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Role of resistant starch and probiotics in colon inflammation
Author:Amansec, Sarah Gracielle
Publication Date:2005
DOI:https://doi.org/10.26190/unsworks/23022
License:https://creativecommons.org/licenses/by-nc-nd/3.0/au/Link to license to see what you are allowed to do with this resource.
Downloaded from http://hdl.handle.net/1959.4/23041 in https://unsworks.unsw.edu.au on 2022-09-11
i
ROLE OF RESISTANT STARCH AND PROBIOTICS
IN COLON INFLAMMATION
By
Sarah Gracielle Amansec
A thesis submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy
School of Biotechnology and Biomolecular Sciences Faculty of Sciences
The University of New South Wales
April 2005
ii
Certificate of Originality
I hereby declare that this submission is my own work and to the best of my knowledge it
contains no material previously published or written by another person, nor material
which to a substantial extent has been accepted for the award of any other degree or
diploma at UNSW or any other educational institution, except where due
acknowledgement is made in the thesis. Any contribution made to the research by others,
with whom I have worked at UNSW or elsewhere is explicitly acknowledged in the thesis.
I also declare that the intellectual content of this thesis is the product of my own work,
except to the extent that assistance from others in the project’s design and conception or
in style, presentation and linguistic expression is acknowledged.
Sarah Gracielle Amansec
iii
Table of Contents
Title Page…………………………………………………………………………………… i Certificate of Originality...................................................................................................... ii Acknowledgements………………………………………………………………………… vi List of Publications………………………………………………………………………… vii List of Tables………………………………………………………………………………. viii List of Figures……………………………………………………………………………… ix List of Abbreviations............................................................................................................. xiii
Abstract…………………………………………………………………………………….. 1 Chapter 1. General Introduction…………………………………………………………. 3 1.1. Inflammatory Bowel Diseases…………………………………………………………. 3 1.2. Gut Microbes: Friend or Foe in Bowel Inflammation…………………………………. 3 1.2.1. Functions of the Gut Microflora…………………………………………………... 7 1.2.2. Microorganisms and Bowel Inflammation………………………………………… 13 1.2.3. Diversity of the Mucosa-Associated Bacteria in Bowel Inflammation……………. 18 1.3. Mucosal Immunity and Bacteria in Bowel Inflammation……………………………… 22 1.3.1. Gut Mucosal Barrier Function in Inflammation…………………………………… 22 1.3.2. Mucosal Immune Response in Intestinal Inflammation…………………………… 22 1.3.3. T cell Responses in Inflammatory Bowel Diseases……………………………….. 24 1.4. Treatments in IBD…………………………………………………………………….. 28 1.5. Use of Functional Foods in IBD……………………………………………………….. 32 1.5.1. Probiotics………………………………………………………………………….. 32 1.5.2. Prebiotics………………………………………………………………………….. 37 1.5.2.1. Role of Resistant Starch in Bowel Health and Inflammation…………………. 39 1.5.3. Synbiotics…………………………………………………………………………. 43 1.6. Hypothesis……………………………………………………………………………… 44 1.7. Aims……………………………………………………………………………………. 45
Chapter 2. Optimization of the Trinitrobenzene Sulfonic Acid Murine Model of Colitis in BALB/c Mice…………………………………………………………………….
46
2.1. Introduction…………………………………………………………………………….. 46 2.2. Materials and Methods…………………………………………………………………. 48 2.2.1. Animals……………………………………………………………………………. 48 2.2.2. Induction of experimental colitis………………………………………………….. 48 2.2.3. Assessment of severity of colon inflammation……………………………………. 49 2.2.4. Histological analysis of colon tissues……………………………………………... 49 2.2.5. Organ culture conditions and cytokine assays…………………………………….. 49 2.2.6. Determination of colonic bacterial concentration…………………………………. 50 2.2.7. Statistical analyses………………………………………………………………… 51 2.3. Results………………………………………………………………………………… 52 2.3.1. Comparison of colitis activity in BALB/c mice induced with 2.5 mg and 2.0 mg TNBS in 50% ethanol……………………………………………………………………….
52
2.3.2. Effect of varying the ethanol concentration affects the intensity of disease activity in BALB/c mice…………………………………………………………………………….
52
2.3.3. Development of TNBS-induced colitis in BALB/c mice…………………………. 56 2.3.4. Colonic bacterial concentrations in BALB/c mice induced with TNBS………….. 63 2.4. Discussion……………………………………………………………………………… 66
iv
Chapter 3. Feeding of High Amylose Maize Resistant Starch Diet to BALB/c Mice with Experimental Induced Colon Inflammation………………………………………..
70
3.1. Introduction……………………………………………….............................................. 71 3.2. Materials and Methods…………………………………………………………………. 73 3.2.1. Animals……………………………………………………………………………. 73 3.2.2. High amylose maize resistant starch diet………………………………………….. 73 3.2.3. Experimental design……………………………………………………………….. 75 3.2.4. Microscopic assessment of colonic tissue…………………………………………. 78 3.2.5. Isolation of lamina propria mononuclear cells (LPMCs)………………………….. 78 3.2.6. Determination of mucosal cytokine production in colon tissues………………….. 79 3.2.7. Cytokine assay by ELISA…………………………………………………………. 79 3.2.8. RNA extraction and RT-PCR analyses of colonic lymphocytes………………….. 80 3.2.9. Short chain fatty acid quantification………………………………………………. 81 3.2.10. Enumeration of colonic microorganisms using selective media…………………. 81 3.2.11. Extraction of nucleic acid from colonic pellet…………………………………… 82 3.2.12. PCR amplification of colonic pellet DNA……………………………………….. 83 3.2.13. Denaturing gradient gel electrophoresis (DGGE) analysis………………………. 83 3.2.14. DNA sequencing analysis………………………………………………………... 83 3.2.15. Statistical analyses……………………………………………………………….. 84 3.3. Results………………………………………………………………………………….. 85 3.3.1. Assessment of disease activity of colitic mice fed with 30% unmodified high amylose maize resistant starch diet………………………………………………………….
