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Characterisation of the Potential of Probiotics Or Their Extracts As Therapy for Skin

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Characterisation of the Potential of Probiotics Or Their Extracts As Therapy for Skin Characterisation of the potential of probiotics or their extracts as therapy for skin A thesis submitted to the University of Manchester for the Degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences 2014 Walaa Mohammedsaeed, Master of Science (MSc) School of Medicine Table of Contents Contents Table of Contents .............................................................................................................. 2 Table of Figures ................................................................................................................ 5 List of Tables .................................................................................................................... 8 List of Abbreviations ........................................................................................................ 9 1 Abstract ....................................................................................................................... 11 2 Declaration .................................................................................................................. 12 3 Copyright Statement .................................................................................................... 13 4 Acknowledgements ...................................................................................................... 14 5 The author ................................................................................................................... 15 6 Publications arising from this Thesis ............................................................................ 15 Chapter One .................................................................................................................... 16 Introduction ................................................................................................................... 16 1.1Skin structure and barrier function .............................................................. 16 1.2 Wounding and the immune response .......................................................... 26 1.3 Probiotics ................................................................................................... 40 1.4 Aims and Hypotheis ................................................................................... 55 Chapter Two ................................................................................................................... 57 Methods and Materials .................................................................................................. 57 2.0 Reagents and materials ............................................................. ….………..57 2.1 Bacterial cell culture .................................................................................. 57 2.2 Screening of inhibitory activity of L. rhamnosus GG .................................. 60 2.3 Fractionation of the L. rhamnosus GG lysate by Reverse Phase Liquid Chromatography .............................................................................................. 62 2.4 Mammalian cell culture .............................................................................. 68 2.5 Wound healing study in vitro and ex vivo models ....................................... 69 2.6 Microarray study ........................................................................................ 75 2.7 Enzyme Linked Immunosorbent Assays (ELISA) ...................................... 77 2.8Statisticalanalysis............................................................................................77 Chapter Three ................................................................................................................. 78 An in vitro investigation into the effects of species of Lactobacilli on toxicity of S. aureus to primary human keratinocytes ................................................................... 78 3.0 Bacterial growth curves .............................................................................. 79 3.1 The effect of S. aureus on the viability of keratinocyte monolayers ........... 81 3.2 The effect of probiotic species on the viability of keratinocytes .................. 82 2 3.3 Specific species of Lactobacilli protect keratinocyte from the pathogenic effects of S. aureus........................................................................................... 84 3.4 The effect of L. rhamnosus GG lysates and spent culture fluid on keratinocyte monolayers infected with S. aureus. ............................................. 86 3.5 The protective effect of L. rhamnosus GG or lysate is not time-dependent.. 88 3.6 Mechanisms of protection by L. rhamnosus GG ......................................... 91 3.7 Discussion ................................................................................................ 101 Chapter Four ................................................................................................................. 106 Fractionation of Lactobacillus rhamnosus GG lysate ................................................. 107 4.0 Heat-killed or protease-treated lysate does not protect keratinocytes from S. aureus. ........................................................................................................... 107 4.1 Separation of L. rhamnosus GG lysate proteins by Reverse Phase Liquid Chromatography (RP-LC) and analysis by SDS-PAGE .................................. 109 4.2 Specific L. rhamnosus GG lysate fractions protected keratinocyte from S. aureus infection ............................................................................................. 112 4.3 Specific fractions of of L. rhamnosus GG lysate inhibit the growth of S. aureus ............................................................................................................ 113 4.4 Specific fractions of of L. rhamnosus GG lysate have anti-adhesive action against S. aureus ............................................................................................ 114 4.5 Anti-adhesive activity of the 50% acetonitrile fraction from L. rhamnosus GG lysate ....................................................................................................... 116 4.6 Further purification of the 50% and 60% acetonitrile fractions from L. rhamnosus GG lysate using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) ......................................................................... 117 4.7 Identification of proteins from the 50% and 60% acetonitrile fractions using Tandem Mass spectroscopy (MS/MS) ............................................................ 125 4.8 Discussion ................................................................................................ 128 Chapter Five ................................................................................................................. 133 An in vitro study to investigate the potential use of probiotic lysates as a therapy for wound healing ........................................................................................... 133 5.0 Probiotic lysates modulate the re-epithelialisation of keratinocyte monolayers in a species-dependent manner ....................................................................... 134 5.1Effect of L. rhamnosus GG and L. reuteri lysates on the rate of keratinocyte migration ....................................................................................................... 136 5.2 Effect of L. rhamnosus GG lysate and L. reuteri lysate on the rate of keratinocyte proliferation ............................................................................... 138 5.3 Effect of the L. rhamnosus GG lysate and L. reuteri lysate on keratinocyte re- epithelialisation in the presence of Mitomycin C ............................................ 139 5.4 Genome-wide Affymetrix microarray study of the effects of the L. rhamnosus GG lysate on keratinocyte gene expression and quantitative RT-PCR studies............................................................................................................ 142 3 5.5 Direct measurement of CXCL2 and FGF7 secretion by keratinocyte cultures ...................................................................................................................... 148 5.6 L. rhamnosus GG lysate alters the expression of CXCR2 and FGFR2 protein receptors in keratinocyte cultures ................................................................... 150 5.7 Effect of CXCR2& FGFR2 neutralizing antibodies on the healing of scratched keratinocyte cultures ....................................................................... 151 5.8 Discussion ................................................................................................ 155 Chapter Six ................................................................................................................... 161 An investigation into the effects of Lactobacillus rhamnosus GG lysate on the re-epithelialisation of ex vivo wounded skin ................................................................ 161 6.0 L. rhamnosus GG lysate stimulated re-epithelialisation of human skin maintained in organ culture ............................................................................ 162 6.1 L. rhamnosus GG lysate increases thickness of the epidermis in wounded skin ................................................................................................................ 164 6.2 L. rhamnosus GG lysate stimulates cell migration in wounded skin .......... 167 6.3 L. rhamnosus GG lysate
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