Präbiotikasupplementation Schwangerer und ihre Wirkung auf die mütterliche und kindliche Darmflora sowie auf ausgewählte fetale Immunparameter [Elektronische Ressource] : eine randomisierte, doppelblinde, placebo-kontrollierte Pilotstudie / vorgelegt von Rania Shadid

Rania Shadid

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 26
• Langue: Deutsch

Extrait

Aus der Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen Kinderspital der
Ludwig-Maximilians-Universität München
Direktor: Prof. Dr. med. D. Reinhardt
&
dem GSF Forschungszentrum für Umwelt und Gesundheit
Institut für Molekulare Immunologie, München
Institutsleiter: Prof. Dr. D. J. Schendel


Präbiotikasupplementation Schwangerer und ihre Wirkung
auf die mütterliche und kindliche Darmflora sowie
auf ausgewählte fetale Immunparameter -
eine randomisierte, doppelblinde, placebo-kontrollierte Pilotstudie







Dissertation
zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München


vorgelegt von

Rania SHADID

aus

Tripolis

2007 From the Children’s Clinic and Children’s Policlinic at the Dr. von Hauner Children’s Hospital of
the Ludwig-Maximilians-University Munich
Director: Prof. Dr. med. D. Reinhardt
&
the GSF-National Research Center for Environment and Health
Institute of Molecular Immunology, Munich
Director: Prof. Dr. D. J. Schendel


Effects of prebiotic supplementation during pregnancy
on maternal & neonatal gut microbiota, as well as
on selected foetal immune parameters -
a randomised, double-blind, placebo-controlled pilot study







Thesis
for obtaining the doctoral degree in human biology
at the faculty of medicine
Ludwig-Maximilians-University in Munich


submitted by

Rania SHADID

from

Tripolis

2007 Mit Genehmigung der Medizinischen Fakultät
der Universität München






1. Berichterstatter: Herr Prof. Dr. med. B. Koletzko

Mitberichterstatter: Herr Prof. Dr. med. G. Enders
Herr Prof. Dr. med. A. Wagner

Mitbetreuung durch den
promovierten Mitarbeiter: Frau Dr. med. S. Krauss-Etschmann



Dekan: Herr Prof. Dr. med. D. Reinhardt

Tag der mündlichen Prüfung: 12.06.2007
Table of contents

1. INTRODUCTION …………………………………………...………………………....
1
1.1 The gastrointestinal tract ………………………...………………….…………….. 1
1.2 Probiotics …………………………………...…………………………………...….. 3
1.3 Prebiotics .…...…………………………………………………………………..…... 5
1.4 The immune system - an overview ……………...………………………………..... 6
1.5 Chemokines & chemokine receptors ……………………..……………………...... 8
1.6 Immune modulatory effects of probiotics & prebiotics ………….………..……... 10

2. AIMS ………………………………………………………..……………………....... 11
2.1 Objectives ………………………………………………………..…………..……… 11
2.2 Outcome parameters ………………………………………………….……............. 11

3. STUDY DESIGN, MATERIAL & METHODS …………...………………………… 12
3.1 Study design ………………………………………………………………………… 12
3.1.1 Study participants ………………………………………………..……….... 12
3.1.2 Dietary intervention ……………………………………………...………… 13
3.1.3 Randomisation & allocation .………………………………..……..……..... 14
3.1.4 Questionnaires ……………………………………………………………... 14
3.1.5 Study samples ...……………..…………………………...……………….... 15
3.2 Materials ……………………………………………..………...…………………… 16
3.2.1 Instruments & software …………………………………….…...…............. 16
3.2.2 Consumables ……………………………………………………..…...…..... 16
3.2.3 Reagents …………………………………………….……………..……..... 17
3.2.4 Antibodies ………………………………………...………..…………........ 18
3.3 Methods ………………………………………………..............……………………. 19
3.3.1 Microbial stool analyses ………………………….….…………..………… 19
3.3.2 Flow cytometry ……………………………………………..…………….... 20
3.3.3 Stimulation assay ………….…………………………..………………........ 26
3.3.4 Multiplex cytokine array ……………………………………………..…..... 26
3.3.5 Statistical analysis …………………………………………………………. 27

4. RESULTS ………………………………………………..…………………………… 28
4.1 Recruitment of study participants & sample collection…………………………... 28
4.2 Analyses of questionnaires …………...…………………………………………...... 30
4.2.1 Baseline characteristics …………………….…………………..………....... 30
4.2.2 Study population at delivery ………………….……………………….….... 30
4.2.3 Follow-up interview ………………………….……………….…………… 34
4.3 Analyses of stool samples …………………………………………….…………….. 35
4.3.1 Maternal stool samples ….…………………………………………………. 36
4.3.2 Neonatal stool samples …………………………………………………….. 43
4.3.3 Microbiota in maternal & neonatal samples …..………………………….... 49
4.3.3.1 Diversity index ….…………………………….………………….. 49
4.3.3.2 Correlations ……………………………..…...……………............ 51
4.3.3.3 Similarity index ……………………………..…...……………...... 53
4.4 Analyses of CB samples …………………………………………...……………….. 54
4.4.1 Flow cytometry data ………………………………………..……………… 54
+ +4.4.1.1 Chemokine receptor expression on CD4 & CD8 T cell subsets …...…... 54
high +4.4.1.2 CD25 expression on CD4 T cell subsets …………………………….. 57
I