85
3.3.2. Evaluation of colon pathology of colitic mice on 30% unmodified high amylose maize resistant starch diet…………………………………………………………………..
88
3.3.3. Effect of feeding 30% unmodified high amylose maize resistant starch diet on cytokine gene expression in the lamina propria mononuclear cells of colitic mice…………
91
3.3.4. Cytokine production from cultured LPMC of colitic mice fed with 30% unmodified high amylose maize resistant starch diet……………………………………….
91
3.3.5. Changes in the gut microbial populations of colitic mice on 30% unmodified high amylose maize resistant starch diet…………………………………………………….
94
3.3.6. Effects of concentration of unmodified high amylose maize resistant starch affects amelioration of colon inflammation…………………………………………………
103
3.3.7. Supplementation of different concentrations of unmodified and modified high amylose maize resistant starch diet to colitic mice………………………………………….
111
3.4 Discussion………………………………………………………………………………. 117 3.4.1. Recovery from colon inflammation observed in low concentration high amylose maize resistant……………………………………………………………………………….
117
3.4.2. Microbial profile of colonic contents of BALB/c mice induced with TNBS colitis 119 3.4.3. Pro-inflammatory and anti-inflammatory cytokine production in colitic mice supplemented with high amylose maize starch diet…………………………………………
122
3.4.4. Effect of high amylose maize resistant starch on short chain fatty acid production in TNBS-induced colitis…………………………………………………………………….
123
Chapter 4. Effects of Selected Probiotic Bifidobacterium and Lactobacillus Strains on the TNBS-colitis Murine Model…………………………………………………………...
125
4.1. Introduction…………………………………………………………………………….. 125 4.2. Materials and Methods…………………………………………………………………. 127 4.2.1. Bacteria and culture conditions……………………………………………………. 127 4.2.2. Induction of colitis………………………………………………………………… 127 4.2.3. Experimental designs……………………………………………………………… 128 4.2.4. Generation of lamina propria mononuclear cells (LPMCs)………………………. 130
v
4.2.5. Cytokine assay…………………………………………………………………….. 130 4.2.6. Assessment of colon inflammation………………………………………………... 131 4.2.7. Bacterial translocation from colon………………………………………………… 131 4.2.8. Enumeration of colonic bacteria…………………………………………………... 131 4.2.9. Statistical analyses………………………………………………………………… 132 4.3 Results………………………………………………………………………………….. 133 4.3.1. Protection provided by probiotic strains against TNBS induced murine colitis….. 133 4.3.2. Dose response effects of L. fermentum in colon inflammation…………………… 138 4.3.3. Administration of L. fermentum after induction of experimental colitis using TNBS………………………………………………………………………………………..
144
4.4. Discussion……………………………………………………………………………… 147
Chapter 5. Effects of Lactobacillus fermentum VRI 003 on the Symptoms, Cytokine Immune Responses and Faecal Profile of UC Patients in Remission…………………...
154
5.1. Introduction…………………………………………………………………………….. 154 5.2. Materials and Methods…………………………………………………………………. 156 5.2.1. Participants………………………………………………………………………… 156 5.2.2. Study design……………………………………………………………………….. 156 5.2.3. Evaluation of symptom scores…………………………………………………….. 158 5.2.4. Measurement of cytokine levels in the serum……………………………………... 158 5.2.5. Enumeration of faecal bacteria from UC patients…………………………………. 159 5.2.6. Statistical analyses………………………………………………………………… 160 5.3. Results………………………………………………………………………………….. 161 5.3.1. Study population characteristics and progress in the study……………………….. 161 5.3.2. Participants’subjective evaluation of their symptoms…………………………….. 164 5.3.3. Effects of L. fermentum VRI 003 on serum cytokine production of UC patients in remission…………………………………………………………………………………….
172
5.3.4. Bacterial analysis of faeces of UC patients in remission prior to and after treatment with L. fermentum VRI 003………………………………………………………
175
5.4. Discussion……………………………………………………………………………… 180
Chapter 6. Concluding Discussion……………………………………………………….. 185 References………………………………………………………………………………….. 192 Appendix I. Case Reports…………………………………………………………………. 217 Appendix II. Effects of placebo treatment on serum IL-10…………………………….. 242
vi
Acknowledgements
My sincerest gratitude to A/Professor Patricia Conway for her supervision, guidance and enthusiastic encouragement in this study. Thank you for showing me the significance of intestinal microbiology. I now realize that “to look good in the outside, it should be right inside”.
A/Professor Michael Grimm, my co-supervisor, for his timely and constructive comments. Also, for sharing his time in this study and knowledge on animal and clinical inflammatory bowel diseases. Thank you as well for his assistance with the TNBS model of IBD. I not only gained scientific training but have formed friendship with Susie and Rosie, who both helped optimize the model for use in our group.
A/Professor Mark Tanaka for patiently explaining to me the ins and outs of the statistical tests to be used in this study.
I like to thank Meera Karunakaran, Ping Wang, Dai Yu and A/Professor Gerald Pang for coming early with me to the animal house and for helping me process animal samples. Thanks for their advice in the immunological aspects of the study.
I am indebted to Dr Mike Taylor and Dr Johnny Queck for sharing their expertise in molecular techniques/methods used in this study.
I also like to thank Jo and Dr Fairley for their warm accommodation whenever I go up to Townsville. My sincerest thank you for their assistance in recruiting patients. Thanks are also due to Ms Ilona Kindinger who assisted in the analysis of serum samples of the patients.
Many thanks to Nedhal Elkaid, John Wilson and Kath Kimpton for accomodating me and my experiments in the animal house facilities in the School as well as for providing company and sharing funny stories during the long hours of animal experiments.
Thank you to Commonwealth of Australia, Starch Australasia and Probiomics Ltd for providing me with the scholarship and other means of assistance at various stages of this work. The work was financially supported by an ARC-SPIRT grant and Probiomics Ltd.
