CROSS-REFERENCE TO RELATED APPLICATION

[01]. This application claims the benefit of Singapore Patent Application No. 201006156-2 filed on 24 August 2010, the entire contents of which are incorporated herein by reference.

FIELD OF INVENTION

[02]. The invention relates generally to methods and kits for determining predisposition or diagnosis of inflammatory disorders such as wheezing / eczema.

BACKGROUND

[03]. The following discussion of the background to the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was published, known or part of the common general knowledge in any jurisdiction as at the priority date of the application.

[04]. Asthma and Eczema are both caused by allergens and so are termed an allergic reaction. Other such allergic reactions include hay fever. Allergic / inflammatory disorders are commonly associated with an immature immune response

[05]. Early childhood wheeze represents the most common cause of hospitalization of infants Studies show that wheezing before the age of 3 years and persistent childhood wheezing are more frequent in infants that have a family history of atopy ^{1 3} . The pathology of wheeze that influences the airway function involves epithelial sloughing , increased mucus production in airways , developmental and mechanical factors in the neuromuscular apparatus ⁴ , and aberrant immune responses are likely to be involved ^{5 ?} . The intrinsic differences in the immune system not only predisposes infants to the development of wheeze but could be involved in increasing its severity as well ⁶

[06]. Wheezing in infants is a common symptom and is predominantly caused by viral infections. Some children will outgrow these symptoms but a subset of viral infections may play a role in the inception and exacerbation of asthma in older childhood (1). Several possible predisposing factors that lead to the development of wheeze include prenatal smoking, maternal asthma, premature lung development and the hosts' immune responses.

[07]. Eczema, a chronic or intermittent skin disease characterized by the cutaneous reactivity and pruritus, is commonly diagnosed during infancy and is considered a significant risk factor for wheezing and the subsequent development of asthma (4). There is a lack of published reports on the cord blood study in association with infant eczema. The main focus of cord blood studies till now has been in relation to the atopic dermatitis (4-7).

[08]. It has been suggested certain childhood allergic / inflammatory disorders are commonly associated with an immature immune response at birth with a general impairment or genetic predisposition in cytokine production. Literature on soluble mediators released by stimulated cord blood mononuclear cells and its association with eczema or wheeze development is scarce (8).

[09]. The innate immune response is of interest as the immaturity of the infant immune system is capable of altering the outcome of microbial, particularly viral, infections; leading to severe forms of illness due to enhanced immunological responses. Through secretion of cytokines and interaction of ligands ¹ , these enhanced immunological responses are responsible for the generation of various lineages of T-helper cells such as Thl, Th2, and Thl7 contribute to the inflammatory process in the airways ⁸ . In this regard, the importance of the innate immune regulation in immunopathology following viral infection has been highlighted in a study showing that RSV infected STAT-1 KO mice with lower levels of regulatory type I IFN(a^8) was associated with increased mucus production and airway hyper-responsiveness along with increased pro-inflammatory cytokines IL-17 and IL-23 ⁹ . These data suggests that the IL-23/IL-17 cytokine axis plays a role in the pathogenesis of viral induced airway inflammation. In particular, this axis is a recognized pathway that drives pro-inflammatory Thl 7 cells, which are also known to participate in the airway inflammation in bronchial asthma ¹⁰ .

SUMMARY

[010]. One objective is to identify the relationships between inflammatory disorders such as wheezing / eczema phenotypes and the intrinsic differences on the production of cytokine, chemokine and soluble mediators from cord blood mononuclear cells (CBMCs). These cytokine, chemokine and mediators secreted by CBMCs are analysed by combined multi- factor analysis approach to corelate with the susceptibility of wheeze or eczema during infancy. This early diagnosis at birth will be useful for early preventive or management measures to control the disease progression in early childhood.

[Oil]. Accordingly an aspect of the invention includes a method of diagnosing risk of an individual to suffer from an inflammatory disorder, the method comprising:

a) measuring in a sample from an individual a level of Interleukin 10 (IL-10) and a level of at least two substances representative for the quantity of respective marker proteins, wherein the marker proteins are selected from the group consisting of cluster of differentiation 14 (CD 14) and tumor necrosis factor receptor type II (TNF-RII) or isoforms or fragments of TNF-RII and CD 14; tissue inhibitor of matrix metalloproteinase-1 (TIMP1) and TNF-RII or isoforms or fragments of TIMP1 and TNF-RII; and CD 14, TIMP 1 and TNF-RII or isoforms or fragments of CD 14, TIMP1 and TNF-RII; and

b) comparing the measured level of IL-10 with a level measured in a control sample; c) comparing the measured level of the at least two substances with a level measured in a control sample;

wherein the individual is diagnosed with the risk to suffer from an inflammatory disorder, if the measured level of IL-10 is lower than the level measured in the control sample and the level of the at least two substances are higher than the level measured in the control sample.

[012]. In one embodiment the sample from the individual is derived from one selected from the group consisting of blood, body fluid, whole blood, blood serum, pus, extracellular fluid and cord blood mononuclear cells (CBMCs).

[013]. In another embodiment the sample is serum free.

[014]. Preferably the substance representative for the quantity of the respective marker protein is the protein itself or a nucleic acid molecule encoding for the protein.

[015]. In one embodiment the nucleic acid is RNA comprising, mRNA or microRNA or siRNA. [016]. The control sample is from an individual not suffering from an inflammatory disorder or not having the risk to suffer from an inflammatory disorder.

[017]. Preferably, the individual is of an age of 0 days, or less than 100 years, or less than 90 years, or less than 80 years, or less than 70 years, or less than 60 years, or less than 50 years, or less than 40 years, or less than 30 years, or 20 years, or 15 years, or 10 years, or 9 years, or 8 years, or 7 years, or 6 years, or 5 years, or 4 years, or 3 years.

[018]. Preferably, the individual is a new-born baby or an infant, or a toddler (1 to 3 years), or a teenager (10 to 19 years) or an adult (more than 20 years).

[019]. In one embodiment the inflammatory disorder is inflammatory skin disease. Such as dermatitis, or eczema selected from the group consisting of atopic eczema, contact dermatitis, xerotic eczema, seborrhoeic dermatitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis, neurodermatitis, autoeczematisation, nummular dermatitis, statis dermatitis, perioral dermatitis and combinations thereof.

[020]. In another embodiment the dermatitis/eczema is selected from the group consisting of dryness of skin, crusting, flaking, blistering, cracking, oozing, bleeding, recurring skin rashes, skin redness, skin edema (swelling) and combinations thereof.

[021]. Preferably, the marker proteins further comprise any of the proteins selected from the group consisting of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-a (TNF-O!), growth regulated oncogene-a (GRO-o;), urokinase-type plasminogen activator receptor (u-PAR), basic fibroblast growth factor (bFGF), epithelial neutrophil-activating protein-78 (ENA-78), Agouti-related peptide (AgRP), Sialic acid-binding Ig-like lectin 5 (Siglec-5), platelet-derived growth factor AA (PDGF-AA), Activin A, tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie-1), matrix metalloproteinase-1 (MMP-1), or isoforms or fragments thereof. [022]. In one embodiment the marker protein CD 14 is a soluble form of CD 14.

[023]. In another embodiment the marker protein TNF-RII is a soluble form of TNF-RII.

[024]. Preferably, the level of at least two substances representative for the quantity of respective marker proteins selected from the group consisting of CD 14, TIMP1, TNF-RII, isoforms and fragments is increased by at least 1 fold, or at least 2 folds, or at least 3 folds, or at least 4 folds, or at least 5 folds, or at least 6 folds, or at least 7 folds or at least 8 folds or at least 9 folds or at least 10 folds or between about 1 to about 10 folds, as compared to the control group.

[025]. Preferably, the level of at least two substances representative for the quantity of respective marker proteins selected from the group consisting of CD 14, TIMP1, TNF-RII, isoforms and fragments is increased by 1 fold, or 1.5 folds, or 2 folds, or 2.5 folds, or 3 folds, or 3.5 folds, or 4 folds, or 4.5 folds, or 5 folds, or 5.5 folds, or 6 folds, or 6.5 folds, or 7 folds, or 7.5 folds, or 8 folds, or 8.5 folds, or 9 folds, or 9.5 folds, or 10 folds, as compared to the control group.

[026]. In one embodiment the method further comprises an increased level of the substance representative for the quantity of IL-6 or an isoform or fragment thereof, compared to the control group.

[027]. Preferably, a decreased level of the substances representative for the quantity of respective marker proteins selected from the group consisting of IL-8, TNF-α, isoforms and fragments thereof, compared to the control sample is indicative of the risk of an individual to suffer from an inflammatory skin disease.

[028]. In one embodiment the inflammatory disorder is a respiratory tract-related disorder such as asthma.

[029]. Preferably, the method may further comprise the steps of: a) measuring in the sample from the individual a level of a further substance selected from the group consisting of interleukin 1-/3 (IL-1/3) and interleukin 23 (IL-23); or isoforms or fragments of IL-1/3 and IL-23; interleukin-6 (IL-6) and IL-23 or isoforms or fragments of IL-6 and IL-23; and IL-1/3, IL-23, IL-6 or isoforms or fragments of IL-1/3, IL-23 and IL-6; and

b) comparing the measured level of the further substance with a level measured in a control sample;

wherein the individual is diagnosed with the risk to suffer from a respiratory tract-related disorder, if the measured level is increased from the level measured in the control sample.

[030]. Preferably, the fragment of IL-23 is IL-23p40 or IL23pl9 or IL-23p40 homodimer.

[031]. Preferably, the marker proteins may further comprise any of the proteins selected from the group consisting of, IL-8, IL-2, interferon-γ (IFN-γ), IL-5, tumor necrosis factor-a (TNF-a), CC chemokine 1-309, IL-16, macrophage derived chemokine (MDC), platelet derived growth factor-BB (PDGF-BB), FMS-like tyrosine kinase 3 ligand (FU-3L), IL-la, insulin-like growth factor-binding protein 6 (IGFBP-6), macrophage migration inhibitory factor (MIF), or isoforms or fragments thereof.

