SOLUBLE MEDIATOR PROFILES OF CORD BLOOD MONONUCLEAR CELLS IN EARLY ONSET CHILDHOOD ALLERGIC DISORDERS QUAH PHAIK LING B.Sc. (Biotech) (Hons.), Monash A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PAEDIATRICS NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ______________________________________ Quah Phaik Ling 21 January 2013 i ACKNOWLEDGMENTS Completing a PhD is truly a marathon event, and I would not have been able to complete this journey without the aid and support of countless people over the past six years. Firstly, I am very fortunate to have not only one but three remarkable supervisors. It is difficult to overstate my gratitude to my PhD supervisor, the late Professor Chua Kaw Yan whom I was fortunate enough to have as an advisor. Thank you for always believing in me, this project, for giving me the opportunity to pursue my PhD as a part-time student in NUS, and last but not least for setting the direction and focus for my research. I am also very much indebted to my supervisor Professor Lee Bee Wah whose enthusiasm, inspiration, dedication and constructive criticism has allowed me to challenge myself to achieve something greater than what I thought I was capable of. To Dr Kuo I-Chun, I thank you for supporting me throughout my thesis-writing period, for reviewing my manuscripts, providing me encouragement, sound advice, good teaching, good company, and lots of good ideas. I thank the Principal Investigator of this study, A/Prof Lynette Shek for trusting me with the cord blood samples that has been collected for the PROMPT study which made this dissertation possible. I am grateful to the members of the PhD Qualifying Examination panel, Professor Hugo van Bever, Professor Koh Dow Rhoon, for their critical suggestions and advice. I wish to thank biostatisticians Dr. Chan Yiong Huak and Dr. Shen Liang for the providing me with statistical advice at times of critical need. A big thank you goes out to the PROMPT (PRObiotic in Milk for the Prevention of aTopy trial) team – Corinne Kwek Poh Lian, Dr. Genevieve Llanora, Dr. Irvin Gerez and Bautista Fatima Yturriaga who assisted in the follow-up data of the subjects, and on top of that every one of you made hanging out in the Porta Cabin and compiling data a lot more fun. A special thank you goes out to all the occupants in the asthma and allergy laboratory, and to the wonderful people that I have spent the past years with in the Clinical Research Center of NUS. I feel truly blessed to say that we are all more than just working colleagues, we are friends. You have filled my years here with so much laughter and joy and never ending encouragement, I wouldn’t have made it till the end without each and every one of you. Last but not least, I would also like to thank my family for the relentless support they provided me through my entire life and in particular, I must acknowledge my husband and best friend, Justin, without whose love, encouragement and patience, I would not have finished this dissertation. In conclusion, I recognize that this research would not have been possible without the financial assistance of the National Medical Research Council, Singapore (NMRC /0674/2002). ii iii TABLE OF CONTENTS Summary xi List of tables xiv List of figures xiv List of abbreviations xix Chapter 1: Introduction and literature review 1.1 Introduction 1.2 Atopy and allergic disorders. 1.2.1 Definitions 1.2.1.1 Atopy 1.2.1.2 Eczema 1.2.1.3 Asthma and wheeze 1.2.1.4 Rhinitis 1.2.2 The atopic march. 