Thank you to the School of Biotechnology and Biomolecular Sciences for providing the exposure and opportunities to enhance my scientific skills.
Cheers to my friends! Simply for putting up with me during these stressful times! Thanks to Arvin who did all the illustrations used in this study. Thanks to Doralyn, Karen, Glecy, Volts, Maria, Trisha, Adam and my sister Anna who have been constantly humouring me to keep me sane until I finish this endeavour.
Last but not the least I want to thank my family for their continued patience, love and support.
vii
List of Publications
Papers
1. Lactobacillus fermentum VRI 003 ameliorates murine colitis by changing the nature of the mucosal immune response Submitted for consideration for publication to Gastroenterology
2. Feeding of high amylose maize resistant starch to TNBS colitic BALB/c mice Submitted for consideration for publication to Gut
3. Effects of different concentrations of high amylose maize resistant starch on experimental murine colitis Submitted for consideration for publication to Alimentary Pharmacology and Therapeutics
4. Mucosal T cell responses, microbial profiles and disease activity indices in TNBS colitic BALB/c mice Submitted for consideration for publication to FEMS Microbiology and Immunology
Conferences
1. Digestive Diseases Week (DDW) in Georgia, Atlanta, USA (2000) Oral Presentation
2. 9th International Symposium for Microbial Ecology (ISME) in Amsterdam, The Netherlands (2001) Poster Presentation
3. 4th Australasian Dietary Fibre Workshop in Newcastle, Australia (2001)Oral Presentation
4. Southpacific Digestive Diseases Symposium in Sydney, Australia (2001) Oral Presentation
5. Australian Society for Microbiology in Melbourne, Australia (2002) Oral Presentation
6. European Union (EU) and Microfunction Meeting, London, Great Britain (2004) Oral Presentation
viii
List of Tables
Table 1.1. Differentiation of the two common forms of inflammatory bowel diseases
5
Table 1.2. Potential mechanisms by which microbial constituents could cause chronic intestinal inflammation.
21
Table 1.3. Targets of biological therapy in IBD. 29 Table 1.4. Potential mode of actions of probiotics to reduce colon
inflammation.35
Table 1.5. Efficacy of probiotic strains used in IBD. 36 Table 1.6. Functional potential of resistant starches 42Table 2.1. Comparison of disease activity index (DAI) of BALB/c
induced with 2.5 mg TNBS and 2.0 mg TNBS in 50% ethanol (n=10).
53
Table 3.1. Composition of high amylose maize resistant starch diet. Three diets, 0%, 5% and 30% were prepared and reflect the concentration of resistant starch used.
74
Table 3.2. Confirmation of the presence of bifidobacteria in the colonic contents of colitic mice on 30% unmodified high amylose maize resistant starch diet.
99
Table 3.3. Identification of isolates from DGGE profile of colitic mice on 30% unmodified high amylose resistant starch diet.
101
Table 3.4. Acetate, butyrate and propionate levels 7 days after colitis induction in colitic mice fed two varieties of high amylose maize starch diet and healthy mice fed with normal mouse diet.
116
Table 4.1. Body weight profile and survival of TNBS-induced colitic mice at the day of sacrifice. Bifidobacterium and Lactobacilluswere administered for 7 days prior to colitis induction with TNBS. Values are significantly different from colitic control group at p<0.05*
135
Table 4.2. Levels of enteric bacteria in the spleens of mice after dosage with either 1x109, 1x108 or 1x107 CFU of L. fermentum or PBS every other day for 2 weeks.
142
Table 5.1. Baseline characteristics of participants (n=12). Values are expressed as frequencies, except for age.
163
Table 5.2. Concentrations (CFU g-1) of endospores detected in the faeces of UC patients in remission during placebo and L. fermentumtreatment. Results are expressed as mean concentration ± SD.
179
ix
List of Figures
Figure 1.1. Vicious cycle of inflammatory bowel disease pathogenesis. 6 Figure 1.2. Human gut microbiota (adapted from Fuller and Gibson,
1997).10
Figure 1.3. Functions of the microbiota. 11 Figure 1.4. T helper (Th) cytokine responses in inflammatory bowel
diseases. 27
Figure 2.1. Colitis activity in BALB/c mice (n=10) induced with 2.5 mg and 2.0 mg TNBS in 50% ethanol. Results are expressed as the mean DAI of surviving animals in each group ± SD.
54
Figure 2.2. Colon histology of BALB/c mice exposed to A) 2.5 mg TNBS in 50% ethanol, B) 2.0 mg TNBS in 50% ethanol and C) 50% ethanol. Colon tissues were stained with Geimsa and assessed at a magnification of 40X. Blocked arrow indicates appearance of granuloma.
55
Figure 2.3. Disease activity index (DAI) of BALB/c mice a week after exposure to different concentrations of ethanol (n=10 per group). Results presented as mean DAI and error bars correspond to SD.* significantly different from other treatments at p 0.01.
57
Figure 2.4. Histopathological changes in H&E stained sections of the colon of BALB/c mice 7 days after exposure to different concentrations of ethanol. Colons of BALB/c mice induced with a) 0%, b) 20%, c) 30% and d) 45% ethanol.
58
Figure 2.5. Disease activity index (DAI) of BALB/c mice (n=10) induced with colitis using different doses of TNBS in 45% ethanol for up to six days after induction.
59
Figure 2.6. Histological features of TNBS-induced colitis in BALB/c mice receiving different doses of TNBS in 45% ethanol; a) 1.5 mg TNBS; b) 2.0 mg TNBS, c) 2.5 mg TNBS and d) 45% ethanol. Magnification was at 40X.
61
Figure 2.7. IL-12 and IL-10 production in organ cultures of colons from BALB/c mice induced with different concentrations of TNBS at 7 days post-induction of colitis.
