Method for assisting prediction of onset of atopic dermatitis

Abstract

To provide a marker which can be utilized for predicting infant's early onset atopic dermatitis, and to provide a prediction method using the marker.SOLUTION: The method for assisting in predicting the onset of atopic dermatitis in an infant based on the amount of IL-2 in the stratum corneum of the invention is a method based on the amount of IL-2 in the stratum corneum of the skin, and the kit for predicting the onset of atopic dermatitis in an infant of the invention comprises a reagent for measuring the amount of IL-2 in the stratum corneum of the skin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for assisting in predicting the onset of atopic dermatitis. [Background technology]

[0002] Eczema often appears on the face of infants around two weeks of age. While infantile eczema may resolve spontaneously, symptoms may persist and then develop into early-onset atopic dermatitis. Objective diagnostic methods for infantile atopic dermatitis include blood tests to measure TARC and SCCA2 levels, but blood sampling is invasive for infants, so these tests are currently rarely performed. The Japanese Dermatological Association's atopic dermatitis diagnostic guidelines stipulate that atopic dermatitis in infants is diagnosed based on pruritus, characteristic rash and distribution, and a chronic, recurrent course lasting for more than two months. Therefore, diagnosis takes time, and because there are no objective, non-invasive diagnostic methods, the current situation is that it is largely dependent on the physician's subjective judgment.

[0003] Here, as a study on atopic dermatitis in infants, Non-Patent Document 1 discloses that the amount of TARC / CCL17 in skin collected from 2-month-old infants using the tape strip method correlates with the onset of early atopic dermatitis (within 2 years of age). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J Allergy Clin Immunol. 2023 151(6):1550-1557 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the marker TARC / CCL17 described in Non-Patent Document 1 is the result of a study conducted on patients without eczema, and the inventors have confirmed that TARC / CCL17 is insufficient as a marker for predicting early-onset atopic dermatitis in infants with eczema. Therefore, an object of the present invention is to provide a marker that can be used to predict early-infantile atopic dermatitis in infants with eczema, and a prediction method using the marker. [Means for solving the problem]

[0006] The present inventors comprehensively analyzed various cytokines in proteins extracted from the stratum corneum of infants and discovered that early-onset atopic dermatitis can be predicted by measuring IL-2, leading to the completion of the present invention.

[0007] That is, the present invention includes, for example, the following. [1] A method to assist in predicting the onset of atopic dermatitis in infants based on the amount of IL-2 in the stratum corneum. [2] The method according to [1], wherein the stratum corneum is derived from skin affected by eczema. [3] The method according to [1] or [2], wherein the infant is an infant aged 2 weeks to 6 months. [4] The method according to any one of [1] to [3], wherein the stratum corneum of the skin is derived from a sample collected by a tape strip. [5] The method according to any one of [1] to [4], wherein, when the amount of IL-2 in the stratum corneum of the skin exceeds a predetermined cutoff value, the infant is judged to be suffering from atopic dermatitis or to be highly likely to develop atopic dermatitis in the future. [6] The method according to [5], wherein the cutoff value is 4.6 pg / μg total protein relative to the total protein in the stratum corneum. [7] A kit for predicting the onset of atopic dermatitis in infants, comprising a reagent for measuring the amount of IL-2 in the stratum corneum of the skin. [8] The kit according to [7], wherein the reagent comprises an anti-IL-2 antibody. [9] The kit according to [7] or [8], further comprising a surfactant that solubilizes the protein.

[10] The kit according to any one of [7] to [9], further comprising a tape for peeling off the stratum corneum of the skin.

[11] A method for diagnosing the risk of developing atopic dermatitis in infants based on the amount of IL-2 in the stratum corneum. [Effects of the Invention]

