published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 234-252) Also available in print.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Leung, Wan Lun Fiona. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 179-204). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.v / Acknowledgement --- p.viii / List of Abbreviations and Symbols x --- p.vi / List of Tables --- p.xx / List of Figures --- p.xxi / Chapter CHAPTER ONE --- Introduction --- p.1 / Chapter 1.1 --- General Overview --- p.1 / Chapter 1.2 --- Necrotizing Enterocolitis (NEC) --- p.3 / Chapter 1.2.1 --- Epidemiology of NEC --- p.3 / Chapter 1.2.2 --- "Clinical Presentation, Diagnosis and Management of NEC" --- p.5 / Chapter 1.2.3 --- Pathophysiology of NEC --- p.9 / Chapter 1.2.3.1 --- Prematurity --- p.9 / Chapter 1.2.3.2 --- Bacterial Colonization --- p.12 / Chapter 1.2.3.3 --- Enteral Feeding --- p.15 / Chapter 1.2.3.4 --- Hypoxia and Ischemia --- p.16 / Chapter 1.2.3.5 --- Genetic Polymorphism --- p.17 / Chapter 1.2.3.6 --- Inflammatory Mediators --- p.20 / Chapter 1.3 --- Spontaneous Intestinal Perforation (SIP) --- p.24 / Chapter 1.3.1 --- Epidemiology of SIP --- p.24 / Chapter 1.3.2 --- "Clinical Presentation, Diagnosis and Management of SIP" --- p.26 / Chapter 1.3.3 --- Risk Factors of SIP --- p.28 / Chapter 1.3.3.1 --- Prematurity --- p.29 / Chapter 1.3.3.2 --- Use of Drugs --- p.30 / Chapter 1.4 --- Comparison between NEC and SIP --- p.32 / Chapter 1.5 --- Role of Cytokines in Pathogenesis of NEC and SIP --- p.38 / Chapter 1.6 --- Immunoregulatory Molecules of Interest in This Study --- p.46 / Chapter 1.6.1 --- Angiopoietin-2 (Ang-2) --- p.46 / Chapter 1.6.2 --- v-erb-b2 Erythroblastic Leukemia Viral Oncogene Homolog 2 (avian) (ErbB3) --- p.48 / Chapter 1.6.3 --- Type II Interleukin-1 Receptor (IL-1RII) --- p.52 / Chapter 1.6.4 --- Urokinase Plasminogen Activator Receptor (uPAR) --- p.54 / Chapter CHAPTER TWO --- Objectives --- p.57 / Chapter CHAPTER THREE --- Materials and Methodology --- p.58 / Chapter 3.1 --- Overview of the Experimental Procedures --- p.58 / Chapter 3.1.1 --- Investigation on the Profile of Circulatory Immunoregulatory Proteins in Plasma of NEC and SIP High Risk Neonates --- p.58 / Chapter 3.1.2 --- Investigation on the mRNA Expression Level of Targeted Immunoregulatory Molecules on Resected Intestinal Tissues in NEC and SIP Neonates --- p.58 / Chapter 3.1.3 --- Investigation on the mRNA and Protein Expression Levels of Targeted Immunoregulatory Molecules in Human Intestinal Cell Lines --- p.60 / Chapter 3.2 --- Reagents and Lab-wares with Their Sources --- p.61 / Chapter 3.3 --- Study Population --- p.63 / Chapter 3.4 --- Collection of Neonatal Whole Blood Samples --- p.65 / Chapter 3.5 --- Cytokine Antibody Array Analyses --- p.67 / Chapter 3.6 --- Enzyme-linked Immunosorbant Assays (ELISA) --- p.69 / Chapter 3.6.1 --- Angiopoietin-2 --- p.69 / Chapter 3.6.2 --- sErbB3 --- p.71 / Chapter 3.6.3 --- sIL-lRII --- p.72 / Chapter 3.6.4 --- suPAR --- p.74 / Chapter 3.7 --- Collection of Neonatal Resected Intestinal Tissues --- p.76 / Chapter 3.8 --- Resected Intestinal Tissue RNA Isolation --- p.78 / Chapter 3.9 --- Purity Assessment of the Purified Tissue RNA Samples --- p.80 / Chapter 3.10 --- Integrity Assessment of the Purified Tissue RNA Samples --- p.81 / Chapter 3.11 --- In vitro Stimulation of Human Enterocytes by Lipopolysaccharides (LPS) and/or Platelet Activating Factor (PAF) --- p.84 / Chapter 3.12 --- mRNA Expression Level Assessment of Selected Target Genes in Resected Intestinal Tissues and Human Intestinal Cell Lines --- p.86 / Chapter 3.12.1 --- Synthesis of