JP2012233777A - Prediction of development of infant atopic dermatitis on basis of cytokine/chemokine in breast milk - Google Patents

Abstract

To clarify the relationship between the level of various cytokines / chemokines contained in breast milk and the onset of atopic dermatitis, and based on this, the onset of infant atopic dermatitis based on cytokine / chemokine levels in breast milk Provide a prediction method.
A method for determining the risk of developing atopic dermatitis in an infant, comprising:
(1) At least one cytokine selected from the group consisting of IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum taken by the infant, or the infant Selected from the group consisting of IL-1α, IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α in mature milk Measuring the concentration of at least one cytokine or chemokine; and (2) correlating the cytokine or chemokine concentration measured in (1) with the risk of developing atopic dermatitis in the infant .
[Selection figure] None

Description

  The present invention relates to a method for predicting the onset of infant atopic dermatitis based on cytokine / chemokine levels in breast milk, and a diagnostic agent used therefor.

  Breastfeeding is one factor associated with the development of many allergic diseases such as atopic dermatitis (AD) in infants. A meta-analysis of 17 past studies in 2001 shows that breastfeeding during the first three months of life has a protective effect on the formation of atopic dermatitis (Non-Patent Document 1). However, one report reports that breastfeeding has no protective effect (Non-Patent Document 2), and other reports suggest that it increases the risk of developing atopic dermatitis ( Non-patent documents 3 and 4). Thus, no studies have shown that breastfeeding has prevented infants from developing atopic dermatitis. While breastfeeding may prevent atopic dermatitis, the relationship between breastfeeding and the development of atopic dermatitis has not been established. One of the reasons that studies on the effects of breastfeeding associated with atopic dermatitis and other allergic diseases have not concluded is that the complexity of breast milk components and the intestinal environment and the immune system in infants There will be in the complexity of the interaction between.

  Human breast milk contains various cytokines / chemokines (Non-Patent Document 5). Those levels greatly differ among individual women depending on the allergic state of the mother (Non-Patent Documents 6 to 11). Of particular importance is the fact that some cytokines present in human breast milk are not digested in the stomach but rather maintain their biological activity in the infant's gastrointestinal tract (Non-Patent Documents 5 and 12). Given that many inflammatory and anti-inflammatory cytokines that may exert immunomodulatory effects through breast milk are contained in breast milk, some cytokines / chemokines in breast milk are atopic in infants Although thought to act to prevent the development of dermatitis, other cytokines / chemokines may work in reverse.

  In recent years, many researchers have investigated breast milk cytokines to clarify the allergy-related effects of breastfeeding. Some researchers have shown that certain cytokines contained in breast milk may be related to the establishment of allergic diseases (Non-Patent Documents 13 and 14). However, the results of these studies are not clear. Past studies have also used breast milk collected at different times. Few studies have examined cytokines / chemokines in both colostrum and mature milk associated with atopic dermatitis.

Gdalevich M, Mimouni D, David M, Mimouni M. Breast-feeding and the onset of atopic dermatitis in childhood: a systematic review and meta-analysis of prospective studies.J Am Acad Dermatol. 2001; 45 (4): 520-527 . Ludvigsson JF, Mostrom M, Ludvigsson J, Duchen K. Exclusive breastfeeding and risk of atopic dermatitis in some 8300 infants. Pediatr Allergy Immunol. 2005; 16 (3): 201-208. Benn CS, Wohlfahrt J, Aaby P, Westergaard T, Benfeldt E, Michaelsen KF, et al. Breastfeeding and risk of atopic dermatitis, by parental history of allergy, during the first 18 months of life.Am J Epidemiol. 2004; 160 ( 3): 217-223. Pesonen M, Kallio MJ, Ranki A, Siimes MA.Prolonged exclusive breastfeeding is associated with increased atopic dermatitis: a prospective follow-up study of unselected healthy newborns from birth to age 20 years.Clin Exp Allergy. 2006; 36 (8): 1011-1018. Field CJ. The immunological components of human milk and their effect on immune development in infants.J Nutr. 2005; 135 (1): 1-4. Bottcher MF, Jenmalm MC, Bjorksten B, Garofalo RP. Chemoattractant factors in breast milk from allergic and nonallergic mothers. Pediatr Res. 2000; 47 (5): 592-597. Bottcher MF, Jenmalm MC, Garofalo RP, Bjorksten B. Cytokines in breast milk from allergic and nonallergic mothers. Pediatr Res. 2000; 47 (1): 157-162. Laiho K, Lampi AM, Hamalainen M, Moilanen E, Piironen V, Arvola T, et al. Breast milk fatty acids, eicosanoids, and cytokines in mothers with and without allergic disease.Pediatr Res. 2003; 53 (4): 642- 647. Prokesova L, Lodinova-Zadnikova R, Zizka J, Kocourkova I, Novotna O, Petraskova P, et al. Cytokine levels in healthy and allergic mothers and their children during the first year of life.Pediatr Allergy Immunol. 2006; 17 (3) : 175-183. Rigotti E, Piacentini GL, Ress M, Pigozzi R, Boner AL, Peroni DG.Transforming growth factor-beta and interleukin-10 in breast milk and development of atopic diseases in infants.Clin Exp Allergy. 2006; 36 (5): 614 -618. Marek A, Zagierski M, Liberek A, Aleksandrowicz E, Korzon M, Krzykowski G, et al. TGF-beta (1), IL-10 and IL-4 in colostrum of allergic and nonallergic mothers. Acta Biochim Pol. 2009; 56 (3): 411-414. Calhoun DA, Lunoe M, Du Y, Staba SL, Christensen RD. Concentrations of granulocyte colony-stimulating factor in human milk after in vitro simulations of digestion. Pediatr Res. 1999; 46 (6): 767-771. Kalliomaki M, Ouwehand A, Arvilommi H, Kero P, Isolauri E. Transforming growth factor-beta in breast milk: a potential regulator of atopic disease at an early age.J Allergy Clin Immunol. 1999; 104 (6): 1251-1257 . Oddy WH, Halonen M, Martinez FD, Lohman IC, Stern DA, Kurzius-Spencer M, et al.TGF-beta in human milk is associated with wheeze in infancy.J Allergy Clin Immunol. 2003; 112 (4): 723- 728.