[0008] According to the present invention, it is possible to predict the onset of atopic dermatitis as early as one month after birth using a non-invasive and simple method, which will enable early treatment and prevent the condition from becoming severe, and may also help prevent food allergies in infants. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a flow chart showing the condition of infants who participated in the study at 1 month and 5 months in Example 1. [Figure 2] 1 is a graph showing the results of an analysis using the Jonckheere-Terpstra test of IL-2 (a), CCL26 (b), and CCL20 (c) in the stratum corneum of three groups of one-month-old infants (healthy group, spontaneous remission group, and advanced atopic dermatitis group) in Example 1. The graph also shows the results of pairwise comparison using the Mann-Whitney U test. [Figure 3] 1 shows receiver operating characteristic curves showing the predictive ability of IL-2, CCL26, and CCL20 levels in the stratum corneum of eczema-affected 1-month-old infants in Example 1 to predict the onset of atopic dermatitis (AD) up to 5 months of age. [Figure 4]1 is a graph showing the results of a logistic regression analysis conducted in Example 1 to examine the relationship between IL-2 levels in the stratum corneum of 1-month-old infants and the presence or absence of atopic dermatitis (AD) at 5 months of age. [Figure 5] 1 is a graph showing the results of an analysis using the Jonckheere-Terpstra test of IL-2 (a), CCL26 (b), and CCL20 (c) in the stratum corneum of three groups of 1-month-old infants (spontaneous improvement group, mild atopic dermatitis group, and moderate / severe atopic dermatitis group) in Example 2. The graph also shows the results of pairwise comparison using the Mann-Whitney U test. [Figure 6] 1 is a graph showing the results of a logistic regression analysis conducted in Example 2 to examine the stratum corneum IL-2, CCL26, and CCL20 levels of 1-month-old infants and the presence or absence of moderate to severe atopic dermatitis (AD) in 5-month-old infants. [Figure 7] FIG. 10 is a graph showing the results of a logistic regression analysis conducted in Example 3 to examine the stratum corneum IL-2 levels of 1-month-old infants and the presence or absence of atopic dermatitis (AD) onset in 5-month-old infants, including a healthy control group in addition to a group with remission of eczema and a group with progression of atopic dermatitis. DETAILED DESCRIPTION OF THE INVENTION

[0010] [Prediction method] The method of the present invention for assisting in the prediction of the onset of atopic dermatitis in infants is based on the amount of IL-2 in the stratum corneum of the skin. This method is an in vitro method.

[0011] Infants refer to children from newborn to less than one year old. Infants to be predicted may be children from 0 days after birth to less than one year old, and are preferably newborn to six months old, two weeks to six months old, two weeks to four months old, or one month to four months old, which are of particular clinical significance. Furthermore, the method of the present invention is a method for assisting in the prediction of atopic dermatitis in infants, which is of great clinical significance, but can also be used for infants aged one year or older, for example, infants aged one to three years old.

[0012] Atopic dermatitis (AD) is a condition characterized by itchy eczema that repeatedly worsens and improves. It often begins in infancy, and symptoms generally improve with age. However, some patients experience persistent symptoms into adulthood. Atopic dermatitis is multifactorial, with a complex combination of factors thought to contribute to its development, including a predisposition to allergies (atopic predisposition), a weakened barrier function, and genetics. Treatment involves topical anti-inflammatory medications and moisturizers. However, if these are ineffective and severe inflammation develops over a wide area, systemic treatment with dupilumab, an IL-4Rα receptor antibody, is considered an option. Its indication has recently been expanded beyond adults to include children aged 6 months and older.

[0013] In one embodiment of the present invention, the skin may be skin without eczema or skin with eczema, preferably skin with eczema, i.e., skin derived from an area affected by eczema. The skin may be skin from any part of the body, such as the face (cheeks, forehead, chin, nose, etc.), upper arms, lower legs, chest, abdomen, or buttocks, but is preferably facial skin, particularly cheek skin.

[0014] The stratum corneum, located at the outermost layer of the skin's epidermis, has a "moisturizing function" that maintains moisture and flexibility, and a "barrier function" that protects against external stimuli such as dryness, irritants, and allergens. While any non-invasive method can be used to collect the stratum corneum, the simple and safe tape strip method is preferred. This method allows the stratum corneum to be collected by applying and peeling off adhesive tape to the skin. In other words, it is preferable that the stratum corneum is derived from the sample collected using the tape strip. The adhesive tape used is not particularly limited as long as it can collect the stratum corneum, and adhesive tape that is less painful is preferred. Examples of adhesive tape include medical tapes such as Cellophane® tape, D-squame tape, and film dressings. The area of ​​the tape is not particularly limited as long as it can collect enough stratum corneum to measure IL-2, but it can be, for example, 1 to 50 cm. 2 , especially 5-40cm 2 , or 10 to 30 cm 2 It may be.