[032]. In one embodiment an increased level of at least two substances representative for the quantity of respective marker proteins selected from the group consisting of IL-1/3, IL-23, IL- 6, isoforms and fragments thereof compared to the control sample, is indicative of the risk of an individual to suffer from a respiratory tract-related disorder.

[033]. In another embodiment an increased level of the substances representative for the quantity of respective marker proteins selected from the group consisting of IL-8, IL-2, IFN- γ, IL-5, TNF-a, isoforms and fragments thereof compared to the control sample, is indicative of the risk of an individual to suffer from a respiratory tract-related disorder.

[034]. The respiratory tract-related disorder may be caused by a viral infection. [035]. The respiratory tract-related disorder may be a wheezing disorder, or asthma or bronchiolitis or combinations thereof.

[036]. Another aspect of the invention includes a method of identifying a compound which can modulate the inflammatory response caused by an inflammatory disorder in an individual, the method comprising

a) measuring in a sample obtained from the individual a level of at least two substances representative for the quantity of respective marker proteins, wherein the marker proteins are selected from the group consisting of Interleukin 10 (IL-10), CD14 and TNF-RII or isoforms or fragments of CD 14 and TNF-RII; TIMP1 and TNF-RII or isoforms or fragments of TIMP1 and TNF-RII; and CD 14, TIMP1 and TNF-RII or isoforms or fragments of CD 14, TIMP1 and TNF-RII; wherein the individual is suffering from an inflammatory skin disease or has a risk to suffer from an inflammatory skin disease and has been exposed with the compound prior to obtaining the sample from the individual; and

b) comparing the measured marker level of the at least two substances with a marker level measured in a control sample,

wherein the compound can inhibit the inflammatory response caused by the inflammatory disorder if the measured marker level deviates from a marker level of the individual prior to being exposed with the compound or the measured marker level is substantially the same as the marker level measured in the control sample.

[037]. The measured marker level of the individual exposed with the compound may be lower than the marker level of the individual prior to being exposed with the compound.

[038]. Preferably, the method may be carried out in an array such as a protein array or a gene array.

[039]. The protein array may be a multiplex-beads-based assay, or a plate-based ELISA, or a protein array on biochips. [040]. The gene array may be a mRNA microarray, or a microRNA array, or a methylated DNA microarray or the gene array is used in a quantitative PCR method.

[041]. In one embodiment prior to measuring in the sample from the individual the sample is exposed to a stimulator substance such as an adjuvant, lipopolysaccharide (LPS) or phytohaemagglutinin (PHA).

[042]. Preferably, the method may further comprise the steps of:

a) measuring in the sample obtained from the individual a level of a further substance selected from the group consisting of IL-1/3 and IL-23 or isoforms or fragments of IL- 1/3 and IL-23; IL-6 and IL-23 or isoforms or fragments of IL-6 and IL-23; and IL-1/3, IL-23, IL-6 or isoforms or fragments of IL-1/3, IL-23 and IL-6; and wherein the individual is suffering from a respiratory tract-related disorder or has a risk to suffer from a respiratory tract-related disorder and wherein the individual has been exposed with the compound prior to obtaining the sample from the individual; and

b) comparing the measured marker level of the further substance with a marker level measured in a control sample, wherein the compound can modulate the inflammatory response caused by a respiratory tract-related disorder if the measured marker level deviates from a marker level of the individual prior to being exposed with the compound or the measured marker level is substantially the same as the marker level measured in the control sample.

[043]. Another aspect of the invention includes a protein array kit for carrying out the methods of any one of claims 1 to 38, the protein array kit comprising

a) an array support;

b) a first detection probe that is adapted to be immobilized on the array support; and c) a second detection probe that is bound to a detectable marker;

wherein the first and second detection probes are selected from the group consisting of Interleukin 10 (IL-10), CD14 and TNF-RII or isoforms or fragments of CD14 and TNF-RII; TIMPl and TNF-RII or isoforms or fragments of TIMPl and TNF-RII; and CD 14, TIMPl, and TNF-RII or isoforms or fragments of CD 14, TIMPl, and TNF-RII. [044]. In one embodiment the protein array kit is used in a sandwich ELISA.

[045]. Preferably, the protein array kit may further comprise a third detection probe that is adapted to be immobilized on the array support; and a forth detection probe that is bound to a second detectable marker; wherein the third and forth detection probes are adapted to bind to a substance selected from the group consisting of IL-1|8 and IL-23 or isoforms or fragments of IL-Ιβ and IL-23; IL-6 and IL-23 or isoforms or fragments of IL-6 and IL-23; and IL-1/3, IL-23, IL-6 or isoforms or fragments of IL-1/8, IL-23 and IL-6.

BREIF DESCRIPTION OF THE DRAWINGS

[046]. Preferred embodiments of the invention will be described with reference to the following drawings of which:

[047]. Figure 1: IL-8 production profile from CBMCs of healthy (n=65), eczema (n=29) and wheeze (n=34) subjects. (A) CBMCs were stimulated with 1 ug/mL of LPS. (B) CBMCs were stimulated with 5 ug/mL PHA.

[048]. Figure 2: IL-6 production profile from CBMCs of healthy (n=65), eczema (n=29) and wheeze (n=34) subjects in response to 1 ug/mL LPS.

[049]. Figure 3: CBMCs derived cytokine profiles. (A) to (E) Cord blood T-cell cytokine profiles in healthy (n=65) and wheeze (n=34) subjects in response to PHA. (F) CBMCs IL-23 p40 cytokine profile in response to 1 ug/mL of LPS in the wheeze (n=34) and healthy subjects (n=65).

[050]. Figure 4: IL-10 production profile from CBMCs of wheeze (n=34) and healthy subjects (n=65). (A) IL-10 production from CBMCs stimulated with 1 ug/mL of LPS (B) IL- 10 production from CBMCs in response to 5 ug/mL of PHA stimulation.

[051]. Figure 5. Results from a cytokine antibody array have identified soluble mediators in LPS stimulated culture supernatant associated with eczema, the soluble mediators CD14, sTNFRII and TIMP-1 are expressed at higher levels in the CBMCs of eczema subjects compared to the healthy controls and wheeze subjects. What is interesting about these soluble mediators is that Scdl4 and STFRII have been reported to be downregulators of IL-8, which might explain the attenuated IL-8 cytokine profile we saw in the eczema subjects. [052]. Figure 6. Flow chart of study profile.

[053]. Figure 7. A- E, Differential CBMCs cytokine profile of healthy (n = 65), eczema (n = 29) and wheeze (n = 34) subjects in response to 1 μ§/πιί of LPS. Closed circles ( # ) represents non-allergen sensitized subjects, while the open circles (O ) represents allergen sensitized subjects. The band in the middle of the boxplot represents the median value, and the ends of the whiskers represent the minimum and maximum value of the data.

[054]. Figure 8. A-H, CBMCs cytokine profile in of healthy (n = 65), eczema (n = 29) and wheeze (n = 34) in response to 5 μg/mL of PHA. The closed circles ( · ) represents non- allergen sensitized subjects, while the open circles ( O ) represent the allergen sensitized subjects. The band in the middle of the boxplot represents the median value, and the ends of the whiskers represent the minimum and maximum value of the data.

[055]. Figure 9. 3D Scatter plot of the healthy control and in wheeze group in respect to their correlation to the variables from the factor analysis. Factor analysis of LPS stimulated CBMCs showed that infant wheeze has higher combined responses from cytokines IL-6, IL- 23p40 and IL-1 3 compared to healthy controls. Figure was drawn with Sigma Plot (Systat Software, Inc., Chicago, IL).

[056]. Figure 10. Intracellular cytokine staining of LPS stimulated CBMCs show CD 14+ cell is main IL-6 producer.

[057]. Figure 11. Intracellular cytokine staining of LPS stimulated CBMCs show CD14+ cell is main IL-8 producer.

[058]. Figure 12. LPS stimulated IL-23p40 increase in wheeze subjects. Significant increase in IL-23p40 in the wheeze compared to healthy control subjects. New IL-12 member composed of a pi 9 subunit specific for IL-23 and a p40 subunit shared with IL-12.

[059]. Figure 13. Screen for 180 soluble mediators in LPS stimulated CBMC supernatant with a Qualitative assay by comparing the signal intensities, relative expression levels of cytokines can be made

DETAILED DESCRIPTION

[060]. Similar approaches have been tried to look for potential biomarkers correlated with childhood wheeze or eczema. Most attempts were not successful so far because they only analysed one biomarker/mediator at a time. Hence the correlation of biomarkers with disease phenotypes was not good enough. The advantage of our current method by combined factor analysis is the stronger predictive power than the single factor analysis method. Our study has identified a group of mediators, namely IL-Ι β, IL-6 and IL23p40, as the predictive biomarkers for the susceptibility of the wheeze phenotype. In the similar experimental setup, another group of mediators, namely soluble CD 14, soluble TNFRII and TIMP-1, were up- regulated as the predictive biomarkers for the eczema phenotype and the wheeze phenotype.

[061]. The technology allows the early diagnosis of common childhood diseases wheeze and eczema. The assay can be done immediately afterbirth with cord blood mononuclear cells. The readout of these predictive mediators can be done with a mini-protein array in the future. The results generated can be used for preventive managements of these diseases in high risk babies during the first few years of life to reduce the overall medical cost spent in the healthcare system.

[062]. Neonates with a family history of atopy are at higher risk for developing wheezing in early life. Certain inherent immunological factors have been shown to predispose to wheezing and atopic disorders. Although wheezing and eczema are clinically related, there are distinct pre-symptomatic innate risk factors which predispose infants to develop early life wheeze disorders. There are intrinsic pre-existent differences in the innate immune responses of infants with wheeze that point towards a possible role of TH17 in the pathogenesis of early wheeze.