  The possible link between the presence of various cytokines / chemokines in breast milk and the development of atopic dermatitis in infants fed breast milk is very interesting. Several cytokines and chemokines function in the network and may be associated with atopic dermatitis by integrating abnormal activation of the infant's immune system. Because various cytokines / chemokines contribute to the pathology of atopic dermatitis, it is not efficient to evaluate individual cytokines and chemokines in breast milk that affect the establishment of atopic dermatitis. Therefore, it is worthwhile to evaluate the amount of many types of breast milk cytokines / chemokines simultaneously.

  The object of the present invention is to clarify the relationship between the levels of various cytokines / chemokines contained in breast milk and the onset of atopic dermatitis, and based on this, infant atopic skin based on cytokine / chemokine levels in breast milk It is to provide a method for predicting the onset of inflammation and a diagnostic agent used therefor.

  The present inventors have intensively studied to solve the above problems, and at 6 months of age, between the breast milk taken by an infant who has developed atopic dermatitis and the breast milk taken by a control infant, IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α concentrations in colostrum and IL-1α, IL-4, IL-6, IL-7 in mature milk , IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2, and MIP-1α were found to be significantly different in concentration. Logistic regression analysis revealed that IL-6, IL-12p40 and IL-13 in high levels of colostrum, eotaxin and IFN-α2 in high levels of mature milk, and IL-1α in low levels of mature milk. , A risk factor for the development of AD. By setting the concentration of chemokines / cytokines including eotaxin, IFN-α2 and IL-1α in mature milk to a specific cut-off point, infant atopy with a very high area under the passive operating characteristic curve (ROC) A model for predicting dermatitis could be constructed. As a result of further studies based on these findings, the present invention was completed.

That is, the present invention relates to the following.
[1] A method for determining the risk of developing atopic dermatitis in an infant,
(1) At least one cytokine selected from the group consisting of IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum taken by the infant, or the infant Selected from the group consisting of IL-1α, IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α in mature milk Measuring the concentration of at least one cytokine or chemokine; and (2) correlating the cytokine or chemokine concentration measured in (1) with the risk of developing atopic dermatitis in the infant .
[2] A method for determining the risk of developing atopic dermatitis in an infant,
(1) measuring IL-6, IL-12p40 and IL-13 in colostrum taken by the infant or eotaxin, IFN-α2 and IL-1α in mature milk; and (2) ( Correlating the cytokine or chemokine concentration measured in 1) with the risk of developing atopic dermatitis in the infant.
[3] Anti-human IL-1β antibody, anti-human IL-1α antibody, anti-human IL-4 antibody, anti-human IL-6 antibody, anti-human IL-7 antibody, anti-human IL-12p40 antibody, anti-human IL-13 An infant comprising at least one antibody selected from the group consisting of an antibody, an anti-human eotaxin antibody, an anti-human G-CSF antibody, an anti-human GM-CSF antibody, an anti-human IFN-α2 antibody and an anti-human MIP-1α antibody A diagnostic agent for determining the risk of developing atopic dermatitis in an infant based on cytokine or chemokine concentration in ingested colostrum or mature milk.
[4] Including anti-human IL-6 antibody, anti-human IL-12p40 antibody and anti-human IL-13 antibody combination, or anti-human eotaxin antibody, anti-human IFN-α2 antibody and anti-human IL-1α antibody The diagnostic agent according to [3], comprising a combination of antibodies.
[5] IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum, or IL-4, IL-6, IL in mature milk -7, whether IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 or MIP-1α levels can be suppressed, or whether IL-1α levels in mature milk can be increased To evaluate and IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum, or IL-4, IL-6, IL-7 in mature milk, A test substance in which the concentration of IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 or MIP-1α is suppressed, or a test substance in which the IL-1α concentration in mature milk is increased, is applied to the infant. Prevent the onset of atopic dermatitis Comprising selecting for a screening method for a substance to prevent the development of infant atopic dermatitis.

  According to the present invention, it is possible to evaluate the onset risk of infant atopic dermatitis with high accuracy based on cytokine / chemokine values in breast milk. In addition, according to the present invention, a candidate substance that prevents the onset of atopic dermatitis in an infant can be screened.

The result of having compared the level of the cytokine and chemokine in colostrum between AD group and a control group is shown. Minimum values, 25th, 50th, 75th and maximum values were shown (n = number of samples). The cytokine concentration in the sample below the detection limit was defined as 1.6 pg / ml. The result of having compared the level of cytokine and chemokine in mature milk between AD group and a control group is shown. Minimum values, 25th, 50th, 75th and maximum values were shown (n = number of samples). The cytokine concentration in the sample below the detection limit was defined as 1.6 pg / ml. ROC curve for prediction of AD onset at 6 months of age with breast milk cytokine / chemokine concentration. These curves were generated by the sum of the normalized values of each cytokine in colostrum or each cytokine / chemokine in mature milk. The area under the curve was determined by statistical analysis.