First Strand cDNA --- p.86 / Chapter 3.12.2 --- Quantitative Polymerase Chain Reaction (qPCR) --- p.87 / Chapter 3.13 --- Statistical Analysis --- p.89 / Chapter CHAPTER FOUR --- Screening of Immunoregulatory Target Protein Molecules in Plasma of NEC and SIP Patients by Cytokine Array Analyses --- p.104 / Chapter 4.1 --- Results --- p.104 / Chapter 4.1.1 --- Screening of Detectable Immunoregulatory Target Molecules --- p.104 / Chapter 4.1.2 --- Selection of Target Molecules Based on the Fold Change in NEC or SIP Compared with Control Samples --- p.105 / Chapter 4.1.2.1 --- Similar Regulation of Target Molecules in Both NEC and SIP patients --- p.105 / Chapter 4.1.2.2 --- Differential regulation of Target Molecules in NEC and SIP Patients --- p.106 / Chapter 4.1.2.3 --- "Relative Normalized Expressions of Selected Circulatory Immunoregulatory Protein Molecules in NEC, SIP and Control Neonates" --- p.108 / Chapter 4.1.2.3.1 --- Anti-inflammation --- p.108 / Chapter 4.1.2.3.2 --- Pro-inflammation --- p.109 / Chapter 4.1.2.3.3 --- Cell Growth --- p.110 / Chapter 4.1.2.3.4 --- Wound Healing --- p.110 / Chapter 4.1.2.3.5 --- Angiogenesis --- p.111 / Chapter 4.1.2.3.6 --- "Anti-apoptosis, Cell Adhesion and Extracellular Matrix Organization" --- p.112 / Chapter 4.1.3 --- Further Selection of Novel Target Molecules Based on Statistical Significance and Fold Change of NEC versus SIP --- p.113 / Chapter 4.2 --- Discussion --- p.115 / Chapter CHAPTER FIVE --- Validation of Target Proteins in Plasma of NEC and SIP Patients by Enzyme-linked Immunosorbant Assay --- p.132 / Chapter 5.1 --- Results --- p.133 / Chapter 5.1.1 --- Demographic Data of the Study Group --- p.133 / Chapter 5.1.2 --- "Comparison of Plasma Levels of Target Proteins between NEC, SIP and Respective Controls" --- p.134 / Chapter 5.1.3 --- Longitudinal Study of the Pre- and Post-operative Target Proteins Levels in Plasma --- p.136 / Chapter 5.2 --- Discussion --- p.138 / Chapter CHAPTER SIX --- Investigation on mRNA Expression Levels of Target Immunoregulatory Protein Molecules in Intestinal Tissue and Intestinal Cell Lines --- p.151 / Chapter 6.1 --- Results --- p.152 / Chapter 6.1.1 --- mRNA Expression Levels of Target Molecules in the Diseased Margin of Resected Intestinal Tissues of NEC and SIP patients --- p.152 / Chapter 6.1.2 --- mRNA Expression Levels of Target Molecules in the Macroscopically Normal and Diseased Margin of Resected Intestinal Tissues of NEC and SIP patients --- p.154 / Chapter 6.1.3 --- mRNA Expression Levels of Target Molecules in Human Intestinal Cell Lines upon LPS and PAF Challenge --- p.156 / Chapter 6.1.3.1 --- FHs-74 Int Cell Line --- p.156 / Chapter 6.1.3.2 --- Caco-2 Cell Line --- p.157 / Chapter 6.2 --- Discussion --- p.158 / Chapter CHAPTER SEVEN --- General Discussion --- p.171 / Chapter 7.1 --- Overall Findings --- p.171 / Chapter 7.2 --- Limitations of Study --- p.174 / Chapter 7.3 --- Future Investigations --- p.177 / References --- p.179

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: An association between maternal human immunodeficiency virus (HIV) infection and Necrotizing Enterocolitis (NEC) in preterm infants has been reported. The impact of probiotics in an HIV-exposed very low birth weight (VLBW) infant on the occurrence of NEC is uncertain at present; however it is known that probiotics have protective effects against inflammation and prevent NEC. Postnatal growth restriction is a major issue in preterm, especially extremely-low-birth-weight (ELBW) infants and probiotics have been found to improve feeding tolerance in preterm infants. Human milk oligosaccharides (HMO) also