1.2.2.1 Birth cohort studies 1.2.2.2 Role of atopy 1.2.3 Prenatal influence on atopic disorders 10 1.3 Innate immunity 12 1.3.1 Toll-Like-Receptors 13 1.3.1.1 Bridging the innate and adaptive immunity with Toll-Like-Receptors 14 1.4 Cord blood mononuclear cells (CBMCs) responses and atopic disorders 15 1.4.1 Fetal responses to allergens 16 1.4.2 CBMC proliferative responses and atopic disorders. 16 1.4.3 Cord blood IgE in allergic disorders. 18 1.4.4 Cord blood mononuclear cell (CBMC) cytokine responses. 19 1.4.4.1 Network of cytokines determines CD4+ T-cell fates 19 iv 1.4.4.2 Cord blood mononuclear cell (CBMCs) cytokine profiles in infants at risk for atopy 22 1.4.4.3 Cord blood mononuclear cell (CBMC) cytokine profiles at birth and subsequent allergic disorders 24 1.4.4.4 Inconsistencies in cord blood studies and Th1/Th2 cytokines 28 1.5 Chemokines in the role of allergic disorders 32 1.5.1 Chemokines in innate immunity 32 1.5.2 Chemokines in the pathogenesis of eczema 32 1.5.3 Chemokines in the pathogenesis of asthma 33 1.5.4 Chemokines and other soluble mediators in the role of allergic disorders 33 1.6 Rationale of study 36 1.7 Study hypothesis and aims of study. 39 Chapter 2: Materials and methods 2.1 Subject recruitment and cord blood collection 42 2.2 Details of inclusion/exclusion criteria 42 2.2.1 Inclusion criteria 42 2.2.1.1 Pre-delivery evaluation 42 2.2.1.2 Post-delivery evaluation 43 2.2.2 Exclusion criteria. 43 2.3 Background on study 43 2.4 Processing and storage of cord blood mononuclear cells from cord blood samples collected 44 2.5 Clinical assessment 45 2.6 Skin prick test and serum collection. 46 2.6.1 Skin prick test 46 2.6.2 Serum collection and IgE measurement 47 2.7 Selecting cord blood samples for further studies 47 v Chapter 3: Development of allergic phenotypes from at risk for atopy Singapore cohort in a five year follow-up study- Early life risk factors and progression of allergic phenotypes 3.1 Introduction 48 3.2 Methods 49 3.2.1 Subject recruitment, clinical assessment and skin prick test 49 3.2.2 Statistical analysis. 49 3.3 Results 52 3.3.1 Demographic data of the allergic phenotypes at years of age 52 3.3.2 Manifestations of allergic disorders up to years of age 56 3.3.3 Co-existent of allergic phenotypes at year 58 3.3.4 Phenotypes of eczema and wheeze 58 3.3.5 Allergic disorders and allergen sensitization 61 3.3.6 Association of allergen sensitization to allergic phenotypes at and years of age 63 3.3.7 Progression of early allergic phenotypes up to years of age 66 3.3.7.1 Progression of early allergic phenotypes 66 3.3.7.2 Association of risk factors at years and clinical phenotype outcomes at years 68 3.4 Discussion 70 Chapter 4: Optimizing in vitro cell culture experiments with cord blood mononuclear cells in serum-free medium 4.1 Introduction 76 4.2 Materials and methods 78 4.2.1 Preparation and storage of cord blood mononuclear cells (CBMCs) and peripheral blood mononuclear cells (PBMC) 78 4.2.2 Stimulation of cord blood cells with lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) for dosage and kinetic studies 78 vi 4.2.3 Cytokine protein detection 79 4.2.3.1 Fluorescence activated cell sorter (FACS) –Array (Becton Dickinson) 79 4.2.4 Comparison of cell culture conditions 80 4.2.4.1 Cell culture of CBMC in serum-free AIM-V medium 80 4.2.4.2 Cell culture of CBMC in RPMI media supplemented with fetal calf serum (FCS) 80 4.2.4.3 Cell culture of CBMC with the recombinant IFN-γ ( rIFN-γ) priming 81 4.2.5 Polymyxin B and TLR4 blocking experiment 81 4.2.6 Statistical analysis 82 4.3 Results 82 4.3.1 Lipopolysaccharide (LPS) stimulated CBMC cytokine response in serum