1. The present invention relates to a method for determining the risk of developing atopic dermatitis in an infant,
(1) At least one cytokine selected from the group consisting of IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum taken by the infant, or the infant Selected from the group consisting of IL-1α, IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α in mature milk Measuring the concentration of at least one cytokine or chemokine; and (2) correlating the cytokine or chemokine concentration measured in (1) with the risk of developing atopic dermatitis in the infant Is to provide.

  As used herein, an infant means a child up to one year after the neonatal period. By the method of the present invention, it is possible to determine the risk that an infant will develop atopic dermatitis, usually by 12 months after birth, preferably by 6 months after birth.

  The subject of the determination method of the present invention is usually a human infant who has received breastfeeding.

  As used herein, colostrum means milk secreted from the mother within 7 days at the most after delivery, preferably within 5 days. In one aspect, the present invention uses colostrum secreted from the mother 4-5 days after delivery.

  Mature milk generally means milk secreted from the mother after 3 weeks or more after delivery, preferably after 4 weeks or more. The mature milk used in the present invention is preferably mature milk secreted from the mother within 2 months of delivery, more preferably after 1 month of delivery.

  In the method of the present invention, at least one selected from the group consisting of IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum taken by the target infant ( Preferably 2 or more, more preferably 3 or more) cytokines, or IL-1α, IL-4, IL-6, IL-7, IL-12p40, eotaxin, G- in mature milk taken by the infant The concentration of at least one (preferably two or more, more preferably three or more) cytokine or chemokine selected from the group consisting of CSF, GM-CSF, IFN-α2 and MIP-1α is measured.

  In one embodiment, the concentrations of IL-6, IL-12p40 and IL-13 in colostrum or eotaxin, IFN-α2 and IL-1α in mature milk are measured. By combining these specific cytokine or chemokine concentrations, a more accurate determination of the risk of developing atopic dermatitis can be expected.

IL-1α, IL-1β, IL-4, IL-6, IL-7, IL-12p40, IL-13, eotaxin, G-CSF, GM-CSF, IFN-α2, and MIP-1α are all well known Of cytokines or chemokines. Representative amino acid sequences of these human cytokines or chemokines and their NCBI accession numbers are as follows.
IL-1α: NP — 000566.3 region 113..271 (SEQ ID NO: 1)
IL-1β: NP — 000567.1 region 117..269 (SEQ ID NO: 2)
IL-4: NP — 000580.1 region 25..153 (SEQ ID NO: 3)
IL-6: NP — 000591.1 region 30..212 (SEQ ID NO: 4)
IL-7: NP — 000871.1 region 26..177 (SEQ ID NO: 5)
IL-12p40: NP_002178.2 region 23..328 (SEQ ID NO: 6)
IL-13: NP_002179.2 region 35..132 (sequence number 7)
Eotaxin: NP_002977.1 region 24..97 (SEQ ID NO: 8)
G-CSF: NP_000750.1 region 31..207 (SEQ ID NO: 9)
GM-CSF: NP_000749.2 region 18..144 (SEQ ID NO: 10)
IFN-α2: NP — 000596.2 region 24..188 (SEQ ID NO: 11)
MIP-1α: NP — 002974.1 region 24..92 (SEQ ID NO: 12)

  The concentration of each cytokine or chemokine in breast milk can be measured by an immunological technique using an antibody that specifically recognizes each cytokine or chemokine. Immunological methods include antibody array, flow cytometry analysis, radioisotope immunoassay (RIA method), ELISA method (Methods in Enzymol. 70: 419-439 (1980)), Western blotting, immunohistochemical staining, etc. Can be mentioned.

  Since breast milk is rich in lipids, it is preferable to use non-fat whey obtained by centrifuging in advance and removing the lipid layer in the immunological measurement of cytokine or chemokine concentration.

  “Specific recognition” of the antigen X by the antibody means that the affinity of the antibody for the antigen X in the antigen-antibody reaction is stronger than the affinity for an antigen other than the antigen X. In this specification, an antibody that specifically recognizes antigen X is abbreviated as “anti-X antibody”.

An antibody that specifically recognizes each cytokine or chemokine can be produced by an existing general production method using the cytokine or chemokine or a partial peptide having antigenicity as an immunogen. In the present specification, the antibody includes a polyclonal antibody, a natural antibody such as a monoclonal antibody (mAb), a chimeric antibody that can be produced using a gene recombination technique, a humanized antibody or a single chain antibody, and binding properties thereof. Fragments are included, but are not limited to these. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof. The binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes, for example, F (ab ′) ₂ , Fab ′, Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol.6, No.5, p.441-456, 1996). The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification.

The antibody may be an appropriate labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, ³² P, ³³ P, ³⁵ S, etc.), an enzyme (eg, β-galactosidase, β -Glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin, etc.), biotin It may be labeled with.

  Antibodies that specifically recognize each of the cytokines or chemokines described above may be bound on a suitable support and provided as an antibody array. The support is not particularly limited as long as it is a support that is usually used in the art, and examples thereof include membranes (for example, nylon membrane), beads, glass, plastics, metals, and the like.

  Next, each cytokine or chemokine concentration in the colostrum or mature milk measured in the step (1) is correlated with the risk of developing atopic dermatitis in the infant. For example, the measured cytokine or chemokine concentration in colostrum or mature milk is the corresponding cytokine or chemokine concentration in colostrum or mature milk taken by an infant who has developed atopic dermatitis, and atopic dermatitis. Compare to the corresponding cytokine or chemokine concentration in colostrum or mature milk taken by an infant who does not develop. Alternatively, the concentration of each cytokine or chemokine in the measured colostrum or mature milk was determined in advance, and the corresponding cytokine or chemokine in the colostrum or mature milk for a large number of infants who developed atopic dermatitis Mean concentration, mean value of the corresponding cytokine or chemokine concentration in colostrum or mature milk for a large number of infants who have not developed atopic dermatitis, many who have / not developed atopic dermatitis You may compare with the distribution map etc. of the corresponding cytokine or chemokine concentration in colostrum or mature milk about the infant of an individual. The comparison of each cytokine or chemokine concentration is preferably performed based on the presence or absence of a significant difference.