[0015] IL-2 (interleukin-2) is a type of cytokine. IL-2 is produced by immature T cells and type 1 helper T cells differentiated from naive T cells in response to stimulation with interferon-γ or IL-12, and is classified as a group of cytokines known as Th1 cytokines. IL-2 is known to be involved in cell-mediated immunity, but its involvement in the pathogenesis of atopic dermatitis in both children and adults has not been clearly established, and it is not known as a predictive marker for the onset of atopic dermatitis.

[0016] The amount of IL-2 in the collected stratum corneum used to predict the onset of atopic dermatitis in infants is preferably the amount relative to total protein. Alternatively, the amount may be relative to an internal standard such as β-actin, β-tubulin, or GAPDH. The amount of IL-2 can be measured according to methods known to those skilled in the art, such as immunological methods such as ELISA using an anti-IL-2 antibody described below. Furthermore, the amount of total protein can be measured by a conventional method using a reagent capable of measuring the amount of total protein described below.

[0017] In predicting the onset of atopic dermatitis in infants, if the amount of IL-2 in the stratum corneum exceeds a predetermined cutoff value, it is determined that the infant has atopic dermatitis or is likely to develop atopic dermatitis in the future. That is, the onset prediction method of the present invention also includes an aspect of a diagnostic method. Here, if the infant is likely to have atopic dermatitis or is likely to develop atopic dermatitis in the future, it is preferable to intervene early by conducting further tests, treatment, or follow-up. Regarding the possibility of future onset of atopic dermatitis, if the amount of IL-2 in the stratum corneum exceeds a predetermined cutoff value, it can be determined that the infant is likely to develop atopic dermatitis at least by 6 months of age or by 2 years of age.

[0018] The cutoff value may vary depending on the infant's eczema symptoms, age, sampling site, etc. The cutoff value can be determined statistically by comparing the amount of IL-2 in the infant's stratum corneum at a certain point in time with the subsequent onset of atopic dermatitis. The cutoff value may be set differently depending on whether the possibility of onset of atopic dermatitis or the possibility of atopic dermatitis with moderate or severe symptoms is being assessed, and on the reliability setting.

[0019] The cutoff value may be, for example, 4.6 (sensitivity 80.4%, specificity 47%) to 7.4 (sensitivity 71.7%, specificity 82.3%) pg / μg total protein relative to the total protein in the stratum corneum of the skin, for example, 4.6 pg / μg, 5 pg / μg, 5.5 pg / μg, 6 pg / μg, 6.5 pg / μg, 7 pg / μg, or 7.4 pg / μg.

[0020] The above prediction method can provide important information for early detection and prevention strategies of atopic dermatitis in infants, which is expected to contribute to reducing the suffering and treatment costs associated with the onset of atopic dermatitis.

[0021] [kit] The kit of the present invention is a kit for predicting the onset of atopic dermatitis in infants, and comprises a reagent for measuring the amount of IL-2 in the stratum corneum of the skin.

[0022] The reagent for measuring the amount of IL-2 in the stratum corneum of the skin is not particularly limited as long as it is a reagent commonly used by those skilled in the art. For example, it may include an anti-IL-2 antibody or an antigen-binding fragment thereof, particularly preferably an anti-IL-2 antibody. The anti-IL-2 antibody may be a monoclonal or polyclonal antibody, preferably a monoclonal antibody. The anti-IL-2 antibody may include, but is not limited to, IgA, secretory IgA, IgG, IgE, and IgM. The antigen-binding fragment may include heavy chain antibodies, light chain antibodies, VHHs, VNARs, Fabs, Fab's, F(ab')2s, variable regions (Fvs), single-chain antibodies (scFvs), and the like. The antibody may be derived from various animals and utilize human IL-2 as an antigen. Such antibodies may be produced by known methods or commercially available.

[0023] In one embodiment of the present invention, the kit may contain other components, such as a detergent for solubilizing proteins and a reagent for measuring the amount of total protein. Examples of detergents include, but are not limited to, SDES, SDS, Tween, and Triton X. Examples of reagents for measuring the amount of total protein include the BCA method, NPC method, Lowry method, and Bradford method.