free AIM-V medium compared to RPMI-serum supplemented media 82 4.3.2 Kinetic and dosage response of lipopolysaccharide (LPS) stimulated cytokine production from cord blood mononuclear cells in the serum-free AIM-V medium 85 4.3.3 Kinetics and dosage responses of phytohaemagglutinin (PHA) stimulated cytokine production from cord blood mononuclear cells in the serum-free AIM-V medium 87 4.3.4 Comparison of cytokine secretion profiles between cord blood mononuclear cells (CBMCs) and peripheral blood mononuclear cells (PBMCs) in the AIM-V serum free medium 89 4.3.5 Inhibition of lipopolysaccharide (LPS) stimulated cytokine production by polymyxin B and anti-TLR4 antibody 91 4.4 Discussion 93 Chapter 5: Cytokine profiles of lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) stimulated cord blood mononuclear cells (CBMCs) and their correlation with clinical outcomes in the first years of life 5.1 Introduction 97 5.2 Materials and methods 98 5.2.1 Subject recruitment criteria 98 5.2.2 Follow up examination 99 vii 5.2.3 Cord blood collection and clinical outcome assessment 99 5.2.4 Subjects selected for cord blood responses 102 5.2.5 Preparation of cord blood mononuclear cells (CBMCs) 103 5.2.6 Stimulation of cord blood cells with lipopolysaccharide (LPS) and phytohaemaglutinin (PHA) 103 5.2.7 Cytokine protein detection by the Fluorescence activated cell sorter (FACS)–Array (Becton Dickinson) 104 5.2.8 Cytokines measured from cord blood mononuclear cells (CBMCs) and clinical phenotypes of interest 104 5.2.9 Intracellular staining and flow cytometry 106 5.2.10 Statistical analysis 107 5.3Results 110 5.3.1 Cytokine responses in lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) - stimulated cord blood mononuclear cells (CBMCs) assessed in relation to the clinical outcomes of eczema and wheeze at years of age 110 5.3.1.2 LPS and PHA stimulated IL-6, IL-8 and TNF-α 110 5.3.1.3 LPS stimulated IL-12/IL-23p40 and IL-1β 111 5.3.1.4 LPS and PHA stimulated cytokine IL-10 114 5.3.1.5 PHA stimulated T-cell cytokines IL-5, IL-2, IL-3 and IFN γ 114 5.3.2 Cytokine profiles of subjects who are healthy with allergen sensitization at years of age 116 5.3.3 Logistic regression analysis of LPS and PHA stimulated CBMC cytokine profiles of eczema and wheeze subjects 120 5.3.3.1 Logistic regression adjusting for factors confounding cytokine profiles 120 5.3.3.2 Logistic regression to determine main risk factors 120 5.3.4 Factor Analysis of cytokine profiles 124 5.3.4.1 Factor analysis of LPS stimulated cytokine profiles 124 5.3.4.2 Factor analysis on PHA stimulated cytokine profile 127 5.3.5 Monocytes are main producers for LPS stimulated IL-6 and both LPS and PHA IL-8 in LPS stimulated CBMCs 128 viii Appendix D 230 Appendix E Cytokine responses to lipopolysaccharide (LPS) in infants with eczema and wheeze at years of age. P values have been adjusted for risk factors gestational age, paternal eczema using logistic regression analysis with eczema as the outcome and for risk factors birth height, birth weight, birth order, mode of delivery and maternal asthma with wheeze as the outcome . * 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 231 Cytokine responses to phytohaemagglutinin (PHA) in infants with eczema and wheeze at years of age. P values have been adjusted for risk factors gestational age, paternal eczema using logistic regression analysis with eczema as the outcome and for risk factors birth height, birth weight, birth order, mode of delivery and maternal asthma with wheeze as the outcome. * Significant differences were found between