased in the serum of children with KD who are unresponsive to gamma globulin, further verifying the results of the metabolomics analysis.

[0064] Example 3

[0065] This embodiment constructs a KD mouse model that is unresponsive to gamma globulin.

[0066] The build process is as follows Figure 6As shown, a KD mouse model was established by intraperitoneal injection of 500 μL of Lactobacillus caseicen Wall Extract (LCWE) (1 mg / mL) into 3-4 week old male C57BL / 6J mice. Five days after LCWE injection, mice were injected with gamma globulin (IgG, 2 g / kg) as the gamma globulin treatment group. All mice were harvested on day 14 after LCWE injection. Before harvesting, mice were anesthetized with isoflurane inhalation, and the swelling and congestion of the tongue were photographed. Heart and abdominal aortic tissues were then harvested for further experiments. Gamma globulin-unresponsive KD mice were screened based on the phenotypes of tongue swelling and congestion, abdominal aortic dilation, pathological damage to the heart and abdominal aorta, and the expression of cardiac inflammatory factors and vascular damage factors. Results are as follows. Figures 7-9 As shown, some mice (i.e., IgG non-responsive KD mice) still exhibited significant inflammatory and vascular damage characteristics after receiving gamma globulin treatment, such as... Figure 7 The results of HE and EVG staining of abdominal aortic tissues from different mouse models show persistent tongue congestion and abdominal aortic dilation, as shown in the images. Figure 8 As shown in the bar chart, the ratio of abdominal aortic diameter to body weight in different mouse models is analyzed. Figure 9 As shown, this indicates abdominal aortic dilation in IgG-unresponsive KD mice; HE and EVG staining results of cardiac tissues from different mouse models are as follows. Figure 10 As shown, this indicates thickening of the cardiac vascular walls in IgG-unresponsive KD mice. The expression levels of inflammatory factors and vascular damage factors in the cardiac tissues of different mouse models are as follows: Figures 11-12 As shown, the cardiac inflammatory factors TNF-α and IL-1β, as well as vascular injury markers vWF and THBD, remained at high levels in IgG-unresponsive KD mice, and did not decrease significantly due to gamma globulin treatment.

[0067] In this embodiment, KD mice that showed persistent inflammation and vascular damage after gamma globulin treatment were classified as mice that did not respond to gamma globulin treatment. Their inflammatory response was not effectively suppressed, which is consistent with the pathological characteristics of gamma globulin-unresponsive KD.

[0068] Example 4

[0069] This embodiment determines the expression characteristics of L-xylitol in an animal model.

[0070] The concentration of L-xylulose in the serum of gamma-immunoglobulin-sensitive and non-reactive KD mice screened in Example 3 was detected using the ultra-high performance liquid chromatography-mass spectrometry detection method of Example 1. Figure 13As shown, the average concentration of L-xylulose in the serum of IgG-sensitive KD mice was approximately 4 µmol / L, while the average concentration in the serum of IgG-unresponsive KD mice was approximately 35 µmol / L. The concentration of L-xylulose was significantly elevated in IgG-unresponsive KD mice. This result indicates that L-xylulose shows a consistent trend in animal models and human samples, demonstrating cross-species consistency and enhancing its reliability as a diagnostic biomarker. Furthermore, this result suggests that the change in L-xylulose is not accidental but closely related to IgG unresponsiveness. This example further validates the stability and reproducibility of L-xylulose as a diagnostic biomarker for IgG-unresponsive KD.

[0071] Furthermore, the results of this embodiment also show that detecting L-xylulose in animal model samples can be used to construct animal models of Kawasaki disease with poor treatment efficacy. Specifically, after treating a Kawasaki disease animal model, if the concentration of L-xylulose in the animal model sample is detected to be higher than a certain value, the corresponding animal model can be identified as an animal model with poor Kawasaki disease treatment efficacy (especially with gamma globulin therapy).

[0072] Example 5

[0073] This embodiment verifies that L-xylulose can aggravate vascular damage and vascular inflammation in KD mice and weaken the therapeutic effect of gamma immunoglobulin.