62
Figure 2.8 Concentrations (Log CFU.g-1 of colon contents) of the bacterial groups involved in colon inflammation detected in colitic BALB/c mice (n=10). Results are expressed as mean bacterial concentrations ± SD.
64
Figure 2.9 Bacteria detected on colitic tissue of BALB/c mice 3 days post-induction with 2.5 mg TNBS.
65
Figure 3.1. Experimental design to assess effects of high amylose maize resistant starch diet on colon inflammation induced by TNBS.
77
Figure 3.2. Weight loss of colitic mice fed 30% unmodified high amylose maize resistant starch diet or starch free diet. Observations presented 0, 1, 4, 7 and 10 days after induction of colitis with TNBS administered rectally.
86
Figure 3.3. Survival of colitic mice fed with 30% unmodified high amylose maize resistant starch diet over the 10 days after
87
x
induction of colitis with TNBS. Figure 3.4 Colon histological scores of colitic mice on 30% unmodified
high amylose maize resistant starch diet and starch free diet. 89
Figure 3.5 Representative colon sections from healthy control mice (a), ethanol control mice (b), colitic mice fed the resistant starch free diet and the 30% unmodified high amylose maize resistant starch diet 7 days after colitis induction.
90
Figure 3.6. Cytokine gene expression in lamina propria mononuclear cells (LPMCs) of colitic mice fed with 30% unmodified high amylose maize resistant starch diet.
92
Figure 3.7. T cell cytokine production in the LPMCs from colitic mice fed with 30% unmodified high amylose maize resistant starch diet.
93
Figure 3.8. Viable counts of different bacterial types from colon contents of colitic mice fed with 30% unmodified high amylose maize resistant starch diet or starch free diet detected 7 days after colitis was induced.
97
Figure 3.9. Bacterial community profiles of colonic contents from colitic mice on 30% unmodified high amylose maize resistant starch diet as determined by PCR-DGGE. Colon contents were collected 1, 4, 7 and 10 days after colitis induction with TNBS.
98
Figure 3.10. Identified DGGE bands of DNA from colonic contents of healthy control mice fed with normal mouse diet (Lanes A1, A7), ethanol control mice fed with normal mouse diet (Lanes B1, B7), colitic mice fed with resistant starch free diet (Lanes C1, C7) and colitic mice fed the 30% unmodified high amylose maize resistant starch diet (Lanes D1, D7). Colonic contents were collected from 1st (Lanes A1, B1, C1, D1) and 7th day (Lanes A7, B7, C7, D7) of colitic period.
100
Figure 3.11. Changes in weight (%) during colitic period of colitic BALB/c mice (mean ± SE of 10 mice per group) treated with different concentrations of unmodified high amylose maize starch diet.
105
Figure 3.12. Colonic damage scores of the colon from mice induced with TNBS colitis and fed different concentrations of unmodified high amylose maize resistant starch.
106
Figure 3.13a. Gene expression of pro-inflammatory and anti-inflammatory cytokines determined by RT-PCR in the colonic tissues of mice with or without treatment of low and high concentration of unmodified high amylose maize resistant starch.
107
Figure 3.13b. Mean ratio mucosal cytokine secretion of IL-4 and IFN- in the colons of colitic mice (n=10) fed the low and high concentration of unmodified high amylose maize resistant starch (mean ± SD).
107
Figure 3.14. Effect of different concentrations of unmodified high amylose maize starch on colonic content counts of different types of bacteria.
108
Figure 3.15. Total enteric counts in the colonic contents of colitic mice fed different concentrations of unmodified high amylose maize resistant starch (a) and the enteric colony types detected in
110
xi
the colonic contents after treatment with unmodified resistant starch given at different concentrations (b).
Figure 3.16. Body weight loss and survival of TNBS-induced colitic mice at the day of sacrifice.
113
Figure 3.17. Mean ratio mucosal cytokine secretion of IL-4 and IFN- in the colons of colitic mice (n=10) fed the of unmodified (UM) and modified (MD) high amylose maize resistant starch (mean ± SD). * significantly different from 30% and 5% UM high amylose maize starch diet at p 0.05.
114
Figure 3.18. Total short chain fatty acid (SCFA) concentration in the colon contents of mice fed the 5% and 30% concentration of unmodified (UM) and modified (MD) high amylose maize resistant starch diet. Colonic contents were collected 1 and 7 days after induction of colitis. Values are expressed as mean ± SD (n=8 per group).
115
Figure 4.1. Experiment design of Chapter 4 129 Figure 4.2. Ratio of IL-10:IFN- cytokines in LPMCs of L. acidophilus
and L. fermentum fed mice 7 days after induction of colitis. Results are expressed as mean ratio ± SD in 10 mice. La = L. acidophilus; Lf = L. fermentum.
136
Figure 4.3. Histological scores of colon tissue of colitic animals fed with L. acidophilus and L. fermentum In all animals, except healthy-control and ethanol-control, colitis was induced with TNBS. Values represent mean±SD. Significance (p<0.01) was observed between colitic-control group and L. fermentumfed colitic mice. La = L. acidophilus and Lf = L. fermentum.
137
Figure 4.4 Body weight loss and survival of colitic animals (n=9) 7 days after induction of colitis with TNBS.
140
Figure 4.5 Ratio of IL-4:IFN- cytokines in the LPMCs of animals fed with different concentrations of L. fermentum 7 days after the induction of colitis with TNBS.
141
Figure 4.6. Concentrations of colonic microorganisms detected 7 days after TNBS induction of colitis in colitic animals (n=9) administered with a range of oral doses of L. fermentum.
143
Figure 4.7. Body weight profile of animals (n=8) therapeutically fed with a daily dose of 1x108 CFU of L. fermentum.
145
Figure 4.8. Ratio of IL-10:IFN- cytokines in LPMCs of colitic mice given L. fermentum or healthy animals given L. fermentumfollowing the induction of colitis with TNBS.
146
Figure 5.1. Randomized, double-blind, cross-over study design using L. fermentum VRI 003 in combination with routine medication in UC patients in remission.