groups with P value < 0.05 ** Significant differences were found between groups with P value < 0.01 232 ND Not detectable 233 Appendix F Demographic characteristics of the children with eczema and wheeze and the healthy control subjects at years of age in the cord blood mononuclear cell study. Healthy (n=65) Eczema ( n= 29) Wheeze (n=34) Healthy vs. Eczema P value Healthy vs. Wheeze P value Gestational age in weeks median (IQR)¶ Infant sex , no (%) 38.7 (1.6) 39.6 (1.7) 39.3 (1.9) 0.021 0.088 Male 30(46) 18(62) 19(56) 0.154 0.358 0.946 0.873 Ethnicity ( %) Chinese Malay Indian Others Mode of delivery , no.(%) 30(46) 27(42) 7(10) 1(2) 14(48) 12(41) 3(10) 0(0) 13(38) 16(47) 4(12) 1(3) Lower segment caesarean section¶ 20(31) 6(21) 4(12) 0.313 0.036 8(12) 12(18) 23(38) 12(18) 5(8) 19(32) 6(8) 7(12) 7(11) 5(17) 4(14) 16(36) 4(14) 7(24) 9(31) 5(17) 5(17) 4(14) 12(35) 4(12) 16(47) 5(15) 2(6) 8(24) 8(24) 6(18) 6(18) 0.522 0.578 0.072 0.578 0.027 0.86 0.264 0.385 0.674 0.007 0.39 0.357 0.638 0.739 0.545 0.053 0.336 0.336 0.771 0.038 >3 Birth Height in median (IQR)¶ 27(42) 13(20) 25(38) 49 (3) 14(45) 6(20) 9(35) 50 ( 2.0) 7(23) 14(45) 13(32) 50 ( 4) 0.076 0.003 Birth weight in median (IQR)¶ 3.0 ( 0.5) 3.1 ( 0.5) 3.3 ( 0.5) 0.147 0.01 Weaning age in months mean ( SD) (1) 6(0.0) ( 2.0) 0.055 0.378 Prenatal maternal smoking 4(6) 2(7) 2(6) 1.000 1.000 Passive smoke exposure in household 19(29) 9(31) 14(41) 0.86 0.231 Keep pets ( %) 18(28) 5(17) 6(18) 0.276 0.268 0.626 0.306 0.187 0.761 0.169 0.636 Family history 1st degree relative (%) Maternal asthma ¶ Maternal eczema Maternal allergic rhinitis Paternal asthma Paternal eczema¶ Paternal allergic rhinitis Sibling asthma Sibling eczema Sibling allergic rhinitis Birth order ¶ Type of housing (%) Public housing 56(86) 26(90) 26(76) Private housing ( Condominium) Landed property 5(8) 4(6) 2(7) 1(3) 8(24) 0(0) 1(2) 47(72) 2(7) 19(66) 0(0) 25(74) Feeding history – birth to month (%) Near total breastfeeding with at least 60mL of Any breastfeeding Total formula 17(26) 8(28) 9(26) Probiotic supplementation 30(46) 9(31) 14(41) *SCORAD index Mean ± SD ( Range) 16.3 ± 8.1 (3.7-35.9) Frequency of wheeze 0 to 0 >3 0 * SCORAD was not recorded for 13 subjects in the eczema group ¶ Statistically significant differences in variables between groups 16 14 Values were presented as median with interquartile range and numbers with percentages for categorical data. Mann – Whitney tests; otherwise χ2 tests were used. 234 Appendix G The levels [median (25th-75th percentiles)] of lipopolysachharide (LPS) stimulated cord blood mononuclear cell (CBMC) chemokines, cytokines, soluble receptors, soluble TIMP/MMP and soluble growth factors in infants who developed eczema, wheeze and those who are healthy at years of age. Eczema Eczema n= Wheeze Healthy Allergen sensitization in healthy subjects Wheeze Healthy Allergen sensitizatio n No allergen sensitization 29 62 32 62 32 62 0.476** 2.873 0.546* 2.873 0.787 2.873 (0.2331.91) (0.5745.747) (0.392-4.14) (0.5745.747) (0.480-1.10) (0.574-5.747) 1.72** 4.906 2.93 4.906 4.62 4.906 (0.3665.24) (1.119.99) (0.875-9.25) (1.11-9.99) (0.521-6.55) (1.11-9.99) 12.8 13.5 (10.3-14.6) 13.8 (1.20-14.9) 17.4** (9.79-14.7) 13.8 (1.2014.9) 13.8 (1.20-14.9) 1.32 2.303 1.59 2.303 17.4** 2.303 (0.3172.88) (0.854.56) (0.439-2.47) (0.85-4.56) (12.9-29.9) (0.85-4.56) 0.0261** 0.064 0.0686 0.064 0.0481 0.064 (0.0150.054) (0.0280.129) (0.0340.143) (0.0280.129) (0.0190.149) (0.028-0.129) 0.0101** 0.044 0.0121 0.044 0.0237 0.044 (0.0090.057) (0.01050.130) (0.0100.097) (0.01050.130) (0.0100.035) (0.01050.130) CCL