  As shown in the examples below, IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α, and maturation in colostrum taken by infants who have developed atopic dermatitis The concentrations of IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α in milk are related to atopic dermatitis. It was higher compared to that in colostrum or mature milk taken by infants who did not develop symptoms. That is, based on a positive correlation between the concentration of each of the above-mentioned cytokines or chemokines contained in colostrum or mature milk and the risk of developing atopic dermatitis in an infant taking the colostrum or mature milk, The risk of developing atopic dermatitis can be determined. For example, at least one cytokine selected from the group consisting of IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum, or IL-4 in mature milk, When the concentration of at least one cytokine or chemokine selected from the group consisting of IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α is relatively high It can be determined that an infant who takes the colostrum or mature milk has a high possibility of developing atopic dermatitis.

  On the other hand, as shown in the Examples described later, the IL-1α concentration in mature milk taken by an infant who developed atopic dermatitis is compared with that in mature milk taken by an infant who does not develop atopic dermatitis. It was low. That is, the risk of developing atopic dermatitis in an infant is determined based on a negative correlation between the concentration of IL-1α contained in mature milk and the risk of developing atopic dermatitis in an infant who takes the mature milk can do. For example, when the IL-1α concentration in mature milk is relatively low, it can be determined that an infant taking the colostrum or mature milk has a high possibility of developing atopic dermatitis.

  In addition, the cutoff value of each cytokine or chemokine concentration in colostrum or mature milk should be set in advance, and the measured cytokine or chemokine concentration in colostrum or mature milk should be compared with this cutoff value. Can also be done. For example, at least one cytokine selected from the group consisting of IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum, or IL-4 in mature milk, The concentration of at least one cytokine or chemokine selected from the group consisting of IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α is equal to or higher than the cut-off value. In this case, it can be determined that an infant who takes the colostrum or mature milk has a high possibility of developing atopic dermatitis. Moreover, when the IL-1α concentration in mature milk is equal to or lower than the cut-off value, it can be determined that an infant who takes the mature milk has a high possibility of developing atopic dermatitis.

  The “cut-off value” is a value that can satisfy both of high diagnostic sensitivity (prevalence of correct diagnosis) and high diagnostic specificity (prevalence of nondiagnostic diagnosis) when a disease is determined based on the value. For example, cut each cytokine or chemokine concentration in colostrum or mature milk that shows a high positive rate in infants with atopic dermatitis and a high negative rate in infants without atopic dermatitis It can be set as an off value.

  The calculation method of the cut-off value is well known in this field. For example, by measuring the above-mentioned cytokine or chemokine concentration in colostrum or mature milk taken by infants who have developed atopic dermatitis and infants who have not developed atopic dermatitis, diagnostic sensitivity and diagnosis at the measured values Specificity is obtained, and a ROC (Receiver Operating Characteristic) curve is created based on these values using commercially available analysis software. Then, a value when the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible is obtained, and the value can be used as a cutoff value. Also, for example, the diagnosis efficiency (the ratio of the total number of diseased and non-diagnosed correctly diagnosed cases to the total number of cases) of the detected value is obtained, and the value at which the highest diagnosis efficiency is calculated is the cut-off value can do.

  In the method of the present invention, at least one selected from the group consisting of IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum taken by the target infant. IL-1α, IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α in mature milk taken by the infant It is sufficient to measure the concentration of at least one cytokine or chemokine selected from the group consisting of: a plurality of (for example, 2 or more, preferably 3 or more) of these cytokines or chemokines, By correlating with the risk of developing atopic dermatitis, a more accurate determination of the risk of developing atopic dermatitis can be expected.

  In a preferred embodiment, the concentrations of IL-6, IL-12p40 and IL-13 in colostrum or eotaxin, IFN-α2 and IL-1α in mature milk are measured. By combining these specific cytokine or chemokine concentrations, a more accurate determination of the risk of developing atopic dermatitis can be expected.

  For example, when the concentrations of IL-6, IL-12p40 and IL-13 in colostrum are relatively high, it is determined that an infant taking the colostrum has a high possibility of developing atopic dermatitis be able to. Alternatively, if the concentrations of eotaxin and IFN-α2 in mature milk are relatively high and the concentration of IL-1α in mature milk is relatively low, the infant who takes the mature milk develops atopic dermatitis It is possible to determine that there is a high possibility of doing.

  Alternatively, a cut-off value of IL-6, IL-12p40 and IL-13 concentrations in colostrum or eotaxin, IFN-α2 and IL-1α concentrations in mature milk is set in advance and measured. Or you may carry out by comparing each cytokine or chemokine density | concentration in mature milk with this cut-off value. If the concentrations of IL-6, IL-12p40, and IL-13 in colostrum are each greater than or equal to the cut-off value, infants who take the colostrum or mature milk may develop atopic dermatitis It can be determined that the property is high. Alternatively, when the concentrations of eotaxin and IFN-α2 in mature milk are each equal to or higher than the cut-off value and the IL-1α concentration is equal to or lower than the cut-off value, the infant who takes the mature milk is atopic. It can be determined that there is a high possibility of developing dermatitis.