166
Figure 5.2. Proportion of UC patients that experienced an increase in symptoms, sustained relief of symptoms and experienced reduction of symptoms during active treatment with L. fermentum or placebo treatment (n=12). P value refers to the difference between the incidence of the symptoms in 12 patients between the active and placebo treatments.
167
Figure 5.3a. Frequency of exacerbation of bowel actions in individual patients of the placebo treatment and L. fermentum treatment. Number of bowel actions was evaluated by the patient. A
168
xii
shaded box represents an increase in bowel actions compared to the previous month.
Figure 5.3b. Cumulative exacerbation rates in the two groups (log rank statistical analysis, p=0.0037). At each monthly appointment of patient, frequency of bowel motions were evaluated in comparison with one month earlier. Hatched lines show the month of exacerbation of diarrhea in UC patients in remission.
169
Figure 5.4. Influence of L. fermentum and placebo treatment on the severity score of the abdominal pain symptoms in 12 UC patients in remission.
170
Figure 5.5. Influence of L. fermentum and placebo treatment on the general well being score of 12 UC patients in remission (n=12). L. fermentum significantly improved general well being at p=0.014.
171
Figure 5.6 Serum IL-10 concentration of UC patients in remission prior to and after 3 and 6 months dosage of L. fermentum andplacebo treatments taken concurrently with standard medication.
173
Figure 5.7. Serum IFN- concentration of UC patients in remission prior to and after 3 and 6 months dosage of L. fermentum andplacebo treatments taken concurrently with standard medication.
174
Figure 5.8. Levels of total enterics detected in the faeces of UC patients in remission during placebo and L. fermentum treatments. Results are expressed as mean log CFU per gram of faeces ± SD (n=12).
176
Figure 5.9. Frequency of detection of the various types of enteric bacteria detected in the faeces of UC patients in remission (n=12).
177
Figure 5.10. Levels of total lactobacilli detected in the faeces of UC patients in remission during placebo and L. fermentumtreatments. Results are expressed as mean log CFU per gram of faeces ± SD (n=12).
178
xiii
List of Abbreviations
CD Crohn’s Disease CFU Colony forming units ELISA Enzyme Linked Immunosorbent Assay GIT Gastrointestinal tract IBD Inflammatory Bowel Disease IFN Interferon IL Interleukin LPMC Lamina propria mononuclear cells LPS Lipopolysaccharides MAC Mac Conkey Agar MD Modified high amylose resistant starch MRS de Mann Rogosa Agar RB Raffinose Bifidobacteria Agar RCA Reinforced Clostridia Agar RS Resistant starch SCFA Short chain fatty acids TNBS Trinitrobenzene sulfonic acid TNF Tumor necrosis factor UC Ulcerative Colitis WCA Wilkins Chalgren Agar WCB Wilkins Chalgren Broth YEPD Yeast extract, Peptone, Dextrose Agar
1
Abstract
An imbalance of the T cell immune response is observed in inflammatory bowel
disease. Intestinal microbes have been linked to the disease and the disease process
leads to severe mucosal injury and systemic translocation of bacterial products.
Aminosalicylates, corticosteroids and immunomodulators reduce these aggressive
activities but are associated with potentially serious adverse events. The aim of this
work was to investigate the effects of administration of prebiotics and probiotics that
modulate the gut microflora and modulate the immune response, in ameliorating
severity of colitis.
The prebiotic, high amylose maize resistant starch was used at two different
concentrations. A number of Bifidobacterium and Lactobacillus strains were used as
probiotics. BALB/c mice were administered the prebiotics and probiotics and
intrarectally infused with 2.5 mg trinitrobenzene sulfonic acid (TNBS) in 45%
ethanol, thereby generating colitis. Mucosal cytokine responses, colonic microbial
profiles and disease activity indices were monitored.
The 5% concentration of high amylose maize resistant starch delayed progression of
TNBS colitis as evidenced by reduced weight loss, lesser tissue damage, abrogation of
the expression and synthesis of IFN- and upregulation of IL-4 and IL-10. The 30%
concentration of high amylose maize resistant starch exacerbated the inflammatory
response with an increase in acetic acid, coliforms and endopores in the colonic
contents.
Three strains of bifidobacteria and 3 strains of lactobacilli were individually screened
for their activity against TNBS colitis. Each strain had a distinctive effect on the
course of colon inflammation. Lactobacillus fermentum VRI 003 was selected for
further study as it provided most protection. The ratio of immunosuppressive
cytokines to pro-inflammatory cytokines was restored closer to the normal T cell
cytokine levels. It also reduced the incidence of translocation of enteric bacteria into
the spleens.
2
Dosing a minimum daily dose of 6x109 CFU L. fermentum VRI-003 to ulcerative
colitis patients in remission and maintained on standard therapy for 6 months
prevented the exacerbation of symptoms, including diarrhea and abdominal pain, and
improved the patient general well being. It also suppressed production of IFN- and
sustained IL-10 levels. Moreover, absence of endospores and lower numbers of
coliforms were detected in the faeces of UC patients during L. fermentum VRI-003
treatment.
In summary, 5% high amylose maize resistant starch and L. fermentum VRI 003
prevented colon inflammation by changing the nature of the T cell immune response
and modifying the colonic microflora in the murine model. The clinical evidence
supported these findings.
3
Chapter 1
General Introduction
1. 1. Inflammatory Bowel Diseases
Inflammatory bowel disease (IBD) is a chronic inflammation of the gastrointestinal
tract which occurs in two forms, Ulcerative Colitis (UC) and Crohn’s Disease (CD).
These two forms of IBD are very similar in that both exhibit a recurrent inflammation
of the gut mucosa and involve the T helper cells in regulating the immune response in
the inflamed gut. UC, however, is limited in the large intestine and almost always
affects the mucosa while inflammation seen in CD extends through the full thickness
of the bowel and can involve any section of the gastrointestinal tract. Moreover, the
immune response exhibited by UC and CD distinguishes them as well1, 2. Table 1.1
presents an overview of the distinguishing characteristics of UC and CD. This
difference will be further discussed in the section of “Mucosal Immunity and Bacteria
in Bowel Inflammation” of this chapter.