[0074] Referring to Example 3, KD mouse models and KD mouse models sensitive to gamma immunoglobulin therapy were reconstructed. These mice were then given exogenous L-xylulose supplementation (0.64 mg / mL, 200 μL, administered by gavage daily starting on day 5 after LWCE injection). Figure 14 As shown, mice supplemented with L-xylulose all exhibited significantly increased tongue congestion and increased abdominal aortic dilation. Figure 15 The results of staining the abdominal aorta in different mouse models are shown. Figure 16 The bar chart shows the analysis of the abdominal aortic diameter / body weight ratio for different models. Figure 16 The results showed that after supplementing the abdominal aorta tissue of mice with L-xylulose, the inner diameter of the abdominal aorta increased, and the elastic fibers were disordered or even broken, indicating that the integrity of the vascular structure was damaged. Figure 17 The results of vascular staining in the heart tissue of different mouse models show that L-xylulose supplementation significantly thickened the heart vessel walls and loosened the elastic fiber structure. These results indicate that L-xylulose not only serves as a diagnostic marker but also exacerbates vascular damage, and its elevation may directly participate in the Karnofsky Performance Status (KD) vascular pathogenesis process. Furthermore, even after gamma globulin treatment, this metabolite can further aggravate the lesions, potentially interfering with the anti-inflammatory or immunomodulatory effects of gamma globulin.

[0075] Furthermore, inflammatory factors and vascular damage factors in the heart tissue of KD mice supplemented with L-xylulose were detected. Figures 18-19 The expression of inflammatory and vascular damage factors in the cardiac tissue of KD mice after L-xylulose supplementation was shown. It can be seen that the levels of both inflammatory and vascular damage factors were significantly increased in mice supplemented with L-xylulose. Even after treatment with gamma globulin, L-xylulose could reverse the anti-inflammatory effect of gamma globulin, indicating that L-xylulose can enhance the inflammatory response and aggravate the symptoms in KD mice; demonstrating that L-xylulose also plays an important role at the molecular mechanism level.

[0076] Example 6

[0077] This embodiment analyzed the correlation between L-xylulose concentration and gamma immunoglobulin reactivity in clinical samples.

[0078] Serum samples were collected from 50 children with KD who were sensitive to gamma globulin and 10 children with KD who were unresponsive to gamma globulin. L-xylulose concentration was measured using the method described in Example 1. Figure 20 As shown, the results indicated that the serum L-xylulose level in children with gamma immunoglobulin (IgG) non-responsiveness was significantly higher than that in children with IgG sensitivity. Figure 21 As shown, correlation analysis between L-xylulose concentration and gamma globulin responsiveness in children with koiloma syndrome (KD) revealed a significant positive correlation between L-xylulose concentration and gamma globulin responsiveness (0: sensitive to gamma globulin treatment; 1: unresponsive to gamma globulin treatment) (R=0.54, P<0.0001), indicating that higher L-xylulose concentrations are associated with a greater likelihood of gamma globulin inactivity. This result suggests that L-xylulose can effectively differentiate between the two groups of children and has predictive value.

[0079] Example 7

[0080] This embodiment evaluates the diagnostic efficacy of L-xylinose in children with KD who are unresponsive to gamma globulin.

[0081] ROC (Receiver Operating Characteristic) curve analysis was performed on the clinical data of the children corresponding to the clinical samples collected in Example 6 and the concentration of L-xylulose in their serum. The results are as follows: Figure 22 As shown, the AUC (Area Under Curve) for predicting gamma globulin non-response was 0.9150 for L-xylulose. This result indicates that this indicator has high sensitivity and specificity, can effectively distinguish between gamma globulin-sensitive and non-response children, and has good clinical diagnostic performance.

[0082] Example 8

[0083] This embodiment analyzes the correlation between L-xylulose concentration and coronary artery injury grade in clinical samples.

[0084] The coronary artery injury grade and L-xylulose concentration of the children corresponding to the clinical samples collected in Example 6 were analyzed, and the results are as follows: Figure 23 As shown, L-xylulose concentration increases progressively with the degree of coronary artery injury (0: no abnormalities and intimal thickening; 1: coronary artery dilation; 2: aneurysm). Figure 24 As shown, the correlation analysis between L-xylulose concentration and coronary artery injury grade showed that L-xylulose concentration was significantly positively correlated with the degree of coronary artery injury (R=0.6987), indicating that the concentration of L-xylulose in the serum of children with KD can effectively reflect the severity of vascular injury.

[0085] Example 9

[0086] This embodiment verifies the predictive ability of L-xylinose for coronary artery injury.

[0087] The clinical data (coronary artery injury status) and L-xylulose concentration of the children with KD collected in Example 6 were analyzed by ROC curve analysis, and the results are as follows: Figure 25 As shown, the AUC was 0.9682, indicating that detecting L-xylulose concentration has extremely high accuracy in predicting coronary artery damage. These results further demonstrate that L-xylulose can not only be used to assess the reactivity of KD children to gamma globulin, but also as an indicator of disease severity in KD children, with high diagnostic efficacy, making it suitable as a clinical assessment indicator.