+ + +4.4.1.3 TLR2 & TLR4 expression on CD14 monocytes ….…………………… 58
4.4.2 Multiplex array data ……………………………………………………….... 59
4.4.2.1 Cytokine expression in Der p1, BLG, LPS & OVA stimulated samples ..... 59
4.4.2.2 Cytokine expression in Con A & SEB stimulated samples ..……………... 62

5. DISCUSSION …………………………………………...……………………………. 64
5.1 Microbiota analyses …………………………………………...……………….….... 64
5.1.1 Maternal vaginal pH ………………………………………..……………..... 64
5.1.2 Maternal stool samples …………………………………………………....... 65
5.1.3 Neonatal stool samples ………………………………………..……………. 68
5.1.4 Comparison of maternal & neonatal microbiota ...………….…………….... 70
5.2 CB analyses …………………………………………...……………………………... 72
5.2.1 Phenotypical characterisation of CB ……………………………………….. 73
5.2.2 Functional characterisation of CB …………………….……………………. 77
5.3 Potential confounders & outlook …….……………………………........................... 80

6. SUMMARY ………………………………………...…………………………………. 81

7. ZUSAMMENFASSUNG …..…………………...……………………………..……… 82

8. REFERNECES ..……………………………………...……………………...……….. 83

9. APPENDIX ………………………………………………………………………….... 96
9.1 List of abbreviations ……………………………………...………………..……...... 96
9.2 Index of figures …………………………………...…………………………………. 98
9.3 Index of tables ……………………………………..……………………………….... 100
9.4 Primers and probes used for the duplex 5 ′ nuclease assay ……………………...... 101
9.5 Study sheets ………………………………………………………………………….. 104
9.5.1 Parent information sheet …………………………………………………..... 104
9.5.2 Inclusion & exclusion criteria sheet ...…………………………………….... 106
9.5.3 Informed consent sheet ……………………………………………………... 107
9.5.4 Instruction sheet …………………………………………………………….. 108
9.6 Study questionnaires ………………………………………………………………... 109
9.6.1 Questionnaire 1: Allergies ………………………………………………….. 109
9.6.2 Questionnaire 2: Stool frequency, consistency & regurgitation ……..……... 111
9.6.3 Questionnaire 3: Vaginal pH ……………………………………………….. 112
9.6.4 Questionnaire 4: Compliance & tolerance …...…………………………….. 113
9.6.5 Questionnaire 5: Follow-up interview …………………………………........ 115
9.7 Acknowledgements …………………………………………………………..…….... 117
9.8 Curriculum vitae ……...…………………...………………………………………... 119
9.9 Publication & abstract list ……………...………………...………………………… 120








II
Parts of this work were presented as posters at

ththe 36 annual meeting of the DGfI German Society of Immunology, DGfI Congress 2005
Kiel, Germany
Effect of maternal prebiotic supplementation on selected foetal immune parameters.
R. Shadid, C. S. Falk, D. J. Schendel, C. Beermann, B. Stahl , S. Krauss-Etschmann, B. V.
Koletzko; Immunobiology 201: p 420; 2005.

and at

ththe 39 annual meeting of the European Society of Paediatric Gastroenterology Hepatology
and Nutrition, ESPGHAN Congress 2006, Dresden, Germany
Effect of maternal prebiotic supplementation on maternal and foetal microbiota as well
as on selected neonatal immune parameters.
R. Shadid, M. Haarman, J. Knol, C. Beermann, D. Rjosk-Dendorfer, D. J. Schendel, B. V.
Koletzko, S. Krauss-Etschmann; Journal of Pediatric Gastroenetrology and Nutrition 42(5);
2006.


































III INTRODUCTION
1. INTRODUCTION

1.1 The gastrointestinal tract

For a long time the primary function of the gastrointestinal tract (GIT) had simply been
considered to digest and absorb nutrients and to excrete waste end products [1, 2]. The
assertion of Josh Billings “a good reliable set of bowels is worth more to a man than any
quantity of brains” (1818-1875) represents a milestone in the knowledge of the digestive tract.
Today, the GIT is known to play a central role in general well being and health [3]. With up to
400 m2, it is not only the largest body area interacting with the environment, but also the
largest organ of immune defence in the human body [4]. The GIT is a complex, heavily
populated and diverse eco