  Alternatively, IL-6, IL-12p40 and IL-13 concentrations in colostrum or eotaxin in mature milk taken by infants who have developed atopic dermatitis and infants who have not developed atopic dermatitis, IFN- Measure α2 and IL-1α concentrations, categorize the data for each cytokine concentration to tertiles or medians using commercially available analysis software, and independent of each cytokine or chemokine for the development of atopic dermatitis By constructing multiple logistic regression models to assess the effects and generating ROC curves, IL-6, IL-12p40 and IL-13 concentrations in colostrum, or eotaxin, IFN-α2 and IL in mature milk -1α concentration and the risk of developing atopic dermatitis in infants, and then the IL in colostrum taken by the measured infant 6. By introducing IL-12p40 and IL-13 concentrations or eotaxin, IFN-α2 and IL-1α concentrations in mature milk into this correlation, the risk of developing atopic dermatitis in the infant can be evaluated. it can. Alternatively, IL-6, IL-12p40 and IL-13 concentrations in colostrum or eotaxin, IFN-α2 and IL-1α concentrations in mature milk were measured at different scales by converting to standardized scores The above cytokines and chemokines are scored relative. Sum of scores for IL-6, IL-12p40 and IL-13 in colostrum, sum of scores for eotaxin, IFN-α2 and IL-1α in mature milk (IL-1α developed atopic dermatitis By performing ROC analysis using a variable of a score coefficient of -1 because the value was significantly lower in mature breast milk taken by infants, the higher the score, the higher the atopic property of the infant. It can be evaluated that the risk of developing dermatitis is high.

2. The present invention relates to a diagnostic agent for determining the risk of developing atopic dermatitis in an infant based on cytokine or chemokine concentration in colostrum or mature milk ingested by the infant, comprising anti-human IL-1β Antibody, anti-human IL-1α antibody, anti-human IL-4 antibody, anti-human IL-6 antibody, anti-human IL-7 antibody, anti-human IL-12p40 antibody, anti-human IL-13 antibody, anti-human eotaxin antibody, anti-human Provided is a diagnostic agent comprising at least one antibody selected from the group consisting of a human G-CSF antibody, an anti-human GM-CSF antibody, an anti-human IFN-α2 antibody and an anti-human MIP-1α antibody. If the diagnostic agent of this invention is used, it will become possible to determine the onset risk of the infant atopic dermatitis easily by the method of this invention.

  The definition and embodiment of “antibody” are as described in the above item 1.

  When the diagnostic agent of the present invention is a diagnostic agent for determining the risk of developing atopic dermatitis in an infant based on the cytokine or chemokine concentration in colostrum taken by the infant, the diagnostic agent is anti-human At least one selected from the group consisting of IL-1β antibody, anti-human IL-6 antibody, anti-human IL-7 antibody, anti-human IL-12p40 antibody, anti-human IL-13 antibody, and anti-human MIP-1α antibody (Preferably two or more, more preferably three or more) antibodies. In this case, in one embodiment, the diagnostic agent of the present invention comprises a combination of antibodies including anti-human IL-6 antibody, anti-human IL-12p40 antibody and anti-human IL-13 antibody. By measuring the IL-6, IL-12p40, and IL-13 concentrations in colostrum using the diagnostic agent of this aspect by the method of the present invention, the atopy of an infant who takes the colostrum with high accuracy The risk of developing dermatitis can be determined.

  When the diagnostic agent of the present invention is a diagnostic agent for determining the risk of developing atopic dermatitis in an infant based on the cytokine or chemokine concentration in mature milk taken by the infant, the diagnostic agent is anti-human IL-1α antibody, anti-human IL-4 antibody, anti-human IL-6 antibody, anti-human IL-7 antibody, anti-human IL-12p40 antibody, anti-human eotaxin antibody, anti-human G-CSF antibody, anti-human GM-CSF It includes at least one antibody (preferably two or more, more preferably three or more) selected from the group consisting of an antibody, an anti-human IFN-α2 antibody and an anti-human MIP-1α antibody. In this case, in one embodiment, the diagnostic agent of the present invention comprises a combination of antibodies including anti-human eotaxin antibody, anti-human IFN-α2 antibody and anti-human IL-1α antibody. By measuring the eotaxin, IFN-α2 and IL-1α concentrations in mature milk using the diagnostic agent of the aspect by the method of the present invention, the atopic skin of an infant who takes the mature milk with high accuracy The risk of developing a flame can be determined.

  The antibody contained in the diagnostic agent of the present invention is usually provided in water or an appropriate buffer solution (eg, TE buffer, PBS, etc.) so as to have an appropriate concentration or lyophilized. Is done.

  Alternatively, the antibodies may be bound on a suitable support and provided as an antibody array. The support is not particularly limited as long as it is a support that is usually used in the art, and examples thereof include membranes (for example, nylon membrane), beads, glass, plastics, metals, and the like.

  The diagnostic agent of the present invention can also be provided as a diagnostic kit further comprising other components necessary for carrying out the method according to the type of immunological measurement method. For example, the diagnostic agent of the present invention can further contain a labeled secondary antibody, a chromogenic substrate, a blocking solution, a washing buffer, an ELISA plate, a blotting membrane and the like.

  Further, the diagnostic agent of the present invention may contain a cytokine or chemokine solution containing a known amount of the cytokine or chemokine to be measured in order to prepare a calibration curve. The cytokine or chemokine solution for preparing a calibration curve is preferably a solution of the cytokine or chemokine to be measured in human breast milk (or its whey). When the diagnostic agent of the present invention is a diagnostic agent for determining the risk of developing atopic dermatitis in an infant based on cytokine or chemokine concentration in colostrum taken by the infant, the diagnostic agent is a measurement target A solution of a cytokine or chemokine in human colostrum (or its whey) may be included for the preparation of a calibration curve. When the diagnostic agent of the present invention is a diagnostic agent for determining the risk of developing atopic dermatitis in an infant based on the cytokine or chemokine concentration in mature milk taken by the infant, the diagnostic agent is a measurement object A solution of a cytokine or chemokine in human mature milk (or whey thereof) may be included for the preparation of a calibration curve.