[0024] In one embodiment of the present invention, the kit may further include an adhesive tape for peeling the stratum corneum of the skin, and the adhesive tape may be one described in the above "Prediction method."

[0025] The kit of the present invention is used in the above-mentioned method of the present invention for assisting in predicting the onset of atopic dermatitis in infants and the method of diagnosing the risk of onset of atopic dermatitis in infants based on the amount of IL-2 in the stratum corneum, as described below.

[0026] [Diagnostic method] The diagnostic method of the present invention is a method for diagnosing the risk of developing atopic dermatitis in infants based on the amount of IL-2 in the stratum corneum of the skin. [Example]

[0027] <Sample collection> After wiping the cheeks of one-month-old infants with facial eczema, a 24 mm x 12 cm long piece of cellophane tape (registered trademark, Nichiban Co., Ltd.) was applied to the cheeks to collect the stratum corneum from the eczema area. The cellophane tape with the attached stratum corneum was attached to a glass slide (Matsunami Glass) and stored at -20°C until measurement.

[0028] <Protein extraction from stratum corneum and sample preparation> Protein extraction from the stratum corneum attached to the cellophane tape and sample preparation were carried out as follows. 1. The cellophane tape with the stratum corneum attached was cut into 5 mm wide strips using a cutter. 2.1 mL of PBST (0.05% Tween 20 / PBS) was prepared in a 5 mL tube. 3. The shredded tape was immersed in PBST placed in a 5 mL tube. 4. Using an ultrasonic disrupter (SONICSTAR 85, VIOLAMO), the stratum corneum cells were detached from the tape on ice for 90 seconds, and proteins including intracellular cytokines were extracted. 5. The mixture was centrifuged at 2300 g for 1 minute at 4°C. 6. The PBST was collected and transferred onto the membrane of a filtration tube (Ultrafree®-CL (2 mL) Merck Millipore, UFC40GV00). 7. 400 μl of PBST was added, and the mixture was washed. 8.Vortex processing was performed. 9. Again, the mixture was centrifuged at 2300 g for 1 minute at 4°C. 10. PBST was collected. 11. Using a filtration tube, the solution was centrifuged at 12,000 g for 4 minutes at 4°C. 12. The recovered solution was dispensed into concentration tubes (Amicon Ultra-0.5 Ultracel-3K, Merck Millipore, UFC5003BK). 13. The mixture was centrifuged at 14,000 g for 30 minutes at 4°C to concentrate the mixture approximately 10-fold. 14. The concentrate was collected (1,000 g for 2 minutes, 4°C). 15. Measurements were performed using a multiplex assay kit from R&D Systems or Bio-Rad using the Bio-Plex Cytokine Assay System (Bio-Rad Laboratories, Hercules, CA) according to the protocol. 16. Protein content was measured and corrected using the BCA method.

[0029] (Example 1) Identification of cytokines associated with the development of atopic dermatitis (AD) A prospective cohort study was conducted on 153 1-month-old infants (55 without eczema, 98 with eczema). Measurements and analysis were performed on a total of 82 subjects: 19 in the non-eczema group who had no eczema by 5 months of age (healthy group), 17 in the eczema group whose eczema had completely resolved spontaneously by 5 months of age, and 46 who were diagnosed with atopic dermatitis (AD) (22 mild, 20 moderate, and 4 severe). The subjects' conditions at 1 month and 5 months of age are shown in Figure 1.

[0030] The following 23 cytokines and chemokines were measured: TH1 type markers (IL-2, INF-γ, CXCL9, CXCL10, CXCL11), TH2 type markers (IL-4, IL-5, IL-13, RANTES / CCL5, TARC / CCL17, CCL18, MDC / CCL22, Eotaxin-3 / CCL26), TH17 markers (IL17 / IL17A, CCL20), IL-1b, IL-8 / CXCL8, TNF-α, IL-12p70, IL25, IL31, IL33, TSLP. Of these, CXCL9, IL-5, RANTES / CCL5, TNF-α, IL-12p70, and IL-31 were excluded because their detection sensitivity was insufficient in more than half of the samples. The remaining 17 items were analyzed.