Inflammatory bowel diseases are more prevalent in developed countries and
considered to be the second most common chronic inflammatory disease behind
rheumatoid arthritis3, 4. Other non-intestinal conditions such as arthritis, inflammation
of the eye, dermatitis or psoriasis are also associated with IBD5-7. Epidemiological
data show that gut inflammation affects 30-80 subjects per 100,000 of the worldwide
population. The highest incidence of IBD is observed in western countries such as
United States, Northern Europe and Australia, wherein the highest incidence can be
seen in Jewish people8. It seems that the incidence and prevalence of IBD are
associated with the socio-economic status of the individual as IBD is less frequent in
populations exposed to poverty and war events. This contradicts the infection theory
wherein exposure to these factors leads to disease9, 10.
The specific antigens responsible for IBD remains speculative, but various pathogenic
mechanisms have been proposed to explain how IBD is perpetuated1, 11. According to
4
Sartor (2003), the most widely accepted theory of IBD pathogenesis is that chronic
intestinal inflammation is the consequence of an overly aggressive cell-mediated
immune response to commensal enteric bacteria in a genetically susceptible host12.
IBD pathogenesis is described to be a vicious cycle of unrestrained activation of the
immune system (Figure 1.1) wherein the epithelial barrier is destroyed and this allows
the uncontrolled entry of microbial and dietary components from the intestinal lumen.
This increased permeation of components triggers a local inflammatory cascade.
Immune cells such as macrophages, mast cells, lymphocytes, and natural killer cells
are recruited to eliminate the putative antigen. This recruitment then triggers the
production of excessive amounts of reactive oxygen and nitrogen species which
destroys cell membranes and releases enzymes that further increase intestinal damage
and permeability. As a consequence, more environmental components perpetuate the
destructive events. Indeed, three components of the gastrointestinal system are
obviously involved in the persistence of bowel inflammation and these are the
epithelium, the gut indigenous microflora and gut mucosal immune mediators.
Normal functions of the gastrointestinal system relies on these components, however,
breach of any one of the components’ activities perpetuates the destructive pathology
observed in IBD. The role of each component in intestinal inflammation is further
discussed in the following sections below.
5
Table 1.1. Differentiation of the two common forms of inflammatory bowel diseases
Characteristics Ulcerative Colitis Crohn’s Disease Indication Recurrent inflammation of
gut mucosa Recurrent inflammation of
gut mucosa
Pathology Limited in large intestine Any section of the gastrointestinal tract
Extent of Inflammation Mucosa Full thickness of the bowel
Immune Response T-cell mediated T-cell mediated
Type of T-cell response Th2 Th1
Triggering Factors Bacteria (Commensal or pathogen is yet to be determined)
Bacteria(Commensal or pathogen is yet to be determined)
6
Figure 1.1. Vicious cycle of inflammatory bowel disease pathogenesis.
Antigen exposure
Local immune system activation
Inflammatory cell recruitment
Reactive oxygen species release
Immuno dysregulation
Epithelial damage
Intestinal permeability
7
1. 2. Gut Microbes: Friend or Foe in Bowel Inflammation
1. 2. 1. Functions of the Gut Microflora
It is established that the gut microflora, which is also referred to as the microbiota, is
made up of a diverse range of microorganisms some of which may have positive or
negative effects or even those that have the potential to be either. (Figure 1.2).
However, the data in Figure 1.2 must be interpreted with caution as the gut microflora
is much more complex than what is presented. A lot of factors affect the microbial
profile such as the host strain, age, metabolic activities, environment, the area and
methods of isolation and identification.
The distribution and composition of microbes differ along the gastrointestinal tract.
There is a large population of facultative anaerobes i.e Lactobacillus, that inhabits the
gastric region267 and anaerobes predominate the lower region of the gut268. The spatial
profile of a region also differs significantly. It has been shown segmented,
filamentous bacteria related to clostridia attach to the epithelium269 while fusiform
and spiral shaped bacterial cells predominate the mucus layer of the epithelium
surface270. Moreover, there is a marked difference in the bacteria colonizing the gut of
different animal species271.Age of the host also determines the microflora
composition. Enterobacteria and bifidobacteria are predominant in the microflora of
infants which is thought to be of maternal origin271. Introduction of solid foods into
the diet shifts the profile to that similar of an adult271.
Most of the studies done previously were that of the microbial content of the faeces.
This limits the results as rarely can one infer the association of microbes detected in
the upper part of the gut from the microbiological assay of the faeces267. Except that
of the distal colon of humans, wherein, the faecal microflora reflects that of the
colon272.
Analysis of the microflora has relied on the use of traditional bacteriological methods
such as the use of culture media, biochemical tests and microscopy. These allowed the
enumeration of specific populations but selectivity and inability to cultivate some
microbial populations are the limiting factors of these methods. The advent of
8
molecular techniques such as the use of oligonucleotide probes and PCR-based
methods paved the way to analyse spatial relationships of microbes in the intestinal
ecosystem and identify bacterial species and communities.
A combination of these analytical approaches has confirmed the complexity and
uniqueness of the composition of the gut microflora. This led then to the difficulty of
assigning bacterial groups to just one category as proposed by Dubos and co-workers
(1965) or Fuller and Gibson (1997). Moreover, the concept of microflora is different
with environmental microbiologists and clinicians. The term “normal” microflora is
used in medical microbiology to denote healthy bowel flora which is clinically normal
and characterized by normal defaecation pattern and negative for pathogens. This is
in contrast with the “infected” bowel flora wherein there is an acute colonization
and/or persistence of pathogens and presence of clinical disease (Borody T. personal
communication).