  Each of these components is dissolved individually (or mixed if possible) in water or in an appropriate buffer (eg, TE buffer, PBS, etc.) to an appropriate concentration, Alternatively, it is stored in a suitable container in a lyophilized state.

3. Screening method As described above, IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum taken by infants who have developed atopic dermatitis, and mature milk IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2, and MIP-1α concentrations of cytokines or chemokines do not develop atopic dermatitis Higher than that in colostrum or mature milk taken by infants. Further, the IL-1α concentration in mature milk taken by an infant who has developed atopic dermatitis is lower than that in mature milk taken by an infant who does not develop atopic dermatitis. Therefore, the test substance is IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum, or IL-4, IL-6, IL- 7. Evaluate whether the concentration of IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 or MIP-1α can be suppressed, or increase the concentration of IL-1α in mature milk IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum, or IL-4, IL-6, IL-7, IL in mature milk -12p40, Eotaxin, G-CSF, GM-CSF, IFN-α2, or MIP-1α test substance with suppressed concentration, or test substance with increased IL-1α concentration in mature milk, Substances that prevent the development of atopic dermatitis To can be selected. The present invention provides a screening method for substances that prevent the onset of atopic dermatitis in infants.

  The test substance to be used for the screening method may be any known compound or novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, prepared using combinatorial chemistry technology Examples thereof include a compound library, a random peptide library prepared by solid phase synthesis or a phage display method, or natural components derived from microorganisms, animals and plants, marine organisms, and the like. Foods, beverages, or extracts thereof are also preferred as test substances.

  The screening method of the present invention can be performed by administering a test substance to a mammal. Examples of the animals include humans and mammals other than humans. Examples of mammals other than humans include, for example, laboratory animals such as rodents such as mice, rats, hamsters, and guinea pigs, and rabbits, domestic animals such as pigs, cows, goats, horses, and sheep, pets such as dogs and cats, and monkeys. , Primates such as orangutans and chimpanzees. As the mammal, a female mammal that is pregnant or secretes breast milk is preferable.

  For example, a test substance is administered to a mammal, and IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum secreted by the mammal, or mature milk IL-1α, IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2, or MIP-1α is measured. Next, IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum secreted by a mammal administered the test substance, or IL-1α in mature milk, IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 or MIP-1α concentrations were secreted by mammals not receiving the test substance Compare with the cytokine or chemokine concentration in colostrum or mature milk. Concentration comparison is preferably performed based on the presence or absence of a significant difference. As a result of comparison, IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum, or IL-4, IL-6, IL-7, IL in mature milk -12p40, Eotaxin, G-CSF, GM-CSF, IFN-α2, or MIP-1α test substance with suppressed concentration, or test substance with increased IL-1α concentration in mature milk, Select as a candidate substance to prevent the development of atopic dermatitis.

  In one embodiment, a test substance is administered to a mammal, and IL-6, IL-12p40 and IL-13 concentrations in the colostrum secreted by the mammal are measured. Next, the concentrations of IL-6, IL-12p40 and IL-13 in the colostrum secreted by the mammal to which the test substance was administered were determined according to the concentrations of IL-6 in the colostrum secreted by the mammal to which the test substance was not administered. 6. Compare with IL-12p40 and IL-13 concentrations. Concentration comparison is preferably performed based on the presence or absence of a significant difference. As a result of the comparison, a test substance in which the concentrations of IL-6, IL-12p40, and IL-13 in colostrum are suppressed is selected as a candidate substance that prevents the onset of atopic dermatitis in the infant.

  In one embodiment, a test substance is administered to a mammal, and the concentrations of eotaxin, IFN-α2 and IL-1α in mature milk secreted by the mammal are measured. Next, the concentrations of eotaxin, IFN-α2 and IL-1α in the mature milk secreted by the mammal to which the test substance has been administered are determined according to the concentrations of eotaxin in mature milk secreted by the mammal to which the test substance has not been administered, IFN -Compare with concentrations of α2 and IL-1α. Concentration comparison is preferably performed based on the presence or absence of a significant difference. As a result of comparison, a test substance that suppresses the concentrations of eotaxin and IFN-α2 in mature milk and increases the concentration of IL-1α is selected as a candidate substance for preventing the onset of atopic dermatitis in the infant To do.

  Candidate substances for preventing the onset of infant atopic dermatitis that can be obtained by the screening method of the present invention can be used as candidate compounds for drug development. Moreover, in order to prevent infantile atopic dermatitis, it can be set as the pharmaceutical composition applied to a mother body by formulating as a pharmaceutical. Or it can also be set as the food composition for a mother ingest in order to prevent atopic dermatitis of an infant.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

Patient and Method Cohort and Study Subjects From January 2007 to May 2008, 500 prospective birth cohorts were set up at Kawatetsu Chiba Hospital. All participants were surveyed before the baby was born. Data on parental allergic diseases and various exposures were obtained. Parents responded to a questionnaire that focused primarily on symptoms related to eczema. When an infant is 6 months old, an infant who has itchy eczema for at least 2 months was defined as atopic dermatitis. The complete response rate for the 6-month questionnaire was 73%. There were 51 infants who developed atopic dermatitis (AD) at 6 months of age, from which 49 AD (+) subjects were selected. Two of the 51 AD infants were excluded from the study because of breast milk loss. Forty-nine AD (-) subjects were randomly selected so that there was no difference in maternal history of AD and other allergic diseases (asthma, allergic rhinitis, food allergies, and hay fever). Twenty of 49 AD (+) babies and 20 of 49 AD (-) babies were mainly breast-fed until one month of age, and the other babies were mixed. It was. A frozen stock of breast milk was subjected to a multiplex cytokine assay. The course of pregnancy was normal in all women. Except for the history of allergic diseases, the mother had no other medical problems. There was no significant background difference between AD (+) infants and AD (-) infants and between the two groups of mothers (Table 1). All participants provided written informed consent to participate in the study. This study was approved by the Ethics Committee of the Chiba University Graduate School of Medicine.