[0031] Logistic regression analysis was performed using JMP Pro (version 15.1.0, SAS Institute Japan), and the results are shown in Table 1. To facilitate interpretation of different cytokine levels, the logistic regression analysis shows the odds ratio of the risk of developing AD per 10-unit increase in stratum corneum cytokine levels. Because CCL20 and TSLP have lower levels than other cytokines, the odds ratio is shown per 1-unit increase. Table 1 shows that stratum corneum IL-2, CCL26, and CCL20 levels in the eczema areas of 1-month-old infants increased the risk of AD by 5 months of age. The odds ratio (OR) was 3.35 (95% CI: 1.31-8.52, P = 0.011), the odds ratio (OR) was 1.67 (95% CI: 1.11-2.83, P = 0.03), and the odds ratio (OR) was 1.8 (95% CI: 1.12-3.54, P = 0.043), respectively. Multivariate logistic regression analysis was performed on family history of allergy, IL-2, CCL26, and CCL20, but only the results of univariate analysis were presented, as IL-2 alone was found to be a strong predictor. [Table 1]

[0032] In addition, a Jonckheere-Terpstra test (testing for increasing or decreasing trends in quantitative variables across multiple groups with an ordered relationship) was performed using IBM SPSS Statistics for Mac (version 29.0.2.0, IBM). The results are shown in Figure 2. A statistically significant increase in stratum corneum IL-2, CCL26, and CCL20 was observed when comparing the healthy control group, the eczema spontaneous remission group (hereafter referred to simply as the "spontaneous remission group" or "eczema spontaneous remission group"), and the atopic dermatitis progression group (hereafter referred to simply as the "AD progression group") at 1 month of age. P < 0.001, P < 0.001, and P = 0.009, respectively. Furthermore, pairwise comparisons using the Mann-Whitney U test revealed statistically significant differences in these cytokines between the eczema spontaneous remission group and the AD progression group, with P < 0.001, P = 0.009, and P = 0.012, respectively. This suggests that markers that can differentiate cytokine expression between the two groups with and without progression of AD could be used as diagnostic markers.Stratum corneum samples were collected from the rash-free area of ​​the cheek in the healthy group, and from the lesioned area of ​​the cheek in the eczema spontaneous remission group and AD group.

[0033] Figure 3 shows the receiver operating characteristic curves (ROCs) for predicting the onset of AD by 5 months of age for IL-2, CCL26, and CCL20 levels in the stratum corneum of eczema-affected infants at 1 month of age. Figure 4 shows the results of a logistic regression analysis examining the correlation between IL-2 levels in the stratum corneum of 1-month-old infants and the onset of AD at 5 months of age. Figures 3 and 4 suggest that IL-2, CCL26, and CCL20 levels at 1 month of age have the ability to predict early-onset atopic dermatitis in infants by 5 months of age. IL-2 showed the best diagnostic characteristic for predicting the progression of AD (AUC = 0.78). A cutoff value of 7.4 pg / μg total protein demonstrated a sensitivity of 72% and a specificity of 82% for predicting AD at 5 months of age in infants with eczema. CCL26 and CCL20 also had good diagnostic ability, second only to IL-2. The AUCs were 0.70, with sensitivity of 71.7% and specificity of 64.7% and 0.69, respectively. The combined effect of IL-2, CCL26, and CCL20 on the overall incidence of AD, including mild, moderate, and severe AD, was not superior to that of IL-2 alone. When IL-2 levels were dichotomized into low and high levels according to the receiver operating characteristic (ROC) cutoff of 7.4 pg / μg total protein, the odds ratio for the risk of AD by 5 months of age was 11.9 (95% confidence interval: 2.91-48.15; P<0.001). In AD prediction tests, higher sensitivity is more clinically useful, and when a sensitivity of 80% was set, the cutoff value was 4.6 pg / μg total protein (specificity 47%), indicating that a cutoff value of 4.6 to 7.4 pg / μg total protein could be used to indicate a high risk of progression to AD.