chemoki nes (ng/mL) CCL1 CCL2 CCL3 CCL4 CCL7 CCL8 235 (12.9-29.9) CCL20 CCL22 CCL24 0.0472 0.0556 0.031** 0.0556 0.0629 0.0556 (0.0170.116) (0.0230.085) (0.0150.042) (0.0230.085) (0.0350.153) (0.023-0.085) 0.165** 0.823 0.525 0.823 1.02 0.823 (0.0460.719) (0.251.547) (0.237-1.17) (0.25-1.547) (0.498-2.28) (0.25-1.547) 1.65** 3.806 3.34 3.806 4.66 3.806 (0.4672.64) (2.3155.575) (2.30-4.23) (2.3155.575) (2.63-6.54) (2.315-5.575) 8.00* 10.5 13.4 (6.91-19.5) 12.8 13.4 (6.91-19.5) (2.74-15.6) 13.4 (6.9119.5) (4.02-16.3) 1.54** 5.900 4.09 5.900 7.89 5.900 (0.2924.49) (1.5298.417) (1.07-5.73) (1.5298.417) (2.56-9.59) (1.529-8.417) 0.007** 0.014 0.968 0.014 0.0172* 0.014 (0.0060.253) (0.0060.017) (0.0060.253) (0.0090.075) (0.006-0.253) CXCL chemoki nes (ng/mL) CXCL1/ 2/3 CXCL5 CXCL6 (0.0030.012) Cytokin 236 (5.02-15.4) es (ng/mL) IL-1α 0.055 0.104 0.056 0.104 0.253 0.104 (0.0330.183) (0.0370.478) (0.0230.142) (0.0370.478) (0.1040.589) (0.037-0.478) 0.554 0.394 0.357 0.394 0.679* 0.394 (0.3070.751) (0.2090.558) (0.2080.551) (0.2090.558) (0.384-1.03) (0.209-0.558) 0.681 0.726 0.634 0.726 1.02** 0.726 (0.5190.787) (0.5460.879) (0.5500.823) (0.5460.879) (0.644-1.24) (0.546-0.879) 0.003 0.002 0.001 0.002 0.0116** 0.002 (0.0010.009) (0.0010.007) (0.0000.003) (0.0010.007) (0.0060.018) (0.001-0.007) 0.0265** 0.045 0.0351 0.045 0.0882** 0.045 (0.0150.056) (0.0280.080) (0.0240.064) (0.0280.080) (0.0440.117) (0.028-0.080) sTNF-α RI 0.0291** 0.033 0.0308 0.033 0.0618** 0.033 (0.0250.045) (0.0260.048) (0.0250.041) (0.0260.048) (0.0490.073) (0.026-0.048) sTNF-α RII 0.165 0.40 0.277 0.40 0.707 0.40 (0.0876- (0.193- (0.147- (0.193- IL-16 Soluble receptor s (ngm/L) sEGFR sIL-2Rα sIL-6R 237 0.561) 0.864) 0.547) 0.864) (0.291-1.58) (0.193-0.864) 1.889* 3.658 3.24 3.658 4.10 3.658 (0.3746.26) (1.4458.655) (0.949-8.95) (1.4458.655) (1.60-13.0) (1.445-8.655) 0.095 0.088 0.105 0.088 0.088 0.02730.175 (0.0160.147) 0.051-0.149 (0.0160.147) (0.016-0.147) 0.0108** 0.0719 0.0344* 0.0719 0.117 0.0719 (0.0040.043) (0.02060.221) (0.0090.077) (0.02060.221) (0.0180.529) (0.02060.221) 0.506** 1.549 1.404 1.549 2.33* 1.549 (0.3041.49) (0.972.928) (0.8092.438) (0.97-2.928) (1.27-4.70) (0.97-2.928) 0.159** 0.297 0.324 0.297 0.426* 0.297 (0.1040.229) (0.1800.424) (0.1790.455) (0.1800.424) (0.256-1.10) (0.180-0.424) 0.015 0.020 0.019 0.020 0.024 0.020 Soluble TIMP/ MMP (ng/mL) TIMP-1 Timp-2 MMP-1 MMP-9 Growth factors (ng/mL) M-CSF PDGFAA 238 (0.008- (0.010- (0.007- (0.017- 0.027) 0.03) 0.031) (0.010-0.03) 0.039) *p value [...]... to elevated chemokine levels xii Conclusions This study emphasizes the importance of early allergic clinical phenotypes as predictors of subsequent allergic disorders The inherent aberrant immunological responses involving soluble mediators from cord blood mononuclear cells influence the development of early onset phenotypes Future work is needed to provide a roadmap to gain more insights on the pathogenesis... response curves of phytohaemagglutinin (PHA) stimulated cord blood mononuclear cells (CBMCs) 88 FIGURE 4-4 Dosage response curves of cytokine responses from lipopolysaccharides (LPS) and phytohaemagglutinin (PHA) stimulated cord blood mononuclear cells (CBMCs) and peripheral blood mononuclear cells (PBMCs) 90 FIGURE 4-5 Inhibition of LPS at 100ng/mL LPS and 1ug/mL LPS using polymyxin B ( 10ug/mL) or... for prognosis or early diagnosis of allergic disorders in early childhood xiii LIST OF TABLES TABLE 1-1 Summary of studies related to in vitro CBMC proliferative responses to subsequent development of atopic disorders 17 TABLE 1-2 Summary