  Maternal and infant characteristics. Maternal age, birth history, pre-pregnancy BMI, pregnant BMI, gestational age and birth weight were analyzed by Student's t test. Other features were analyzed by Pearson's chi-square test.

Sampling and storage of breast milk Breast milk samples were collected 4-5 days after birth and 1 month after delivery and frozen at -80 ° C for long-term storage. Breast milk samples were quickly thawed before testing and centrifuged at 10,000 × g for 10 minutes. After removal of the lipid layer, non-fat whey was extracted with a pipette.

Multiplex Analysis Bio-Plex Suspension Array System (Bio-Rad) with multiple kits purchased from Millipore, containing polystyrene beads coated with antibodies against each of the 26 cytokines and chemokines for cytokines and chemokines in breast milk Laboratory). The advantage of this technique is that only 25 μl of sample is required for simultaneous measurements up to 100 analytes in the sample. 26 cytokines [IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-12p70 IL-13, IL-15, IL-17, IFN-α2, IFN-γ, TNF-α, TNF-β, eotaxin, granulocyte colony stimulating factor (G-CSF), granulocyte / macrophage colony stimulating factor ( GM-CSF), macrophage inflammatory protein (MIP) -1α, MIP-1β, monocyte chemotactic protein (MCP) -1, and interferon gamma inducible protein 10 (IP-10)] For milk and mature milk. The breast milk sample was thawed at room temperature and added in duplicate to a 96-well filter bottom plate. Beads were added to the wells and incubated for 1 hour at room temperature on a plate shaker. After washing the wells by vacuuming through the filter bottom, antibody conjugate was added and stirring continued for an additional hour. Streptavidin-phycoerythrin was added to the wells and a final agitation incubation was performed. The plate was washed as before, 100 μl of sheath fluid was added to the well and the well read. Standard curves and quality controls for all analytes were performed on the plates. Median fluorescence intensity of cytokine-bound beads was converted to concentration (pg / ml) using a five parameter logical model. For all cytokines and chemokines, the detection limit of the kit was 3.2 pg / ml and the maximum detection limit of the kit was 10,000 pg / ml.

Statistical analysis Statistical analysis was performed using the SPSS program (v. 19.0., SPSS Inc.). Comparison of maternal and infant characteristics was assessed by the Peason's chi-square test and the Student's t-test. Non-parametric tests were used because the various cytokine / chemokine data does not have a normal distribution. Differences between groups were analyzed by Mann-Whitney U test, and a probability level of <0.05 was considered statistically significant. In addition, data on breast milk cytokine / chemokine concentrations were categorized and a multiple logistic regression model was constructed to evaluate the independent effects of different breast cytokines / chemokines on the development of atopic dermatitis; the results were expressed as 95% confidence intervals (CIs ) By the odds ratio. A Passive Person Operating Characteristic Curve (ROC) curve was created to assess how much breast milk cytokine / chemokine can predict the onset of atopic dermatitis.

Results Cytokine / chemokine profile in AD and control groups breast milk cytokine / chemokine concentrations were significantly expanded. In colostrum, the frequency of positive IL-1β, IL-12p40 and IL-13 values in the AD group was greater than that of positive IL-1β, IL-12p40 and IL-13 values in the control group (chi-square). Tests, P <0.001, <0.001, and 0.028, respectively, Table 2).

  Cytokine profile in colostrum. A positive result means a concentration of the cytokine above the detection limit. The possibility of generating positive cytokine values between the two groups was examined by chi-square test.

  In mature milk, the frequency of positive IL-4, IL-6, IL-12p40, G-CSF, GM-CSF and MIP-1α values in the AD group was greater than that in the control group (chi-square test, P <0.001, 0.038, 0.014, 0.022, <0.001 and <0.001, respectively, Table 3). IL-8, MCP-1 and IP-10 concentrations exceeded detection limits in most colostrum samples and MCP-1 and IP-10 concentrations exceeded detection limits in some mature milk samples . Because of the lack of carry-over milk, most of these samples could not be remeasured and were excluded from further analysis. Statistical analysis of these chemokines was not performed.

  Cytokine profile in mature milk. A positive result means a concentration of the cytokine above the detection limit. The possibility of generating positive cytokine values between the two groups was examined by chi-square test.

Comparison of cytokine / chemokine levels in colostrum To examine the relationship between cytokine / chemokine levels in breast milk and the onset of AD at 6 months of age, infants who developed AD at 6 months of age ingested The cytokine / chemokine level in breast milk was compared with the cytokine / chemokine level in breast milk taken by an infant who did not develop AD at that time (hereinafter referred to as AD group and control group, respectively). As shown in FIG. 1, in colostrum, the key differences in median concentrations of breast milk cytokine / chemokine were as follows: In colostrum taken by infants in the AD group, IL-1β, IL-6 , IL-7, IL-12p40, IL-13 and MIP-1α were higher compared to the control group ((Mann-Whitney test, P <0.001, 0.005, 0.024, <0.001, 0.018 and 0.029).