known as the prebiotics of human milk, are known to have bifidogenic and anti-adhesive effects. Infants that receive human milk show a reduced incidence of NEC compared to those who receive infant formula. Very little is known about the composition of breast milk in the HIV-infected mother. Objective: The primary objective of the study was to assess the effect of probiotics on the incidence and severity of NEC in high-risk infants born to HIV-positive and HIV-negative women. The secondary objectives were to assess the effect of probiotic administration on feeding tolerance and growth outcomes of HIV-exposed but uninfected preterm infants, to describe the HMO composition of HIV-infected mothers breast milk and lastly to determine if HMO composition affects the incidence of NEC in HIV-exposed preterm very low birth weight infants. Patients and Methods: A randomized, double blind, placebo controlled trial was conducted for the period July 2011 to August 2012. HIV-exposed and HIV-unexposed premature (<34 weeks gestation) infants with a birth weight of ≥500g and ≤1250g were randomized to receive either a probiotic or a placebo. The probiotic consisted of 1x109 CFU, L. rhamnosus GG and B. infantis per day and was administered for 28 days. NEC was graded according to Bell’s criteria. Anthropometrical parameters and daily intakes were monitored. Breats milk samples were analysed for oligosaccharide content. Results: 74 HIV-exposed and 110 HIV-unexposed infants were enrolled and randomized (mean birth-weight, 987g; mean gestational 28.7 weeks). The incidence of death and NEC did not differ significantly between the HIV-exposed and unexposed groups but a significantly higher NEC incidence was found in the control group. There was no difference in the average daily weight gain for treatment groups or HIV exposure. The HIV-exposed group achieved significantly higher z-scores for length and head circumference at day 28 than the unexposed group (p<0.01 and p=0.03, respectively). There were no differences in the incidence of any signs of feeding intolerance and abdominal distension between the groups. Our results show significantly higher absolute concentrations of 2’-fucosyllactose, laco-N-tetraose and lacto-N-fucopentaose 1 and higher relative abundance of 3’-sialyllactose, difucosyl-lacto-N-tetraose and fucosyl-disialyllacto-N-hexaose in HIV-infected compared to -uninfected Secretor women. DSLNT concentrations were significantly lower in the breast milk of mothers whose infants developed NEC compared to infants without NEC. Conclusion: Probiotic supplementation reduced the incidence of NEC in the premature infants; however results failed to show a lower incidence of NEC in HIV-exposed premature infants. Probiotic supplementation did not affect growth outcomes or the incidence of any signs of feeding intolerance in HIV-exposure. The data confirms previous reports that HIV-infected mothers have higher 3’sialyllactose milk concentrations. Most intriguing though, the data also indicates that low levels of DSLNT in the mother’s milk increase the infant’s risk for NEC, which is in accordance with results from previously published animal studies and warrants further investigation. / AFRIKAANSE OPSOMMING: Inleiding: ʼn Verwantskap tussen moederlike menslike immuniteitsgebreksvirus (MIV) en nekrotiserende enterokolitis (NEK) in premature babas is aangemeld. Die impak van probiotika in ʼn MIV-blootgestelde baie lae geboortemassa (BLGM) baba op die voorkoms van NEK is tans nog onseker, maar dit is wel bekend dat probiotika ʼn beskermende effek het teen inflammasie en die voorkoms van NEK. Nageboortelike