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Title Page

Contents

Chapter 1. A Lactic Acid Bacterium Isolated from Kimchi Ameliorates Intestinal Inflammation in DSS-induced Colitis 8

ABSTRACT 9

INTRODUCTION 10

MATERIALS AND METHODS 12

Isolation of lactic acid bacteria 12

Identification of lactic acid bacteria 12

Mice 13

Induction of experimental colitis and administration of LS2 13

Determination of disease activity index (DAI) 14

Histological assessment 14

Assay of myeloperoxidase activity 14

Isolation of lamina propria lymphocytes 15

Flow cytometry and measurement of cytokine 16

RESULTS and DISCUSSION 17

LS2 ameliorates DSS-induced colitis 17

Administration of LS2 attenuates histological damage 18

The effect of LAB on myeloperoxidase activity in DSS-induced colitis 18

The induction of inflammatory cytokines is reduced by LS2 administration 19

The Th1 cell population and macrophages are reduced by LS2 administration 19

Figures 22

REFERENCES 28

Chapter 2. Involvement of TR2 in the pathogenesis of TNBS-induced colitis in mice 33

ABSTRACT 34

INTRODUCTION 35

MATERIALS AND METHODS 38

Mice 38

Induction of colitis 38

Treatment with monoclonal antibody 39

Assessment of inflammation and histologic grading of colitis 39

Cytokines 40

Measurement of serum IgG subclasses 40

Assay of MPO activity 41

RESULTS 42

TR2-/- mice are protected from TNBS-induced colitis(이미지참조) 42

Lack of increased proinflammatory cytokines and humoral responses against TNBS-induced Colitis in TR2-/- mice(이미지참조) 42

TR2 mAb reduces the symptoms of TNBS-induced colitis 43

DISCUSSION 44

Figures 47

REFERENCE 54

Chapter 3. The bactericidal effect of an ionizer under low concentration of ozone 59

ABSTRACT 60

INTRODUCTION 61

MATERIALS AND METHODS 62

Generating air ions 62

Bacterial strains and culture conditions 62

Exposure to negative and positive ions 62

Ozone quantification 63

Ion capture 64

ROS assay 64

Measurement of 8-Oxo-dG 65

Assay of aconitase activity 66

Adenylate energy charge (AEC) 66

RESULTS 68

Survival of bacteria after ionizer exposure 68

ROS 69

Oxidative DNA damage 70

Aconitase 70

Energy charge values 70

DISCUSSION 72

Figures 74

REFERENCES 81

초록보기

Chapter 1. A Lactic Acid Bacterium Isolated from Kimchi Ameliorates Intestinal Inflammation in DSS-induced Colitis

Some species of lactic acid bacteria have been shown to be beneficial in inflammatory bowel disease (IBD). In the present study, a strain of lactic acid bacterium (Lactobacillus paracaseiLS2) was isolated from the Korean food, kimchi, and was shown to inhibit the development of experimental colitis induced by dextran sulfate sodium (DSS). To investigate the role of LS2 in IBD, mice were fed DSS in drinking water for seven days along withLS2 bacteria which were administered intragastrically to some of the mice, while phosphate-buffered saline (PBS) was administered to others (the controls). The administration of LS2 reduced body weight loss and increased survival, and disease activity indexes (DAI) and histological scores indicated that the severity of colitis was significantly reduced. The production of inflammatory cytokines and myeloperoxidase (MPO) activity also decreased. Flow cytometry analysis showed that the number of Th1 (IFN-y) population cells was significantly reduced in the LS2-administered mice compared with the controls. The administration of LS2 induced the increase of CD4+FOXP3+ Treg cells, which are responsible for IL-10. Numbers of macrophages (CD11b+ F4/80+), and neutrophils (CD11b+ Gr-1+) among lamina propria lymphocytes (LPL) were also reduced. These results indicate that LS2 has an anti-inflammatory effect and ameliorates DSS-induced colitis.

Chapter 2. Involvement of TR2 in the pathogenesis of TNBS-induced colitis in mice

The TNFR-related 2 (TR2, HVEM, TNFRSF14), induces bidirectional signals via Ig superfamily members B and T lymphocyte attenuator (BTLA) and CD160. It also activates T cells through the TNF family member LIGHT and promotes the development of inflammation. We used hapten 2,4,6-trinitrobenzene sulfonic acid(TNBS)-induced inflammatory bowel disease (IBD) to investigate the role of TR2 in IBD as well as in the increased myeloperoxidase (MPO) activity and increased levels of proinflammatory and Th1 cytokines accompanying tissue damage, goblet cell loss, crypt distortion and immune cell infiltration. Remarkably, the effects of TNBS on TR2-/- mice were much milder. Administration of antagonist TR2 mAb to TR2+/+ mice also protected them from all these consequences of TNBS treatment. We conclude that TR2 plays an important role in the development of TNBS-induced experimental colitis.

Chapter 3. The bactericidal effect of an ionizer under low concentration of ozone

Several mechanisms have been suggested for the bactericidal action of ionizers including electrical phenomena, effects of negative and positive ions and electrostatic repulsion. Negative and positive ions have indeed been shown to have bactericidal effects. In addition, since ozone is generated along with ions, these may contribute to the bacterial killing. In this study, we used a newly developed ionizer to establish that ions generated by the ionizer are bactericidal. The effect of ions on bacterial killing was compared with that of ozone using ion trap. In our experiments, operation of ionizers caused increasing the level of 8-oxo-dG, which indicate the oxidative damage of DNA. And ionizer caused decreasing the aconitase activity, which is known to be sensitive to the ROS. The ionizer further affected on the adenylate energy charge of the cell. These results indicated that negative and positive ions generated by ionizer induced oxidative stress which can damage to bacterial survival. In this study, we used an ion generator with the aim of clarifying whether its effect on bacterial cells was due to ions or ozone, and, if ions, how ions exerted their effects.

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