[063]. Other than cytokines affecting the balance of immune responses at birth and possibly driving the development of childhood diseases, soluble mediators from cells at birth also can be analyzed in association with the development of childhood diseases. Cord blood mononuclear cells, (CBMCs) are cells isolated from umbilical cord blood at birth.

[064]. In order to determine whether distinct differential innate immune responses, including IL-23, preexists in infants with wheezing disorders, this study set out to evaluate cord blood mononuclear cell (CBMC) immune responses in a clinically well defined birth cohort of at risk infants. Subjects were grouped according to their clinic outcomes at 2 years of age: wheeze, eczema (disease-related control), and healthy controls. Cytokine, chemokine and soluble mediator responses of CBMC to both the Toll- like- receptor 4 (TLR4) agonist lipopolysaccharide (LPS) and T cell mitogen phytohaemagglutinin (PHA) were assessed to target both the innate antigen presenting cells and naive T cells, respectively.

[065]. Taken together, the data from our study support a new notion that an intrinsic hyper responsive LPS-induced innate cytokine profile involving up-regulation of combined responses of IL-1/3, IL-6 and IL-23p40, is characteristic of the wheezing but not the eczema phenotype. These findings have implications on the IL-23/IL-17 axis and therefore the probable role of TH17 immune deviation in early life wheeze disorders.

[066]. Accordingly a method of diagnosing risk of an individual to suffer from an inflammatory disorder, is described where measuring in a sample from an individual a level of Interleukin 10 (IL-10) and a level of at least two substances representative for the quantity of respective marker proteins, wherein the marker proteins are selected from the group consisting of cluster of differentiation 14 (CD 14) and tumor necrosis factor receptor type II (TNF-RII) or isoforms or fragments of TNF-RII and CD 14; tissue inhibitor of matrix metalloproteinase-1 (TIMP1) and TNF-RII or isoforms or fragments of TIMP1 and TNF-RII; and CD 14, TIMP1 and TNF-RII or isoforms or fragments of CD 14, TIMP1 and TNF-RII; and comparing the measured level of IL-10 with a level measured in a control sample; comparing the measured level of the at least two substances with a level measured in a control sample; wherein the individual is diagnosed with the risk to suffer from an inflammatory disorder, if the measured level of IL-10 is lower than the level measured in the control sample and the level of the at least two substances are higher than the level measured in the control sample.

[067]. In a similar manner a method of diagnosing risk of an individual to suffer from a respiratory tract-related disorder, is described where measuring in a sample from an individual a level of at least two substances representative for the quantity of respective marker proteins, wherein the marker proteins is selected from the group consisting of interleukin 1-/3 (IL-1 3) and interleukin 23 (IL-23); or isoforms or fragments of IL-1/3 and IL- 23; interleukin-6 (IL-6) and IL-23 or isoforms or fragments of IL-6 and IL-23; and IL-1/3, IL- 23, IL-6 or isoforms or fragments of IL-1/3, IL-23 and IL-6; and comparing the measured level of the at least two substances with a level measured in a control sample; wherein the individual is diagnosed with the risk to suffer from a respiratory tract-related disorder, if the measured level deviates from the level measured in the control sample. [068]. The measurement of at least two substances representative for the quantity of respective marker proteins, wherein the marker proteins is selected from the group consisting of interleukin 1-/3 (IL-1/3) and interleukin 23 (IL-23); or isoforms or fragments of IL-l S and IL-23; interleukin-6 (IL-6) and IL-23 or isoforms or fragments of IL-6 and IL-23; and IL-1/3, IL-23, IL-6 or isoforms or fragments of IL-1/3, IL-23 and IL-6; can also be used in a method of identifying a compound which can modulate the inflammatory response caused by an inflammatory disorder such as a respiratory tract-related disorder in an individual. Protein array kits are preferably used for carrying out the methods.

Inflammatory disorder

[069]. Abnormalities associated with inflammation comprise a large, officially unrelated group of disorders which underlie a vast variety of human diseases. The immune system is often involved with inflammatory disorders, demonstrated in allergic reactions resulting in abnormal inflammation. A large variety of proteins are involved in inflammation, and any one of them is open to a genetic mutation which impairs or otherwise dysregulates the normal function and expression of that protein. Examples of disorders associated with inflammation include: Asthma;

[070]. Allergy is a hypersensitivity disorder of the immune system whereby normally harmless environmental substances known as allergens cause acquired reactions that are predictable, and rapid. Strictly, allergy is one of four forms of hypersensitivity. It is characterized by excessive activation of certain white blood cells and IgE antibody resulting in an extreme inflamatory response. Common allergic reactions include eczema, hives, hay fever, asthma, food allergies and reaction to stinging insect venom.

[071]. In one embodiment the inflammatory disorder is an inflammatory skin disease such as dermatitis, or eczema. Dermatitis/eczema may be selected from the group consisting of atopic eczema, contact dermatitis, xerotic eczema, seborrhoeic dermatitis, dyshidrosis, discoid eczema, venous eczema, dermatitis ηεφβίϊίοπηΪΒ, neurodermatitis, autoeczematisation, nummular dermatitis, statis dermatitis, perioral dermatitis and combinations thereof. Symptoms may include dryness of skin, crusting, flaking, blistering, cracking, oozing, bleeding, recurring skin rashes, skin redness, skin edema (swelling) and combinations thereof.

[072]. In another embodiment the inflammatory disorder is a respiratory tract-related disorder such as asthma. The respiratory tract-related disorder may be caused by a viral infection. The respiratory tract-related disorder may be a wheezing disorder, or asthma or bronchiolitis or combinations thereof. [073]. Samples can be taken from blood, body fluid, whole blood, blood serum, pus, extracellular fluid and cord blood mononuclear cells (CBMCs). Preferably samples are taken from CBMCs and are examined serum free.

[074]. Our study is one of the few that is using the AIM-V serum free condition to study cytokine expression profile in response to LPS. Previous studies have employed the method of IFN-γ priming cord blood mononuclear cells before stimulating the cells with LPS in RPMI + 10% FCS media (complete RPMI) ^{35, 52} . Pre-priming cells with IFN-γ with complete RPMI as a cell culture media has been known to induce high amounts of cytokine production at low doses of LPS but might be an artificial upregulation of cytokines ⁵² . Pre-priming of CBMC also enables the activation of dendritic cells and the induction of cytokine IL-12 which is not inducible in a serum-free culture condition ⁵² . To investigate if LPS was capable of inducing IL-12p70 cytokine production in a serum- free condition, we have measured LPS induced IL-12p70 but cytokine levels were below detection limits of the assay. A study has shown that IL-12p70 production is indeed defective in neonatal monocyte-derived dendritic cells and failed to be induced by LPS stimuli in a serum-free condition ²³ . The serum free method using AIM-V as the cell culture media helps reduce artificial effects of IFN-γ priming and that of the fetal calf proteins in the serum.

[075]. In one embodiment the substance representative for the quantity of the respective marker protein is the protein itself or a nucleic acid molecule encoding for the protein. The nucleic acid may be RNA such as mRNA or microRNA or siRNA.

[076]. Preferably the control sample is taken from an individual not suffering from an inflammatory disorder or not having the risk to suffer from an inflammatory disorder. Preferably the individual from which the sample or the control sample are taken, are of the same age of 0 days (at birth), or less than 100 years, or less than 90 years, or less than 80 years, or less than 70 years, or less than 60 years, or less than 50 years, or less than 40 years, or less than 30 years, or 20 years, or 15 years, or 10 years, or 9 years, or 8 years, or 7 years, or 6 years, or 5 years, or 4 years, or 3 years. Preferably the individual is a new-born baby or an infant, or a toddler (1 to 3 years), but the individual may also be or a teenager (10 to 19 years) or an adult (more than 20 years).

[077]. Where the method of diagnosing risk of an individual to suffer from an inflammatory disorder is an inflammatory skin disease the marker proteins may further comprise any of the proteins selected from the group consisting of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-a (TNF-a), growth regulated oncogene-a (GRO-a), urokinase-type plasminogen activator receptor (u-PAR), basic fibroblast growth factor (bFGF), epithelial neutrophil-activating protein-78 (ENA-78), Agouti-related peptide (AgRP), Sialic acid- binding Ig-like lectin 5 (Siglec-5), platelet-derived growth factor AA (PDGF-AA), Activin A, tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie-1), matrix metalloproteinase-1 (MMP-1), or isoforms or fragments thereof.

[078]. Preferably the marker protein CD 14 is a soluble form of CD 14 or the marker protein TNF-RII is a soluble form of T F-RII.

[079]. Where the method of diagnosing risk of an individual to suffer from an inflammatory disorder is an inflammatory skin disease preferably the level of at least two substances selected from the group consisting of CD 14, TIMP1, TNF-RII, isoforms and fragments is increased by at least 1 fold, or at least 2 folds, or at least 3 folds, or at least 4 folds, or at least 5 folds, or at least 6 folds, or at least 7 folds or at least 8 folds or at least 9 folds or at least 10 folds or between about 1 to about 10 folds, as compared to the control group.

[080]. The level of at least two substances selected from the group consisting of CD 14, TIMP1, TNF-RII, isoforms and fragments is increased by at 1 fold, or 1.5 folds, or 2 folds, or 2.5 folds, or 3 folds, or 3.5 folds, or 4 folds, or 4.5 folds, or 5 folds, or 5.5 folds, or 6 folds, or 6.5 folds, or 7 folds, or 7.5 folds, or 8 folds, or 8.5 folds, or 9 folds, or 9.5 folds, or 10 folds, as compared to the control group.

[081]. Where the method of diagnosing risk of an individual to suffer from an inflammatory disorder is an inflammatory skin disease preferably the level of the substance representative for the quantity of IL-6 or an isoform or fragment thereof, is also increased compared to the control group.