of studies relating CBMC cytokine responses to a family history of atopic disorders 23 TABLE 1-3 Summary of studies related to in vitro CBMC cytokine responses to... relative with allergic disorder and allergen sensitization) for atopy infants ii) evaluate the influence of intrinsic immunologic responses in cord blood mononuclear cells on eczema and wheezing disorders and allergen sensitization at 2 years and 5 years of age Methods This study is centered in a cohort of 253 newborn infants who participated in a double-blind, placebo-controlled randomized clinical trial... expressions of innate immune receptors TLR5 and TLR9 in cord blood (55) There have been a number of studies on maternal diet in pregnancy and the outcomes of allergic disorders These studies have focused mainly the dietary intake of antioxidants The most recent study conducted in Australia has shown how antioxidant intake in pregnancy may influence fetal immune programming and the risk of allergic disease... antioxidants vitamin E and zinc were found to be inversely associated with any wheezing at 2 y of age (57) Furthermore, low vitamin D intake during pregnancy is also believed to be associated with higher rates of food sensitization in early life in infants (58) and peanut allergy (59) 11 1.3 Innate immunity Innate immunity serves as a first line of defense against infectious agents including viral and bacterial... copper intake was associated with reduced risk of wheeze and development of any early allergic disease in infants at high risk due to family allergy history Furthermore, maternal dietary vitamin C intake in pregnancy was shown to protect against the development of wheeze in the first year of life (56) Similar results were obtained with a study in Boston where maternal total intake of antioxidants vitamin... assessed in relation to the clinical outcomes of wheezing, eczema or allergen sensitization and healthy control at 2 years and 5 years of age xi Results I) Clinical outcomes In this cohort, 63% of the wheeze and 85% of the eczema subjects developed symptoms in the first 2 years of life Early onset is defined as clinical phenotype outcomes in the first 2 years of life Early onset of eczema alone (adjusted... history of allergic disease from the antenatal clinics at the National University Hospital, Singapore Cord blood samples were collected from 195 eligible subjects Soluble mediators from lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) stimulated mononuclear cells were analyzed using the fluorescent activated cell sorting (FACS)array and Bio-Plex 200 instrument These profiles were assessed in relation... FIGURE 6-6 Chemokine profile of healthy (n=41), eczema (n=8) and wheeze (n=10) subjects from unstimulated (M) cord blood mononuclear cells (CBMCs) and in response to 1ug/mL lipopolysaccharide(LPS) 163 FIGURE 6-7 Soluble receptor, cytokine, growth factor, TIMP/MMP profile of healthy (n=41), eczema (n=8) and wheeze (n=10) subjects from unstimulated (M) cord blood mononuclear cells (CBMCs) and in response . cytokine profiles in infants at risk for atopy 22 1.4.4.3 Cord blood mononuclear cell (CBMC) cytokine profiles at birth and subsequent allergic disorders 24 1.4.4.4 Inconsistencies in cord blood. SOLUBLE MEDIATOR PROFILES OF CORD BLOOD MONONUCLEAR CELLS IN EARLY ONSET CHILDHOOD ALLERGIC DISORDERS QUAH PHAIK LING B.Sc. (Biotech) (Hons.), Monash. of early allergic clinical phenotypes as predictors of subsequent allergic disorders. The inherent aberrant immunological responses involving soluble mediators from cord blood mononuclear cells