Comparison of cytokine / chemokine levels in mature milk Significant differences in median concentrations of breast milk cytokines and chemokines in mature milk are as follows: IL-4, IL in mature milk consumed by infants in the AD group The concentrations of -6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α were higher than those in the control group (Mann-Whitney test, respectively, P <0.001, 0.018, 0.025, 0.015, <0.001, <0.001, <0.001, <0.001, and <0.001, FIG. 2). On the other hand, the level of IL-1α in mature milk of the AD group was lower than that of the control group (Mann-Whitney test, P = 0.005, FIG. 2).

Multivariate Logistic Regression Analysis of Breast Milk Cytokines / Chemokines Table 4 shows the results of the correlation between breast cytokine / chemokine levels (classified as tertiles or medians) and infant AD prognosis.

  Logistic analysis was performed for different cytokines and chemokines in colostrum (a) and mature milk (b). OR, odds ratio adjusted for different cytokines / chemokines; CI, confidence interval.

  Infant AD risk was correlated with levels of IL-6, IL-12p40 and IL-13 in colostrum. As shown in Table 4a, the risk of infant AD is increased at intermediate levels of IL-6, with an odds value (OR) of 3.627 and a confidence interval (CI) of 1.04-12.65. There was an increase in high levels of IL-6 with an odds value (OR) of 251 and a confidence interval (CI) of 2.194-31.03. The risk for infant AD is 6.125 OR (CI 1.301-28.828), increased at moderate levels of IL-12p40, and 11.679 OR (CI 3.64-37.85) Elevated at high levels of IL-12p40. Also, the risk for infant AD was elevated at high levels of IL-13 with an OR of 3.627 (CI 1.04-12.65). Furthermore, the risk of infant AD was correlated with the levels of eotaxin, IFN-α2 and IL-1α in mature milk. As shown in Table 4 (b), the risk for infant AD is 78.225 OR (CI 7.957-769.643) and 15.251 OR (high) at high levels of eotaxin and IFN-α2, respectively. CI 2.577-90.248). On the other hand, the risk of infant AD decreased at an OR of 0.035 (CI 0.005-0.268) at high levels of IL-1α.

Evaluation of breast milk cytokine / chemokine biomarkers by ROC analysis Create ROC curves for colostrum cytokines including IL-6, IL-12p40 and IL-13, or mature milk cytokines / chemokines including eotaxin, IFN-α2 and IL-1α did. The prediction of AD onset by breast milk cytokine concentration in colostrum was significant, achieving an area under the curve of 0.802 (95% CI 0.711-0.892, P <0.001). The prediction by cytokine / chemokine concentration in mature milk was also significant, achieving an area under the curve of 0.888 (95% CI 0.814-0.963, P <0.001).

  According to the present invention, it is possible to evaluate the onset risk of infant atopic dermatitis with high accuracy based on cytokine / chemokine values in breast milk. In addition, according to the present invention, a candidate substance that prevents the onset of atopic dermatitis in an infant can be screened.

Claims (5)

A method for determining the risk of developing an atopic dermatitis in an infant,
(1) At least one cytokine selected from the group consisting of IL-1β, IL-6, IL-7, IL-12p40, IL-13 and MIP-1α in colostrum taken by the infant, or the infant Selected from the group consisting of IL-1α, IL-4, IL-6, IL-7, IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 and MIP-1α in mature milk Measuring the concentration of at least one cytokine or chemokine; and (2) correlating the cytokine or chemokine concentration measured in (1) with the risk of developing atopic dermatitis in the infant . A method for determining the risk of developing an atopic dermatitis in an infant,
(1) measuring IL-6, IL-12p40 and IL-13 in colostrum taken by the infant or eotaxin, IFN-α2 and IL-1α in mature milk; and (2) ( Correlating the cytokine or chemokine concentration measured in 1) with the risk of developing atopic dermatitis in the infant.   Anti-human IL-1β antibody, anti-human IL-1α antibody, anti-human IL-4 antibody, anti-human IL-6 antibody, anti-human IL-7 antibody, anti-human IL-12p40 antibody, anti-human IL-13 antibody, anti-human IL-13 antibody First to be taken by an infant comprising at least one antibody selected from the group consisting of human eotaxin antibody, anti-human G-CSF antibody, anti-human GM-CSF antibody, anti-human IFN-α2 antibody and anti-human MIP-1α antibody A diagnostic agent for determining the risk of developing atopic dermatitis in an infant based on cytokine or chemokine concentration in milk or mature milk.   Combination of antibodies including anti-human IL-6 antibody, anti-human IL-12p40 antibody and anti-human IL-13 antibody, or combination of antibodies including anti-human eotaxin antibody, anti-human IFN-α2 antibody and anti-human IL-1α antibody The diagnostic agent of Claim 3 containing this. The test substance is IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum, or IL-4, IL-6, IL-7 in mature milk, To evaluate whether the concentration of IL-12p40, eotaxin, G-CSF, GM-CSF, IFN-α2 or MIP-1α can be suppressed, or whether the concentration of IL-1α in mature milk can be increased And IL-1β, IL-6, IL-7, IL-12p40, IL-13 or MIP-1α in colostrum, or IL-4, IL-6, IL-7, IL-12p40 in mature milk , Eotaxin, G-CSF, GM-CSF, IFN-α2, or MIP-1α test substance, or test substance with increased IL-1α concentration in mature milk, As a substance to prevent the onset of dermatitis Including selecting, screening method of a substance to prevent the development of infant atopic dermatitis.