[082]. Where the method of diagnosing risk of an individual to suffer from an inflammatory disorder is an inflammatory skin disease preferably the substances representative for the quantity of respective marker proteins selected from the group consisting of IL-8, TNF-a, isoforms and fragments thereof, being decreased compared to the control sample is indicative of the risk of an individual to suffer from an inflammatory skin disease.

[083]. Where the method of diagnosing risk of an individual to suffer from an inflammatory disorder is a respiratory tract-related disorder the sample from the individual is measure for a level of a further substance selected from the group consisting of interleukin 1-/3 (IL-1/3) and interleukin 23 (IL-23); or isoforms or fragments of IL-1/3 and IL-23; interleukin-6 (IL-6) and IL-23 or isoforms or fragments of IL-6 and IL-23; and IL-1/3, IL-23, IL-6 or isoforms or fragments of IL-1/3, IL-23 and IL-6; and compared to the measured level of the further substance with a level measured in a control sample; wherein the individual is diagnosed with the risk to suffer from a respiratory tract-related disorder, if the measured level is increased from the level measured in the control sample. Preferably, the fragment of IL-23 is IL-23p40 or IL23pl9.

[084]. Where the method of diagnosing risk of an individual to suffer from an inflammatory disorder is a respiratory tract-related disorder the marker proteins may further comprise any of the proteins selected from the group consisting of, IL-8, IL-2, interferon-γ (IFN-γ), IL-5, tumor necrosis factor-a (TNF-a), CC chemokine 1-309, IL-16, macrophage derived chemokine (MDC), platelet derived growth factor-BB (PDGF-BB), FMS-like tyrosine kinase 3 ligand (Flt-3L), IL-la, insulin-like growth factor-binding protein 6 (IGFBP-6), macrophage migration inhibitory factor (MIF), or isoforms or fragments thereof. Preferably, the level of the substances representative for the quantity of respective marker proteins selected from the group consisting of IL-1/3, IL-23, IL-6, isoforms and fragments thereof is increased compared to the control sample. An increase being indicative of the risk of an individual to suffer from a respiratory tract-related disorder. An increased level of the substances representative for the quantity of respective marker proteins selected from the group consisting of IL-8, IL-2, IFN- γ, IL-5, TNF-a, isoforms and fragments thereof compared to the control sample, may be further indicative of the risk of an individual to suffer from a respiratory tract-related disorder.

[085]. In another embodiment a method of identifying a compound which can modulate the inflammatory response caused by an inflammatory disorder in an individual is described comprising measuring in a sample obtained from the individual a level of at least two substances representative for the quantity of respective marker proteins, wherein the marker proteins are selected from the group consisting of Interleukin 10 (IL-10), CD 14 and TNF-RII or isoforms or fragments of CD 14 and TNF-RII; TIMP1 and TNF-RII or isoforms or fragments of TIMP1 and TNF-RII; and CD 14, TIMP1 and TNF-RII or isoforms or fragments of CD 14, TIMP1 and TNF-RII; wherein the individual is suffering from an inflammatory skin disease or has a risk to suffer from an inflammatory skin disease and has been exposed with the compound prior to obtaining the sample from the individual; and comparing the measured marker level of the at least two substances with a marker level measured in a control sample, wherein the compound can inhibit the inflammatory response caused by the inflammatory disorder if the measured marker level deviates from a marker level of the individual prior to being exposed with the compound or the measured marker level is substantially the same as the marker level measured in the control sample. Preferably, the measured marker level of the individual exposed with the compound is lower than the marker level of the individual prior to being exposed with the compound.

[086]. Preferably, the methods of the invention are carried out in an array. The array may be a protein array or a gene array. Where the array is a protein array the protein array may be a multiplex-beads-based assay, or a plate-based ELISA, or a protein array on biochips. Where the array is a gene array the gene array may be an mRNA microarray, or a microR A array, or a methylated DNA microarray or the gene array is used in a quantitative PCR method. An example of an array system suitable for carrying out the invention is depicted in Figure 13. Other array systems are known to those skilled in the art.

[087]. Preferably, the methods of the invention, prior to measuring in the sample from the individual, include the step of exposing the sample to a stimulator substance. Preferably, the stimulator substance is an adjuvant. Preferably, the stimulator substance is lipopolysaccharide (LPS) or phytohaemagglutinin (PHA).

[088]. Where the method of identifying a compound which can modulate the inflammatory response caused by an inflammatory disorder is to modulate a respiratory tract-related disorder the method may further comprising the steps of: measuring in the sample obtained from the individual a level of a further substance selected from the group consisting of IL-1/3 and IL-23 or isoforms or fragments of IL-1/3 and IL-23; IL-6 and IL-23 or isoforms or fragments of IL-6 and IL-23; and IL-1 3, IL-23, IL-6 or isoforms or fragments of IL-1/3, IL-23 and IL-6; and wherein the individual is suffering from a respiratory tract-related disorder or has a risk to suffer from a respiratory tract-related disorder and wherein the individual has been exposed with the compound prior to obtaining the sample from the individual; and comparing the measured marker level of the further substance with a marker level measured in a control sample, wherein the compound can modulate the inflammatory response caused by a respiratory tract-related disorder if the measured marker level deviates from a marker level of the individual prior to being exposed with the compound or the measured marker level is substantially the same as the marker level measured in the control sample. [089]. Another embodiment comprises a protein array kit for carrying out the methods of the invention, the protein array kit comprising

i) an array support;

ii) a first detection probe that is adapted to be immobilized on the array support; and iii) a second detection probe that is bound to a detectable marker;

wherein the first and second detection probes are selected from the group consisting of Interleukin 10 (IL-10), CD14 and TNF-RII or isoforms or fragments of CD14 and TNF-RII; TIMP1 and TNF-RII or isoforms or fragments of TIMP1 and TNF-RII; and CD 14, TIMP1, and TNF-RII or isoforms or fragments of CD 14, TIMP1, and TNF-RII. The protein array kit may be used in a sandwich ELISA or in other methods known in the art.

[090]. The protein array kit may further comprise a third detection probe that is adapted to be immobilized on the array support; and a forth detection probe that is bound to a second detectable marker; wherein the third and forth detection probes are adapted to bind to a substance selected from the group consisting of IL-1/3 and IL-23 or isoforms or fragments of IL-1/3 and IL-23; IL-6 and IL-23 or isoforms or fragments of IL-6 and IL-23; and IL-1/3, IL- 23, IL-6 or isoforms or fragments of IL-1/3, IL-23 and IL-6.

Detailed description of preferred embodiments and examples

[091]. From a birth cohort of at risk infants (first degree family with atopic disease), this study aimed to evaluate the influence of distinct intrinsic immunologic risk factors on wheezing disorders in the first 2 years of life.

[092]. The study involved 128 eligible subjects of a birth cohort of 253 at risk subjects whose cord blood samples were collected for evaluation. The subjects studied were those who developed wheezing (n = 34) and eczema (n = 29) in the first 2 years of life, and 65 healthy control infants. Cord blood mononuclear cells (CBMCs) were stimulated with either lipopolysaccharide (LPS) or phytohemagglutinin (PHA). Cytokine responses were analyzed from culture supernatants using fluorescent activated cell sorting (FACS)-array and their profiles were evaluated using factor analysis.

[093]. Infants with wheeze had higher combined LPS stimulated production of IL-13, IL-6 and IL-23 compared to the healthy controls and eczema (p = 0.003). Logistic regression analysis, which included potential clinical confounding factors, showed that these combined cytokine responses was an independent risk factor for wheeze (OR,2.45; 95% 1.50-3.93, /» = 0.001). Additionally PHA stimulated CBMCs produced higher levels of pro-inflammatory cytokines (IL-6, IL-8, IFN-γ, IL-2, IL-5 and TNF-a) in the wheeze group compared to healthy controls (IL-6; p = 0.015 and for all other cytokines; p = 0.003) but attenuated (except IFN-γ) in eczema compared to wheeze (IL-6, IL-8 and IL-2; p = 0.003, IL-5 ; p = 0.006, TNF-a; p = 0.0021 and IL-13; p = 0.036).

[094]. In infants at genetic risk of atopy, wheeze but not eczema in the first 2 years of life is associated with intrinsic hyper-responsive innate cytokine responses including a LPS-induced combined profile involving IL-1/3, IL-6 and IL-23. These findings have implications on the IL-23/IL-17 axis and support the probablity of TH17 immune deviation in early life wheeze disorders.

[095]. Association of differential immunophenotypes with the development of eczema or wheeze at 24 months of age was examined.

a) To determine cytokine/chemokine / soluble mediators secretion profiles from

lipopolysaccharide (LPS) or phytohemagglutinin (PHA) stimulated CBMCs and

PBMC at I year.

b) To identify cell type(s) in CBMCs involved in the differential expression of

cytokine\chemokine\ soluble mediators.

c) To elucidate the molecular mechanisms associated with the differential

immunophenotypes by gene microarray approach

METHODS

Preparation of cord blood mononuclear cells

[096]. Umbilical cord blood was collected by withdrawing blood from an umbilical vein after delivery and processed within 24 hours. Cord blood mononuclear cells (CBMCs) were isolated from heparinized cord blood by Ficoll-Hypaque gradient centrifugation. Cells in the interphase were harvested, washed with RPMI with 10% fetal calf serum and used immediately or cryopreserved in liquid nitrogen for further use.

Stimulation of cord blood cells with lipopolysaccharide (LPS) and phytohaemagglutinin ( PHA)

[097]. Our study is one of the few that is using a serum free condition to study cytokine expression profile in response to LPS. Previous studies have employed the method of IFN-γ priming cord blood mononuclear cells before stimulating the cells with LPS (5). CBMCs were seeded at 5 ^χ 10 ⁵ cells/well of a 96 well plate in 200 μΐ AIM-V medium. Cells were either unstimulated (control), stimulated with 5 ug/mL of PHA (Sigma) for 48 hours or stimulated with LPS (Escherichia coli LPS 0111 :B4, Sigma) at concentrations 10, 100 or 1000 ng/ml for 24 hours. All cultures were plated in duplicate in 96-well, round-bottom plates (Nunc) and incubated in a 5% C0 ₂ atmosphere incubator. After culture, the supernatants were acquired and stored at -80°C until required for cytokine analysis by means of FACS-Array. The choice of time points was based on initial experiments that have demonstrated the optimal response.