Nevertheless, the structure and function of the microflora is very stable and a
favourable equilibrium exists between its members and the host13. These properties of
the microflora enable them to perform numerous functions to benefit the host (Figure
1.3). The microflora serves as a mediator between food factors and organ functions14.
This is clearly seen in ruminants where they rely on their gut microbes to digest
dietary plant materials15. Indigenous gut microbes can utilize non-digestible
carbohydrates to produce large quantities of metabolites such as short chain fatty
acids (SCFAs) that provide additional energy to enterocytes, indirectly regulate
peristaltic movement in the gut by stimulating water and salt absorption in the bowel
and lower pH levels to reduce the risk of colonization by pathogenic microorganisms.
The microflora confers protection against potential enteric pathogens and intestinal
diseases and this is referred to as colonization resistance16. This is achieved through
the concerted physiological functions of both host and the microbes17. One of the
proposed prerequisites for colonization resistance is that the microorganisms must be
able to attach onto the mucosa of the host. The ability to adhere can determine the
success of colonization18. It has been reported that if attachment sites are blocked by
indigenous microorganisms the subsequent colonization attempt by a pathogen could
fail. It has been shown in in vitro studies that Lactobacillus fermentum cells and their
9
supernatant reduced adhesion of enterotoxigenic K88ab or K88ac fimbriated
Escherichia coli to porcine ileal mucus19. L. rhamnosus strain GG20 and L.
acidophilus21 were able to inhibit adhesion of diarrhoeagenic bacteria Salmonella
typhimurium, E. coli, Listeria monocytogenes and Yersinia pseudomonocytogenes to
intestinal Caco-2 cells. However, to date this mechanism of protection has not been
proven in vivo.
Furthermore, an intact indigenous microflora is needed to maintain microbial
homeostasis in the gut and to prevent colonization of pathogens. Germ-free animals
and infants are found to be more susceptible to enteric infections compared to their
conventional and adult counterparts22, 23. This is probably because the adult’s
microflora is already completely established, while that of the infant’s is still
developing, thus, their protective effects could be not as effective.
Another protective mechanism that the gut microbes employ is through the production
of antimicrobials. Bifidobacterium and Lactobacillus, which are recognized as the
main health-promoting microorganisms in the gut, produce antimicrobial substances
called bacteriocins24, 25. Bacteriocins are diffusible substances that have an active
protein moiety that exert antimicrobial activity against other bacterial strains but not
against the producing microorganisms itself24. Bacteriocins are commonly used in the
food industry to combat food-borne pathogens. Other types of antimicrobials from
lactobacilli and bifidobacteria that prevent the proliferation of less desirable
microorganisms are SCFAs, ammonia, hydrogen peroxide and bacterial enzymes16, 17,
26. These antimicrobials create a restricted environment in the gut by lowering
oxidation-reduction potential that allows the growth of obligate anaerobes which in
turn suppresses growth and activities of facultative anaerobes and aerobic
microorganisms. The latter mentioned groups are members of normal microflora but
when occurring in high concentrations can be pathogenic16, 26, 27.
11
Figure 1.3. Functions of the microbiota.
The microbiota competitively excludes pathogenic microorganisms by 1) inhibiting attachment to epithelial cells and by 2) producing antimicrobials. It can 3) stimulate the mucosal immune system and 4) maintains the ecology in the gastrointestinal tract while 5) reinforcing the function of the mucosal epithelium barrier. The microbiota also 6) serves as the mediator in the exchange of nutrients between food factors and organ functions and 7) indirectly facilitate the production of energy i.e short chain fatty acids (SCFAs) to provide energy to the enterocytes and thereby maintain integrity of the gut.
13
microorganisms and antigens from the surface of the epithelium. Monoassociation of
germ-free animals with segmented filamentous bacterium (SFB) or Morganella
morganii resulted in an IgA plasma cell profile and activated lamina propria CD4+
cells similar to that seen in conventional animals36, 37. Aside from IgA, epithelial cells
and neutrophils secrete antimicrobial compounds called defensins that assist in the
eradication of pathogens. These defensins cannot be released from Paneth cells unless
there is prior activation by the microflora38. Using immunodeficient mice, it has been
shown that caecal bacteria can influence the secretion of anti-inflammatory regulatory
cytokine IL-10 when exposed to IFN- secreting Th 1 CD4+ T cells and, as a result,
reduce colitis39.
1. 2. 2. Microorganisms and Bowel Inflammation
Despite the beneficial contributions of the microflora to the host, evidence exists that
implicates the gut microflora in the pathogenesis of IBD. As mentioned earlier, IBD
could be due to an abnormal and uncontrolled immune response to a common luminal
antigen which could be of dietary or microbial origin. Brandwein and colleagues40
tested this hypothesis and conducted a study that investigated the role of epithelial
cells, diet and microbial antigens in triggering an immune response seen in IBD. They
tested the reactivity of C3H/HeJBir mice against antigens implicated in IBD such as
epithelial, food and indigenous bacterial antigens. It was demonstrated through
chemiluminescence Western blotting technique that sera from C3H/HeJBir mice did
not demonstrate antibody reactivity against epithelial cell antigens nor did the sera
show any response to food antigens. These results contradict the autoimmune theory
of ulcerative colitis, wherein, the immune response is directed towards the epithelial
cell and is destroyed by the immune effector mechanisms41. The attack on the
epithelial cell is probably an appropriate immune response that is directed to a
lumenal antigen but because of similarities of the proteins on epithelial cells and the
lumenal antigen, the immune response also attacks the epithelial cell. This study also
does not support the idea that IBD is a result of an abnormal immune response
towards dietary antigens since sera from spontaneously colitic mice did not react with
food antigens. Thus, even if IBD patients exhibit high levels of antibodies against
food antigens, especially cow’s milk, this might just reflect a normal response to
dietary proteins. When the epithelial monolayer is broken because of the
14
inflammation, it allows the uncontrolled passage of dietary antigens into the mucosa.