[0088] Through the above embodiments, it was verified that L-xylulose can effectively distinguish between IgG-sensitive and non-responsive Kawasaki disease (KD) patients, with an AUC of 0.9150, indicating its high diagnostic value. Simultaneously, the concentration of L-xylulose was significantly correlated with the degree of coronary artery damage in KD patients (AUC of 0.9682), indicating its important role in prognostic risk assessment and its potential use in evaluating the severity of Kawasaki disease. Animal experiments further demonstrated that exogenous L-xylulose supplementation exacerbated the inflammatory response and vascular damage in KD mice and weakened the therapeutic effect of IgG, suggesting that it may not only serve as a diagnostic biomarker but also participate in the disease progression process. The diagnostic biomarker (L-xylulose) provided by this invention is used to predict and / or assess the treatment effect of Kawasaki disease, with accurate, effective, and highly specific results, making it suitable for clinical application.

[0089] It should be noted that, in addition to the specific embodiments described above, those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Although the description of the present invention is presented in conjunction with preferred embodiments, this does not mean that the features of the invention are limited to these embodiments. On the contrary, the purpose of describing the invention in conjunction with the embodiments is to cover other options or modifications that may be derived based on the claims of the present invention. To provide a deep understanding of the invention, many specific details are included in the above description, and the invention may also be implemented without using these details. Furthermore, to avoid confusion or obscuring the focus of the invention, some specific details will be omitted in the description. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0090] While the present invention has been described and illustrated with reference to certain preferred embodiments, those skilled in the art should understand that the above description is a further detailed explanation of the invention in conjunction with specific embodiments, and should not be construed as limiting the specific implementation of the invention to these descriptions. Various changes in form and detail can be made by those skilled in the art, including several simple deductions or substitutions, without departing from the spirit and scope of the invention.

Claims

1. The use of L-xylitol as a diagnostic marker for children with gamma globulin-unresponsive Kawasaki disease.

2. The application of L-xylulose as a diagnostic biomarker in the preparation of Kawasaki disease diagnostic products, wherein the diagnostic biomarker is a biomarker collected from samples of children with Kawasaki disease, and the Kawasaki disease diagnostic products include products for predicting and / or evaluating the treatment effect of Kawasaki disease.

3. The application as described in claim 2, characterized in that, The prediction and / or assessment of Kawasaki disease treatment efficacy includes predicting and / or assessing the therapeutic effect of gamma globulin on children with Kawasaki disease.

4. The application as described in claim 2, characterized in that, The Kawasaki disease diagnostic products also include products for assessing the severity of Kawasaki disease.

5. The application as described in claim 2, characterized in that, The Kawasaki disease diagnostic products include reagents, kits, systems, models, and chips for detecting the diagnostic markers.

6. The application as described in claim 5, characterized in that, The system for detecting the diagnostic biomarker includes at least one of a liquid chromatography apparatus, a liquid chromatography-mass spectrometry apparatus, an enzyme-linked immunosorbent assay (ELISA) apparatus, and a gas chromatography-mass spectrometry (GC-MS) apparatus.

7. The application as described in claim 6, characterized in that, The system for detecting the diagnostic biomarker includes an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) device, wherein the detection conditions for liquid chromatography include: A C18 bonded phase column was used at a column temperature of 40℃; mobile phase A was 5% acetonitrile aqueous solution, and mobile phase B was 47.5% acetonitrile, 47.5% isopropanol and 5% water. The gradient elution program in positive ion mode includes: 0-3 min, 0-20% B; 3-4.5 min, 20-35% B; 4.5-5 min, 35-100% B; 5-6.3 min, 100% B; 6.3-6.4 min, 100-0% B; 6.4-8 min, 0% B. The gradient elution program in negative ion mode includes: 0-1.5 min, 0-5% B; 1.5-2 min, 5-10% B; 2-4.5 min, 10-30% B; 4.5-5 min, 30-100% B; 5-6.3 min, 100% B; 6.3-6.4 min, 100-0% B; 6.4-8 min, 0% B. The flow rate was 0.4 mL / min.

8. The application as described in claim 7, characterized in that, The mass spectrometry detection conditions included: an electrospray ion source with an ion source voltage of 3.5 kV, a capillary temperature of 326 °C, a heating temperature of 425 °C, a sheath gas flow rate of 50 arb, an auxiliary gas flow rate of 13 arb, a scanning range of 70-1050 Da, a full scan resolution of 60,000, and a secondary mass spectrometry scan resolution of 7,500.

9. The application as described in claim 2, characterized in that, The sample may be serum, plasma, or urine.

10. The application as described in any one of claims 2-9, characterized in that, The diagnostic markers also include at least one of ribulose, phenylalanine, and tryptophan.

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