Cytokine protein detection by FACS-Array

[098]. Detection of cytokine production was performed using the fluorescence activated cell sorter (FACS)-Array (BD Bioscience), which is a new flow cytometry platform for fast and sensitive analysis of cytokine proteins by multiplexed bead assays. The advantage is the usage of small sample volumes; fewer sample dilutions and values are obtained in substantially less time as compared to conventional ELISA. Cytokines measured were IL-10, IL-lft IL-6, IL-8, IL-12p70, TNF-a, IFN-γ, IL-2, IL-5, IL-23p40 and IL-13. The detection limit was 10 pg/mL for all cytokines.

Cytokine/chemokine/soluble mediator detection by protein array

[099]. Multiple protein expression levels released into the supernatant of the cell culture media were detected using the human cytokine antibody microarray (BioRad G series 2000). This antibody array matrix can detect 174 cytokines, chemokines and soluble factors. The microarray glass chip slides were blocked with blocking buffer before cell culture supernatants from the LPS stimulated CBMCs were incubated on the slides. After incubation, the glass chips were washed before the addition of biotin-conjugated antibodies followed by fluorescent dye-conjugated strepavidin. Glass chips were then washed and air dried before scanning with the laser Axon GenePix scanner.

Statistical analysis

[0100]. Statistical analysis was performed using SPSS software (version 16.0 for windows). The data was not normally distributed so the Mann- Whitney U test was performed to asses the differences between in the cytokine profiles based on absence or presence of eczema and wheezing. The cytokine data was then adjusted with possible predictors using the binary logistic regression. Significant predictor's variables were selected for eczema and wheeze outcomes. This test was also used to determine the odds ratio (OR) and the associated 95% confidence interval (CI). A p-value of less than 0.05 was considered statistically significant for all analyses.

[0101]. To determine predictors of cytokines that are associated with wheeze and eczema, factor analysis was used. Cytokines in the physiological setting do not act alone but in concert with other cytokines in a network of groups. The factor analysis is a data reduction technique which takes a large set of variables (cytokines) and groups together inter-correlated sets of variables (cytokines), this summary of data are known as factors. Factors with an eigenvalue greater than 1 were formed using only the healthy control subjects to describe cytokine profile of the healthy children. Factor-specific loading was calculated for each original variable which indicates the degree of correlation between the specific factor formed and the variable. To perform additional analysis by using factors as variables, factor scores were generated by the statistical software SPSS. Factor scores are a linear combination of all the variables weighted by the corresponding factor loadings. These factor scores were then compared between groups by Mann-Whitney U test. Logistic regression was used to determine factor scores as possible predictors of wheeze and eczema while adjusting for confounding factors (9).

Results

[0102]. The main finding of the correlation between the development of wheeze and eczema at 1 year old with the mediators secreted from CBMCs stimulated with LPS is detailed in the examples. The combined factor analysis showed that IL-Ιβ, IL-6 and IL23p40 secreted from the LPS stimulated CBMCs of wheeze subjects was significantly higher than that secreted from eczema and health subjects (p value = 0.001) (Table 1).

Factor 1 Factor 2

Ι1-1β (0.776) TNF-a (0.701)

Cytokine

IL-6 (0.644) IL-10 (0.642)

IL-23p40 (0.691) IL-8 (0.769)

Total variance explained (%) 33.5% 26.5%

Factor 1 p value Factor 2 p value

Comparison between groups

(Higher score group) (Higher score group)

0.181 0.001

Healthy vs eczema

(Healthy) 0.001 0.546

Healthy vs wheeze

(Wheeze)

0.001 0.001

Eczema vs wheeze

(Wheeze) (Wheeze)

• Combine responses from IL-Ιβ, IL-6 and IL-23p40 responses had higher factor scores (higher production) in the wheeze group as compared to the healthy group.

• Combine responses from TNF-a, IL-10 and IL-8 had higher factor scores (higher production) in the healthy group as compared to the eczema group.

Table 1: Summary of factor analysis including factor loadings from a model of cytokine responses in the LPS stimulated CBMCs of healthy control subjects. Comparisons of factor scores between groups were compared using Mann-Whitney U test.

Enhanced and decreased secretion of innate cytokines IL-8 and IL-6 in infants who developed wheeze and eczema respectively

[0103]. LPS and PHA stimulated cytokine production of IL-8 from CBMCs was found to be significantly higher in infants who developed wheeze (Figure la and lb) but interestingly this cytokine secretion was found to be suppressed in infants who developed eczema as compared the healthy infants. The LPS stimulated IL-6 cytokine secretion is significantly higher in infants who developed eczema and wheeze (Figure 2). This observation of increased IL-6 and IL-8 in infants who developed wheeze concurred with a recent study that reported high levels of IL-6 and IL-8 cytokine secretion in cord blood mononuclear cells predicted the severity of respiratory syncytial virus infections which can induce asthma and infant wheeze later on in life (1).

Cytokine profiles specific for infants who developed wheeze

[0104]. The infants who developed eczema and those who developed wheeze have different cytokine secretion profiles. Infants who developed wheeze was also shown to have increased secretion of LPS stimulated IL-23p40 and increased secretion of PHA stimulated TNF-alpha, IL-2,IL-5, IFN-γ and IL-13 (Figure 3). Differences in IL-23p40, IL-2 and IL-5 production remained significant even after p value was adjusted with risk factors (data not shown). To the best of our knowledge, this is the first report of IL-23p40 secretion in cord blood mononuclear cells to be associated with development of wheeze. IL-5 responses at birth predicting risk for respiratory infections in children with family history of atopic disease has been recently reported (3).

Role of immunoregulatory cytokine IL-10 in development of eczema and wheeze

[0105].Immunoregulatory cytokine IL-10 was found to be suppressed in the cord blood mononuclear cells of infants who developed eczema and wheeze later on in life (Figure 4). Screening for novel markers in the cohort using the cytokine/chemokine/soluble mediator antibody array

[0106]. Soluble mediators which were differentially expressed in the eczema and wheeze group were detected in the LPS stimulated CBMCs. Our preliminary studies from a cytokine antibody array shows that LPS stimulated CBMC culture supernatants have higher soluble CD 14, soluble TNF-RII and TIMP-1 in infants who developed eczema compared to infants who developed wheeze and health controls (Figure 5). These targets were selected for further studies to validate the predictive power for early diagnosis of childhood eczema. i) Soluble CD14 (sC 1)14)

[0107]. CD 14 receptor forms a complex with LPS binding protein (LBP) and LPS resulting in the secretion of a variety of cytokines. Natural monocyte activating agents such as LPS are capable of inducing protease mediated shedding of membrane bound CD 14 receptor from cell surface and sCD14 could be released as an acute phase protein. sCD14 may serve as a modulator of the binding of LPS on mCD14 by competing for LPS binding (10,11). Studies have shown that sCD14 measured in cord blood serum have been shown to be present in differential levels in atopic dermatitis and wheeze infants. It is known to be a regulatory factor capable of interfering with CD40 signalling in B cells and inhibiting IL-6 production (12). ii) Soluble Tumour Necrosis Factor Receptor 2 (sTNFRII)

[0108]. Monocytes constitutively release low levels of sTNF-RII that can be measured in human serum, while activation of monocytes by LPS induces rapid shedding of mTNFRII from monocytes. Enzymatic cleavage and shedding of sTNF receptors are capable of binding and neutralizing soluble TNF-a and serve as natural antagonists (13). iii) Tissue inhibitors of metalloproteinase-1 (TIMP-1)

[0109]. TIMP-1 are endogenous inhibitors that inhibit activity of matrix metalloproteinases (MMPs) which are proteolytic enzymes produced by inflammatory cells. TIMP-1 has been shown to bind to pro and active forms of MMP-9. It would be interesting to investigate TIMP-1 as an imbalance of this mediator via the involvement of different cytokines has been reported in inflammatory disease (14). Preliminary results have shown that cord blood supernatants of infants with eczema are capable of producing more TIMP-1 than wheeze patients. Profile

[0110]. Our results have shown that there are natural pre-existing differences in the immune responses of infants that pre-dispose them to the development of childhood diseases such as eczema and wheeze. The cord blood mononuclear cells associated with disease development have an overall hyper-responsive immune response other than production of immunoregulatory IL-10 which is suppressed in disease groups. We have also seen from our study that infants with eczema and infants with wheeze have different cytokine profiles which have not been reported before. On top of that, results from the cytokine antibody array have identified interesting biomarkers that are differentially expressed in the eczema and wheeze groups in the supernatants of LPS stimulated cord blood mononuclear cells.

Subject recruitment criteria

[0111]. Between May 2004 and June 2006, 253 families with a 1 ^st degree relative with allergic disease were recruited from the antenatal clinics at the National University Hospital to this study. The subjects from this cohort were originally participants of a probiotic intervention clinical trial. The details of the inclusion /exclusion criteria and characteristics of the cohorts have been published previously ". A written informed consent was obtained from all families. The study was approved by the National University Hospital's ethics review committee (Ref Code: B/00/322) ⁿ .