groei beperkings is ʼn groot probleem in premature, veral ekstreme lae geboortemassa (ELGM) babas. Daar is gevind dat probiotika voeding toleransie in premature babas kan verbeter. Menslike melk oligosakkariede (MMO), ook bekend as die prebiotika van menslike melk, is bekend om bifidogeniese en anti-kleef effekte te hê. Babas wat moedersmelk ontvang toon ʼn verlaagde voorkoms van NEK in vergelyking met diegene wat baba formule melk ontvang. Baie min inligting is bekend oor die samestelling van borsmelk in die MIV-positiewe moeder. Doel: Die primêre doel van die studie was om die effek van probiotika op die voorkoms en die graad van NEK in hoë risiko babas van MIV-positiewe en MIV-negatiewe vroue te bepaal. Die sekondêre doelwitte was om die effek van probiotika op voeding verdraagsaamheid en groei uitkomste van MIV-blootgestelde, maar nie- geinfekteerde premature babas te evalueer sowel as die MMO samestelling van MIV-positiewe moeders se borsmelk te beskryf en laastens om die invloed van die MMO samestelling op die voorkoms van NEK in baie lae geboortegewig MIV-blootgestelde premature babas te beskryf. Pasiënte en Metodes: ʼn Gerandomiseerde, dubbelblinde, plasebo-beheerde studie is vir die tydperk Julie 2011 tot Augustus 2012 onderneem. MIV-blootgestelde en nie-blootgestelde premature (<34 weke) babas met 'n geboorte gewig van ≥500g en ≤1250g was ewekansig verdeel om probiotika of plasebo te ontvang. Die probiotika het bestaan uit 1x109 kolonie vormende eenhede, L. rhamnosus GG en B. infantis per dag en is toegedien vir 28 dae. NEK is gegradeer volgens Bell se kriteria. Antropometriese parameters en daaglikse inname is gemonitor. Borsmelk monsters is geanaliseer vir oligosakkaried inhoud. Resultate: 74 MIV-blootgestelde en 110 MIV-nie-blootgestelde babas is ingesluit en ewekansig ingedeel (gemiddelde geboorte gewig, 987g, gemiddelde gestasie 28,7 weke). Die voorkoms van die sterftes en NEK het nie beduidend verskil tussen die MIV-blootgestelde en nie-blootgestelde groepe nie, maar 'n beduidende verskil is gevind vir NEK voorkoms tussen die studie en die kontrole groep. Daar was geen verskil in die gemiddelde daaglikse gewigstoename tussen die behandelings groepe of MIV-blootstelling nie. Die MIV-blootgestelde groep het beduidend hoër z-tellings vir lengte en kopomtrek op dag 28 getoon teenoor die nie-blootgestelde groep (p <0.01 en p = 0,03, onderskeidelik). Daar was geen verskille in die voorkoms van voeding onverdraagsaamheid en abdominale distensie tussen die twee groepe nie. Ons resultate dui op aansienlik hoër absolute konsentrasies van 2'-fucosyllactose, laco-N-tetraose en lakto-N-fucopentaose 1 en hoër relatiewe voorkoms van 3'-sialyllactose, difucosyl-lakto-N-tetraose en fucosyl-disialyllacto-N-hexaose in MIV-positiewe vroue in vergelyking met-negatiewe Sekretor vroue. DSLNT konsentrasies was aansienlik laer in die melk van moeders wie se babas NEK ontwikkel het in vergelyking met babas sonder NEK. Gevolgtrekking: Probiotika aanvullings verminder die voorkoms van NEK in premature babas, maar die resultate kon nie ʼn laer voorkoms van NEK in MIV-blootgestelde premature babas bewys nie. Probiotiese aanvulling het geen invloed op groei uitkomste of die voorkoms van voeding onverdraagsaamheid in MIV-blootstelling getoon nie. Die data bevestig vorige verslae wat aandui dat MIV-besmette moeders hoër 3'sialyllactose borsmelk konsentrasies het. ʼn Interessante aspek is dat lae vlakke van DSLNT in die moeder se melk beduidend is van ʼn verhoogde risiko vir NEK, wat in ooreenstemming is met die resultate uit voorheen gepubliseerde dier studies en regverdig verdere ondersoeke.