[0034] (Example 2) Study on prediction of severity of AD We examined the expression of IL-2, CCL26, and CCL20 at one month of age in infants with spontaneous eczema remission, mild AD, and moderate / severe AD (hereafter referred to as the "moderate to severe AD group") at five months of age. Figure 5 shows the results of a Jonckheere-Terpstra test to examine trends among the three groups. Figure 5 shows a statistically significant increase in IL-2, CCL26, and CCL20 in the stratum corneum at one month of age in the spontaneous eczema remission, mild AD, and moderate / severe AD groups. P < 0.001, P = 0.003, and P = 0.033, respectively. Furthermore, between-group tests revealed significant differences in IL-2 between the spontaneous eczema remission group and the mild AD group, and between the spontaneous eczema remission group and the moderate to severe AD group. Significant differences in CCL26 and CCL20 were observed only between the spontaneously improving and moderate to severe AD groups.

[0035] We also investigated the diagnostic characteristics for moderate to severe AD. Figure 6 shows the results of a logistic regression analysis of the stratum corneum IL-2, CCL26, and CCL20 levels in infants at one month of age and the presence or absence of AD at five months of age. In the (remittent eczema + mild AD) vs. moderate AD group, the odds ratio (OR) for IL-2 was 1.47 (0.99-2.17), P = 0.056, ROC = 0.67; for CCL26, the odds ratio (OR) was 1.56 (1.13-2.18), P = 0.008, ROC = 0.70 (cutoff value 34.4 pg / μg total protein, sensitivity 50.0%, specificity 84.6%); and for CCL20, the odds ratio (OR) was 1.47 (1.08-2.13), P = 0.026, ROC = 0.67 (cutoff value 1.9 pg / μg total protein, sensitivity 58.3%, specificity 79.5%). These results suggest that IL-2 is a better predictor of AD, including overall severity, than for moderate to severe AD. On the other hand, the accuracy of CCL26 and CCL20 in predicting moderate to severe AD based on the ROC results was similar to that in predicting AD including overall severity.

[0036] Example 3 Furthermore, infants without eczema (healthy group) were also included in the study. The data for the eczema remission group vs. the AD progression group shown in Example 2 were added to the healthy group. Logistic regression analysis was performed to examine the stratum corneum IL-2 levels of infants at 1 month of age and the presence or absence of AD in infants at 5 months of age. The analysis results for a total of 82 cases, including the healthy group, eczema remission group, and AD group, are shown in Figure 7. Healthy group + eczema remission group (group without AD at 5 months of age) vs In the analysis of the AD progression group, the odds ratio for IL-2 was 3.95 (1.80-8.70), P = 0.0006, ROC = 0.77 (cutoff value 5.6 pg / μg total protein, sensitivity 78.3, specificity 66.7), for CCL26 was 1.67 (1.19-2.35), P = 0.0033, ROC = 0.72 (cutoff value 11.4 pg / μg total protein, sensitivity 84.8, specificity 52.8), and for CCL20 was 1.81 (1.18-2.79), P = 0.0069, ROC = 0.68 (cutoff value 0.84 pg / μg total protein, sensitivity 78.26, specificity 52.8). In the analysis including the healthy group, the odds ratio for eczema remission group vs. The results were almost identical to those of the AD progression group.

Claims

1. A method for assisting in predicting the onset of atopic dermatitis in infants based on the amount of IL-2 in the stratum corneum of the skin.

2. The method according to claim 1, wherein the stratum corneum is derived from skin affected by eczema.

3. 10. The method of claim 1, wherein the infant is between 2 weeks and 6 months old.

4. The method of claim 1, wherein the stratum corneum is derived from a sample taken by tape strips.

5. The method according to claim 1, wherein, when the amount of IL-2 in the stratum corneum exceeds a predetermined cutoff value, the infant is determined to be suffering from atopic dermatitis or to be highly likely to develop atopic dermatitis in the future.

6. The method of claim 5, wherein the cutoff value is 4.6 pg / μg total protein relative to the total protein in the stratum corneum.

7. A kit for predicting the onset of atopic dermatitis in infants, comprising a reagent for measuring the amount of IL-2 in the stratum corneum of the skin.

8. The kit of claim 7, wherein the reagent comprises an anti-IL-2 antibody.

9. The kit of claim 7, further comprising a detergent that solubilizes the protein.

10. The kit according to claim 7, further comprising a tape for peeling the stratum corneum of the skin.

11. A method for diagnosing the risk of developing atopic dermatitis in infants based on the amount of IL-2 in the stratum corneum.

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