Follow up examination

[0112]. The primary clinical outcome measure was development of eczema and episodes of wheezing which were diagnosed by clinicians ¹¹ The secondary outcome measures were allergen sensitization, asthma and allergic rhinitis. Infants were evaluated by a paediatrician at 1, 3, 6, and 12 months of age, which involved a detailed history, recording of anthropometric data and clinical examination. Questionnaires were also administered at these visits to record clinical disease and environmental exposures, including day care, size of sibship, use of antibiotics, and smoking and pets. Biweekly phone calls were performed for the first 6 months, after which monthly phone contacts were carried out to collect data on the health status of the children. Wheezing episodes of each subject was recorded from hospital records for hospitalized infants and clinic records of specialists or family practitioners for those managed as outpatients. Eczema was defined as a pruritic rash over the face and/ or extensors with a chronic relapsing course as described by Hanifin and Rajka and modified by Seymour for infants ¹² , and the severity assessed by The Scoring Atopic Dermatitis (SCORAD) index

[0113]. Serum was collected from cord blood and at 12 months, samples were stored at -70°C before being assayed. The total IgE of the serum at 1 year was measured using the fluoroenzymeimmunoassay method (UniCAPs Phadiatop, Pharmacia Diagnostics, Uppsala, Sweden) with a detection limit of 0.35 kU/L.

[0114]. Skin prick test was performed at 12 months of age using a standardized technique with common allergen extracts such as (Alyostal, Stallergenes Laboratoires, Antony Cedex, France), milk, egg yolk, egg white, dust mite allergens - Dermatophagoides pteronyssinus (Greer Laboratories, Lenoir, NC, USA) and Blomia tropicalis manufactured in-house ¹⁴ . A histamine dihydrochloride solution (10 mg/mL) was used as a positive control and solvent (50% Cocas 50% Gly) as a negative control. A weal >3mm in diameter above the negative control was considered positive ¹⁵ .

Preparation of cord blood mononuclear cells

[0115]. Umbilical cord blood was collected into blood bags (Terumo, Somerset, USA) prepared with heparin and RPMI medium (Gibco Life Technology, Grand Island, NY, USA). Cord blood is drawn from an umbilical vein after delivery and processed within 24 hours. The heparinized blood was centrifuged at 700 rpm to remove plasma layer before cord blood mononuclear cells (CBMCs) were isolated by Ficoll - Hypaque (GE Healthcare, Uppsala, Sweden) gradient centrifugation. Cells in the interphase were harvested , washed with RPMI supplemented with 10% fetal calf serum (Thermo Scientific, Cramlington, UK) and cryopreserved in 14% dimethyl sulfoxide ( Sigma ,St Louis, Mo, USA ) in fetal calf serum before storing in liquid nitrogen (-150°C) for further use.

Stimulation of cord blood cells with lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) [0116].CBMCs were thawed and washed 3 times with Hanks Balanced Salt Solution (HBSS) (Sigma, St Louis, Mo, USA) before mixing the cells with trypan blue to determine cell viability. The cells normally retained 60% cell viability after thawing. The CBMCs were cultured in AIM-V medium (Gibco Life Technology, Grand Island, NY, USA) and 1 ^χ 10 ^"4 M 2-Mercaptoethanol (Gibco Life Technology, Grand Island , NY , USA ) at 5 ^χ 10 ⁵ viable cells/well in 200 μΐ medium. Cells were either cultured alone or stimulated with 5 g/mL PHA (Sigma, St. Louis , Mo, USA) mitogen for 48 hours or LPS (Escherichia coli LPS 0111 :B4, Sigma, St. Louis, Mo, USA) at concentrations lOng/ml or 1 μg/ml for 24 hours. All cultures were plated in duplicates in a 96- well, round-bottom plate and incubated in a 5% C0 ₂ atmosphere incubator. After culture, the supernatants were acquired and stored at -80°C until required for cytokine analysis by means of FACS Array. Optimal concentration of stimulants used and optimal time-points of co-culture with CBMCs were evaluated based on our initial experiments (data not shown).

Cytokine protein detection

[0117]. Detection of cytokine production was performed using the fluorescence activated cell sorter (FACS)-Array (BD Bioscience, Franklin Lakes, NJ , USA). Cytokines measured were IL-10, IL-1 3 , IL-6, IL-8, IL-12p70, TNF-a , IFN-γ , IL-2 , IL-5, IL-23p40 and IL-13 using the BD bead array flexset (BD Bioscience, Franklin Lakes, NJ, USA). The detection limit was lOpg/mL for all cytokines. The responses presented are those evoked by the LPS / PHA stimulus after subtraction by responses from unstimulated cultures.

Statistical analysis

[0118]. Statistical analysis was performed using SPSS software version 16.0 for windows (SPSS, Inc. Chicago IL, USA). The cytokine data was not normally distributed so the Mann- Whitney U test was performed to asses the differences in the cytokine profiles between the 3 groups wheezing and eczema. The p value was adjusted based on the Bonferroni correction to account for multiple comparisons.

[0119]. The Mann Whitney U and the yl tests were used to evaluate the differences in the demographic data. Significant differences in groups at univariate analysis were included into the logistic regression to adjust for these confounders. This test was also used to determine the odds ratio (OR) and the associated 95% confidence interval (CI). A p- value of 0.05 was considered statistically significant for all analyses. All figures were drawn using Sigma Plot (Systat Software, Inc., Chicago, IL, USA).

[0120]. To determine predictors of cytokines that are associated with wheeze and eczema, factor analysis was used. Cytokines in the physiological setting do not act alone but in concert with other cytokines in a network of groups. The factor analysis is a data reduction technique which takes a large set of variables (cytokines) and groups together intercorrelated sets of variables (cytokines), this summary of data are known as factors. Factors with an eigenvalue greater than 1 were formed using only the healthy control subjects to describe cytokine profile of the healthy children. Factor-specific loading was calculated for each original variable which indicates the degree of correlation between the specific factor formed and the variable. To perform additional analysis by using factors as variables, factor scores were generated by the statistical software SPSS. Factor scores are a linear combination of all the variables weighted by the corresponding factor loadings. These factor scores were then compared between groups by Mann- Whitney test. Logistic regression was used to determine factor scores as possible predictors of wheeze and eczema while adjusting for confounding factors ^I6 .

RESULTS

Demographic characteristics

[0121]. Of the 253 subjects at risk of allergy (first degree relative with allergic disorder) recruited a total of 208 cord blood samples were collected for the study. Of these, 178 samples had sufficient cells for evaluation. Based on their clinical outcome, these 178 subjects were divided into 3 groups (Figure 6). Group 1 : healthy control infants (n=65) with no clinical manifestations of eczema, wheeze or any atopic disorder, and were not allergen sensitized to dietary or inhalant allergens; Group 2: infants with wheeze (n=34); and Group 3: infants who developed eczema (n=29). There were 50 subjects excluded from the study. In the Group 2 wheeze subjects, (24%, n= 8) infants developed wheeze below 6 months of age, 11 (32%) between age 13 to 24 months and the majority (44% , n = 15) developed wheeze between 7 to 12 months of age. There were 16 subjects with a single wheeze episode, while 14 subjects had wheezed between 2-3 times, and only 4 subjects wheeze more than 3 times (Table 2). Notably, from the demographic characteristics a higher proportion of wheeze subjects had a family history of maternal asthma compared to the healthy controls (12/34 vs 8/65,/? = 0.007).

Table 2: Demographic characteristics of the children with eczema and wheeze and the healthy control subjects. Values were presented as median with interquartile range and numbers with percentages for categorical data. Mann- Whitney tests; otherwise χ ² tests were used.

* SCORAD was not recorded for 13 subjects In the eczema group

Statistically significant differences in variables between groups [0122]. In the Group 3 eczema subjects, the majority (76%, n=22) developed eczema by the age of 6 months (Table 3). The mean SCORAD index of the infants with eczema at 24 months was 16.3. Additionally a higher proportion of eczema subjects had a family history of paternal eczema compared to the healthy controls (24/29 vs 8/65, p = 0.027) (Table 2).

Table 3: Age of disease onset for the eczema (n = 29) and wheeze ( n = 34) subjects

Age of onset Eczema Wheeze

( Months) n=29 (%) n=34 (%)

< 6 22 (76%) 8 (24%)

7 up to 12 5 (17%) 15 (44%)

13 up to 24 2 (7%) 1 1 (32%)

[0123]. These were subjects who developed allergen sensitized individuals without any clinical manifestation of atopic symptoms (n=32); those with overlap eczema and wheeze (n=7); and 11 subjects who only developed transient eczema in early infancy. There were only 3 (9%) subjects from the wheeze group and 9 (31%) subjects from the eczema group who were sensitized to the common dietary or inhalant allergens (Table 4). Subjects who developed eczema from group 3 was included in this study as a disease control group, with the reasons that eczema is a known risk factor to the development of wheeze in infants ¹⁷ .

Table 4: Allergen sensitization chart of infants at 24 months of age.

Healthy Eczema Wheeze

Allergen sensitization ( %) n=65 (%) n=29 (%) n=34 (%)

Positive skin prick test 0(0) 9(31 ) 3(9)

Dietary ( Any) 0(0) 1(3) 1(3)

Cow's milk 0 0 0

Egg white 0 1 1

Egg yolk 0 0 0

Soy 0 0 0

Inhalant allergens ( Any) 0(0) 8(28) 3(9)

Dermatophagoides pteronyssinus 0 8 3

Blomia tropica lis 0 1 2

Multiple sensitized 0(0) 1(3) 1(3)

Monosensitized 0(0) 8(28) 2(6) The total IgE geometric mean (95% confidence interval) for healthy controls, wheeze and eczema groups was 14.4 ( 18.0 - 45.7 ) kU / L, 26.9 ( 33.6 - 84.7 ) kU/L, and was 22.5 (22.4 - 88.6) kU/L; respectively (p > 0.05).

Cytokine responses in LPS - stimulated cord blood mononuclear cells

[0124]. The cytokine profiles are shown in Figure 7. Both the wheeze and eczema groups produced higher levels of IL-6 compared to healthy controls (47.40 vs 15.93 ng/mL, p = 0.003 and 23.14 vs 15.93 ng/mL, p = 0.036 respectively), with the wheeze group producing the highest levels (compared with eczema, 47.40 vs 23.14 ng/mL, p = 0.024). Likewise IL-8 responses in the wheeze subjects were also significantly higher compared to both healthy controls and eczema subjects (182.40 vs 94.50 ng/mL and 182.40 vs 12.05 ng/mL respectively, /? = 0.003), but in contrast , the eczema subjects had significantly attenuated IL- 8 production compared to the healthy controls and wheeze subjects ( 12.05 vs 94.50 ng/mL, ? = 0.018 and 12.05 vs 182.40 ng/mL, /? = 0.003, respectively). In addition, LPS stimulated IL- 23p40 production was also significantly higher in the subjects with wheeze compared to the healthy controls (0.10 vs 0.040 ng/mL, p = 0.003). Notably, the readout for the related cytokine IL12 (shared p40 subunit), IL-12p70, was undetectable in all groups.

[0125]. There were no significant differences between groups for the production of other cytokines (IL-1/3, TNF-a and IL-10) when evaluated individually. Additionally, T-cell cytokines IFN-γ, IL-13, IL-5, IL-2 were undetectable in the LPS stimulated groups (Table 5 and 6).

Table 5: Cytokine responses to lipopolysaccharide (LPS) and phytoheamagglutinin (PHA) in infants with eczema . P values have been adjus for risk factors gestational age, paternal eczema using logistic regression analysis.

* Significant differences were found between groups with P value < 0.05

** Significant differences were found between groups with P value < 0.01

ND Not detectable

Table 6: Cytokine responses to lipopolysaccharide (LPS) and phytoheamagglutinin (PHA) in infants with wheeze. P values have been adjust for risk factors birth height, birth weight, birth order, mode of delivery and maternal asthma using logistic regression analysis.

* Significant differences were found between groups with P value < 0.05

** Significant differences were found between groups with P value < 0.01

ND Not detectable

Factor Analysis of LPS-stimulated cytokines

[0126]. Factor analysis of LPS stimulated cytokine responses showed that infants with wheeze have higher factor scores with combined IL-1/3, IL-6 and IL-23p40 responses compared to healthy control subjects and eczema subjects (p = 0.003) (Table 7). When the 3 variables selected by the factor analysis were visualized using a 3D plot, the wheeze group showed a positive correlation between these while the healthy controls showed a negative correlation (Figure 8). Whereas combined responses of from TNF- , IL-10 and IL-8 had lower factor scores (lower production) in the eczema group compared to the healthy control group (p = 0.003) and wheeze group (p = 0.003) (Table 7).

Table 7: Summary of factor analysis, including factor loadings from a model of cytokine responses in the LPS and PHA stimulated CBMCs of healthy control subjects

Cytokine responses in LPS stimulated CBMC Cytokine responses in PHA stimulated CB C

Factor 1 Factor 2 Factor 1 Factor 2 Factor 3

1.-1 0 ( 0.776) TNF- ff (0.701) IL-1jS (0.760) TNF- a ( 0.785) IL-10 ( 0.788)

1-6 ( 0.644) IL-10 ( 0.642) IL-6 ( 0.889) IL-13 ( 0.832) 1-5 ( 0.682)

1-23( 0.691) IL-8 ( 0.769) FN- y ( 0.807)

L-2 (0.711)

L-8 ( 0.609)

Total variance explained (%) 33.5 26.5 33.4 20.6 15.1

Standardized factor-score variables derived from the loadings were used in the Mann- Whitney U

and logistic regression analyses.

Logistic Regression Analysis

[0127]. In order to adjust for potential confounding demographic and clinical factors, univariate analysis of the variables (Table 2) was evaluated. Based on factors that were statistically different between groups, the comparison of cytokine profiles were adjusted for the following variables: gestational age (p = 0.021) and paternal eczema (p = 0.027) for eczema and control groups; and mode of delivery (p = 0.036), maternal asthma (p = 0.007), birth order (p = 0.038), and birth weight (p = 0.003) and birth height (p = 0.01) for the wheeze and control groups. Logistic regression analysis including these variables did not affect the differences in cytokine profiles between groups (Table 5 and 6). Similar variables were used for adjustment of factor scores in the logistic regression with eczema or wheeze as dependent variables .Logistic regression analysis showed that the combined cytokine responses (IL-1 3 , IL-6 and IL-23p40) could predict wheeze relative to the scores for the healthy control subjects (OR, 2.45; 95% 1.50 - 3.93, p =0.001).

Cytokine responses in PHA-stimulated cord blood mononuclear cells

[0128]. The PHA stimulated CBMC cytokine profiles are shown in Figure 8. The wheeze subjects compared to both the healthy controls as well as the eczema subjects showed an increased production of IL-6 (2.75 vs 0.86 ng/mL, p = 0.003 and 2.75 vs 0.82 ng/mL, p = 0.015 respectively) and IL-8 (153.80 vs 80.70 ng/mL, p= 0.003 and 153.80 vs 6.58 ng/mL, /? = 0.003 respectively) from PHA stimulated CBMCs. Similar to the LPS stimulated responses, IL-8 production in the eczema group were significantly attenuated compared to the healthy controls and the wheeze group (6.58 vs 80.7 ng/mL, p = 0.003 and 6.58 vs 153.80 ng/mL, p = 0.003, respectively).

[0129]. A hyper- responsive T-cell cytokine profile was seen in the wheeze subjects with increased TNF-a (6.49 vs 4.02 ng/mL, p = 0.003), IL-2 (3.38 vs 1.23 ng/mL, p = 0.003), IL-5 (0.03 vs 0.01 ng/mL, p = 0.006), and IFN-γ (2.71 vs 0.75 ng/mL, ? = 0.003) compared to the healthy controls. The PHA stimulated IL-10 production was decreased among the subjects with wheeze compared to healthy control subjects (p =0.090).

[0130]. In contrast, aside from IL-8, none of the other PHA stimulated cytokines were found to be significantly different in the eczema group when compared to healthy controls. Furthermore, when compared to the wheeze group, the eczema subjects had a significantly more attenuated cytokine response in the cytokine responses for TNF-a (6.49 vs 3.91 ng/mL, p = 0.021), IL-2 (3.38 vs 0.85 ng/mL, p = 0.003), IL-5 (0.03 vs 0.01 ng/mL, ? = 0.003) IL-13 (4.46 vs 3.39 ng/mL, /? = 0.006), IL-6 (2.75 vs 0.82 ng/mL, p = 0.036), and IL-8 (153.80 vs 6.58 ng/mL, /? = 0.015). The PHA stimulated IL-12p70 levels remained undetectable and IL- 23p40 production was below detection limit for most of the subjects in all groups (Table 5 and 6).

[0131]. Combined responses from JL-Ιβ , IL-6 , IFN-γ, IL-2 and IL-8 responses along with combined responses from TNF-a and IL-13 had higher factor scores (higher production) in the wheeze group compared to the healthy group (p = 0.003 and p = 0.003 respectively), but was not significantly related to wheeze when analyzed by the logistic regression (Table 7). This association was not seen in the eczema group.

Cytokine profile for non -atopic eczema and non - atopic wheeze infants.

[0132]. In order to further refine the clinical phenotypes evaluated, the data was analyzed for the group with non-atopic wheeze (absence of allergen sensitization at 24 months) and non- atopic eczema. The multi comparison of the healthy, wheeze and eczema infant cytokine profiles in this subset did not change and maintained a similar trend (the allergen sensitized and non allergen sensitized individuals are represented in Figure 7 and Figure 8 using open circles and closed circles, respectively.

DISCUSSION

[0133]. Functional alterations of the neonatal antigen presenting cells (APCs) such as monocytes/macrophages and dendritic cells (DCs) could lead to secondary effects such as hyper -responsiveness of the adaptive T cell immunity and therefore susceptibility to disease 8. In our at risk for atopy birth cohort, we prospectively assessed the relationship of the immunophenotypes in CBMCs and the subsequent development of wheeze and eczema in their first 2 years of life ¹¹ . Using factor analysis, we made a novel observation that infants with wheeze had higher combined responses of LPS stimulated IL-1/3, IL-6 and IL-23 and this factor could significantly predictive value in the logistic regression analysis (Table 7). The increased combined cytokine production capacity by LPS-stimulated APCs (macrophages/ monocytes/ DCs) is of particular interest because these three cytokines are important determining factors for the differentiation of IL-17-producing Thl7 cells in [0134]. Recently much attention has been focused on cytokine IL-23, a novel member of IL- 12 cytokine family. It is a heterodimeric cytokine produced by antigen presenting cells (monocytes, dendritic cells) which compose of a pi 9 subunit specific for IL-23 and a p40 subunit shared with IL-12 ⁹ . It has been shown that the secretion of IL-12(p70) in neonatal dendritic cells is impaired due to a specific defect in the IL-12(p35) subunit transcription while the IL-12(p40) subunit transcription is preserved thus neonatal monocytes are biased in the production of IL-23 ' . Data from these previous studies showed that LPS-stimulated neonatal monocytes produced high levels of IL-23, which in turn promote the differentiation of neonatal T cells ( CD4+ and CD8+ T), in particular the CD8+ T cell subset, into 1L-17- producing cells ¹¹ . It is now well established that IL-23 is a signature cytokine that is important for the differentiation of CD161 ⁺ precursors cells from cord blood and newborn thymus into Thl7 cells, and for polarizing neonatal CD4 ⁺ and CD8 ⁺ T cells into IL-17 producing cells ¹⁹ ' ²⁰ ' ²² ' ²⁴ . Incidentally, the dominant pathogenesis role of CD8+ T cells in RSV infection has been demonstrated by two animal studies using experimental respiratory syncytial virus infection mouse model ²⁵ ' ²⁶ '. To exert its biological activities, IL-23 requires an interaction with a heterodimeric complex composed of IL-12R/31 and IL23R to allow signal transduction and this polarizes the differentiation of naive T cells into CD4+ Thl7 cells receptor ²² ' ²¹ . To drive Thl7 differentiation, the up regulation of Thl7 specific transcription factor RORC2 is necessary by pro-inflammatory cytokines IL-6 and IL-10 through signal transducer and activator of transcription 3 (STAT3). The role of IL-1/3 was found to be in inducing expression of RORC2 while IL-6 further sustained this expression. Studies have consistently shown that the presence of IL-23 or IL-1 3 was enough to drive Thl7 development from naive CD4+ T cells and IL-6 is able to further enhance this development ' ' ' . Furthermore , monocytes activated by lipopolysaccharide (LPS) which produce IL-1/3 and IL-6 were the most efficient antigen presenting cells (APCs) for TH17 differentiation which lead to the hypothesis that changes in the APC function especially in monocytes precedes inappropriate development and differentiation into Thl7 cells ¹⁸ .

[0135].CBMCs have been shown to produce higher levels of IL-6 or IL-8 in response to RSV ^{29, 30} or LPS ⁶ stimulation. In the physiological condition, the IL-8 is primary neutrophil chemokine attractant molecules, secreted in the epithelial cells upon viral infection. The main function of these neutrophils will be to adhere to epithelial cells and augment epithelial damage and detachment of epithelial cells ³¹ . Other studies have also reported the association of IL-8 promoter polymorphism ³² with non-atopic virus-induced wheezing as IL-8 has been found to selectively inhibit IgE production by inhibiting IL-4 ³³ , individuals carrying the (-) 251 A allele produces greater IL-8 response when stimulated with LPS in vitro ³⁴ . In this study, (31/34) of our high-risk for atopy infants who developed wheeze without allergen sensitization by the age of 24 months, showed a similar cytokine profile of increased IL-8 production by their cord blood mononuclear cells stimulated by LPS (Figure 7). Besides IL-8, the cord blood mononuclear cells of these wheezing infants have higher LPS induced IL-6 and IL-23 individual cytokine responses profiles (Figure 7).

[0136]. Although IL-6 has not been directly associated with wheeze outcome in infants , IL-6 as a pro-inflammatory cytokine has also been reported to be produced in higher levels in CBMC of neonates with high risk of allergy and also those who subsequently had allergic disease ^{35, 36} . IL-6 derived from antigen presenting cells (APCs) is able to polarize CD4+ T cells into Th2 cells and inhibit the development of T regulatory cells which is an important protective arm of the immune response ³⁷ .

[0137]. Additionally, by using PHA as a stimulus in this study we aim to target cytokine production from mainly T cells as well as monocytes, as it is well known that PHA is capable of stimulating cytokine production of IL-8 and MCP-1 from purified monocytes ³⁸ . A hyper- responsive mixed Thl/Th2 cytokine profile in response to PHA as a stimulus was observed in the wheeze infants compared to the eczema and healthy infants with increased PHA stimulated IL-2, IFN-γ, IL-13 ,TNF-a and IL-5 which are cytokines produced by T cells (Figure 8).

[0138]. Previous concepts underlying early wheeze evolved around the Thl/Th2 paradigm with the hypothesis that inflammatory responses of these diseases are associated with Th2 cells and their cytokines ³⁹ ' ⁴⁰ . Other published studies showed that increased IFN-γ production might have a role in contributing to disease outcome. It has been shown that increased IFN-γ produced by CD8 ⁺ T cells from CBMC from a study of infants from a high genetic risk background are more likely to develop allergen sensitization at 2 years of age ⁴¹ . Whole blood cultures from adult asthmatics also have increased levels of IFN-y/CD8 ⁺ T cells and the increased levels of IFN-γ in cord blood might be responsible in the enhancement of a Th2 response later on in life ⁴² . Percentages for CD8+ T cells positive for IFN-γ in the peribronchial region of the lung were found to be significantly higher in fatal asthma associated with rhinovirus infections ⁴³ . It was hypothesized that IFN-γ producing Thl cells can synergize with Th2 cells in the development of respiratory disease ⁴⁴ . But the pathological role of IFN-γ especially in children with high genetic risk for atopy background remains unclear.

[0139]. PHA stimulated IL-6 and IL-8 cytokines were still found to be significantly higher in the wheeze group compared to the healthy group. Notably, intracellular staining that monocytes are the main producers of IL-6 and IL-8 upon stimulation with PHA (data not shown). Together with cytokine profiles derived from LPS stimulated CBMC, this observation supports the notion that the functional alterations of the neonatal monocytes could lead to some secondary immunological defects.

[0140]. LPS and PHA stimulated IL-10 did not appear to be the key cytokine that is differentially expressed in disease and healthy groups in this study despite it's role as a potential immunoregulator by inhibiting the progression or expression of inflammatory diseases ⁷ · ^{45> 46} . Attenuated IL-10 was reported to be associated with the wheezing phenotype at 5 years of age, but the effects of this cytokine was strengthened by generating a IL-10/IL-5 response ratio for each subject ⁷ . This study was also contradictory to another project by COAST on high risk children which also investigated relationships between cord blood cytokine profile responses and susceptibility to viral infections during the first year of life. There were no comparable relationships between the IL-5 and IL-10 responses to subsequent viral infections in that cohort ⁵ .

[0141]. In summary, we observe from this cohort that cytokine responses did not develop according to a strict T helper cell l(Thl) or T helper cell 2 (Th2) polarization pattern but instead shows heterogeneity in the immune response patterns. Combine responses from IL- 1/3, IL-6, IFN-γ, IL-2 and IL-8 responses along with combined responses from TNF-a and IL- 13 had higher factor scores (higher production) in the wheeze group compared to the healthy group but these factors were not significantly related to wheeze susceptibility in the logistic regression analysis (Table 7). In view of this, we propose that upregulation of T cell cytokines may not be the triggering factors for RSV-induced wheeze phenotypes, instead the increased production of Th cytokines may contribute to the severity of the disease. Indeed,

26

this notion has been implicated by a mice study . [0142]. A positive relationship between early eczema and moderate to severe wheezing illnesses early in the first 2 years of life have been reported ^{17, 7} ' ^{48 49} . Despite this close clinical relationship, the differences in the immunophenotypes at birth are poorly understood. It is worth noting that although the eczema and wheeze phenotypes might be related clinically, they have very distinctly different immunophenotypes at birth. The wheeze phenotype is associated with a more hyper-responsiveness cytokine profile in both the innate and T-cell compartment that may be due to reduced regulation of the immune response leading to disease development while the eczema group presented with a more hyporesponsive profile. For example, IL-8 was found to be significantly suppressed in the eczema subjects not only in comparison to the wheeze but also to the healthy subjects. Intriguingly, a combination of LPS stimulated TNF-α, IL-10 and IL-8 cytokines in the eczema group has lower factor scores (lower production) relative to healthy control subjects. The suppression in IL-8 cytokine seen in eczema infants lead us to believe that other soluble factors such as soluble receptors can play a role in regulating this pro-inflammatory cytokine

50, 51

[0143]. Our study is one of the few that is using the AIM-V serum free condition to study cytokine expression profile in response to LPS. Previous studies have employed the method of IFN-γ priming cord blood mononuclear cells before stimulating the cells with LPS in RPMI + 10% FCS media (complete RPMI) ³⁵ ' ⁵² . Pre-priming cells with IFN-γ with complete RPMI as a cell culture media has been known to induce high amounts of cytokine production at low doses of LPS but might be an artificial upregulation of cytokines ⁵² . Pre-priming of CBMC also enables the activation of dendritic cells and the induction of cytokine IL-12 which is not inducible in a serum-free culture condition ⁵² . To investigate if LPS was capable of inducing IL-12p70 cytokine production in a serum- free condition, we have measured LPS induced IL-12p70 but cytokine levels were below detection limits of the assay. A study has shown that IL-12p70 production is indeed defective in neonatal monocyte-derived dendritic cells and failed to be induced by LPS stimuli in a serum-free condition ²³ . The serum free method using AIM-V as the cell culture media helps reduce artificial effects of IFN-γ priming and that of the fetal calf proteins in the serum.

[0144]. Our study design is also quite unique as compared to most published studies as we are doing a parallel immunological comparison between the eczema and wheeze outcomes in infants from the same cohort. Other studies have studied a mixed cohort of allergic disease without subcategorizing the disease outcomes and analyzing them separately to study the possibility of different cytokine profiles existing for the different disease outcomes ⁵ ' ⁵³ ' ⁵⁴ . While other studies have only focused on comparing cord blood mononuclear cells (CBMCs) cytokine profiles of high risk neonates and low risk neonates , without following them with respect to the development of atopy/ allergic diseases ³⁶ ' ^55~57 .

[0145]. Our results might not be similar to what other studies have shown but we have to note the possibility of the variations in clinical phenotypes in atopics / non-atopic childhood diseases that might contribute to the result of differences seen in the immune response profiles ⁴ ' ⁴¹ . Other than that, our study subjects consists of an all high-risk cohort so our data must be interpreted keeping in mind that the cord blood of healthy infants used as controls in our study could be immunologically different from healthy infants with parents without a history of allergic disease .The differences in culture conditions used especially involving the LPS stimulus, could contribute to the difference in the cytokine profile of our study compared to other studies.

[0146]. Due to the subdivision of our original cohort study into individual clinical phenotypes of study interest, our sample size of study is not large enough. A larger sample would be useful for accurately defining and confirming the association of these 2 disease groups and related cytokine profiles.

[0147]. A different cytokine secretion profile in eczema and wheeze subjects such as results seen in our study might influence immune cell types to induce inflammatory responses which will lead to its specific disease phenotype outcomes. Cord blood research is important for identifying markers for childhood inflammatory diseases in the earliest time of life. These findings may have implications for future early intervention studies by helping to identify infants at increased risk for developing childhood wheeze and eczema. Further studies of innate immunity in wheezing/asthma and eczema are required to discover novel mechanisms underlying distinct neonatal immunity that will identify novel ways to prevent and treat inflammatory disease in this relatively high-risk population.

[0148]. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0149]. Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0150]. Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0151]. The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0152]. The invention described herein may include one or more range of values (eg size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

[0153]. Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes",

"included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. [0154]. Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

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