As a result, the immune cells in the lamina propria cannot distinguish, select and
process the real antigens and, therefore, the specific immune response to the
pathological agent is insignificant in comparison with the immune responses directed
to the lumenal components that passed through the damaged epithelium.
In the study by Brandwein and colleagues (1997), the sera did show reactivity with
antigens from the resident enteric bacteria40. This indicates that the resident flora may
contribute to the initiation or perpetuation of IBD. Other studies as well give
compelling evidence that the resident gut microorganisms are involved in the
pathogenesis of experimental and clinical IBD. Chronic inflammation does not
appear in germ free mice but inflammation occurs when the animals are subsequently
transferred to an environment populated with bacteria. For example, IL-10 deficient
mice reared in SPF conditions developed clinical signs of intestinal inflammation as
evidenced by weight loss and diarrhea while IL-10 deficient mice reared in sterile
conditions had no clinical or histological evidence of colitis11. Furthermore, chronic
inflammation can clearly be observed in animals with defined flora, but not without it.
This has been demonstrated in animal models that used chemicals, polymer/microbial
products or impaired genetic animals to induce the colitis42-45. Rats whose colon
segments were recolonized with defined bacteria developed severe inflammation and
increased intestinal permeability to hydrophilic molecules compared to antibiotic
treated rats after induction of IBD with trinitrobenzene sulphonic acid (TNBS)46.
Similarly, the presence of a normal microbiota is needed to produce severe clinically
apparent IBD in severe combined immunodeficient mice infected with H. hepaticus
and H. bilis11.
Another evidence that the resident flora is important in the pathogenesis of IBD is the
finding which showed that the tolerance of the intestine towards resident bacterial
components is broken down in active IBD47, 48. Using different mouse strains
(BALB/c, SJL/J and C3H/HeJ), these workers showed that the mice were tolerant to
their own bacterial sonicates but that an immune response was raised against the
bacterial sonicates coming from foreign gut. This, however, changed when their
tolerance to their own microbiota was abrogated after treatment with TNBS48. Other
workers have shown a very strong immune response directed to the resident flora40.
15
The two most antigenic groups identified in the resident flora were the aerobic
Enterobacteriaceae and Enterococcus spp. 40, 49. Although the most abundant bacteria
in the microflora are the anaerobes such as Bacteroides and Eubacterium, only few of
their antigens reacted with the serum samples. It has been further shown that the
aerobic species, particularly, E. coli, Proteus mirabilis, E. aerogenes and S.
typhimurium were all immunoreactive. Another enteric microorganism found to
persist in human IBD patients but one that did not stimulate an aggressive type of
immune response is Yersinia spp 50.
Another interesting observation is that there appears to be an alteration in the bacterial
colonization of IBD-prone mice with an increase in mucosal associated and
translocated aerobic bacteria wherein inflammation becomes more severe concomitant
with an increase in aerobic bacterial numbers51. This, however, is contradictory to the
findings of Garcia-Lafuente and colleagues (1998) who demonstrated deeper colonic
lesions and more severe inflammatory response in TNBS induced colitic rats
colonized with anaerobes (Clostridium ramnosum, Bacteroides fragilis and B.
uniformis) than those exposed to aerobic bacteria (E. aerogenes, Klebsiella
pneumoniae and Streptococcus viridans)46. This suggests that some anaerobic species
may be critical to the development of transmural inflammation. Other microorganisms
which were able to trigger and perpetuate inflammation both in animal and clinical
studies are Bacteroides vulgatus 52, 53, Enterococcus faecalis54, sulphate-reducing
bacteria such as Desulfovibrio desulfuricans55, and Escherichia coli56.
Lower concentrations of lactobacilli and bifidobacteria are reported in IBD254-255, 261.
These lactic acid bacteria did not induce observable pathology in a murine model of
transmural inflammation57. In another experiment, it was shown that E. faecalis or E.
coli do not cause colitis in HLA-B27 transgenic rats58 but are capable of inducing the
disease in IL-10 knock-out mice54. Moreover, caecal inflammation rapidly developed
in animals monoassociated with E. coli, but inflammation was delayed in the presence
to E. faecalis59. It seems that the host responds differently to these commensal
microorganisms and that the degree of colitis differs when the same host is exposed to
different bacteria. Thus, it is reasonable to conclude that different types of indigenous
bacteria have different capacities of inducing inflammation.
16
A number of microbial constituents have been proposed as inducers of bowel
inflammation, and these include: cell wall components like peptidoglycan from Gram
positive bacteria and lipopolysaccharide (LPS) from Gram negative bacteria60-62,
formylated peptides such as N-formyl-methionyl-leucyl-phenylalanine (fMLP)
synthesized by colonic bacteria63, 64 and nucleic acids like the CpG motifs of bacterial
DNA65. These microbial components have been found to be associated in colonic
inflammation as well as in other extraintestinal manifestations of the disease such as
arthritis and granulomatous arthritis. However, whether the bacteria or bacterial
products abnormally interacts with the mucosal immune system or whether bacterial
invasion is a secondary result of dysregulated functions of the mucosal barrier and
immune response, thereby, perpetuating the inflammation is still not completely
understood and remains a controversial issue.
In addition to the indigenous bacterial species above, there are infectious
microorganisms that have been implicated in IBD and this includes Mycobacterium
paratuberculosis. It has been proposed that M. paratuberculosis is involved in the
pathogenesis of IBD as it has been isolated in some studies from tissues and stools of
patients with UC and CD66. Furthermore, histological analysis and clinical
examination has revealed features which resemble Johne’s Disease, a chronic
granulomatous enteritis in ruminants which is caused by M. paratuberculosis. There
are still doubts as to whether M. paratuberculosis is the causative agent of IBD for a
number of reasons: the mycobacteria can also be isolated from patients with other
intestinal diseases, patients with IBD can improve after treatment with
immunosuppressives which is not expected in a disease caused by mycobacteria; and
because polymerase chain reaction (PCR) analyses showed that mycobacteria species
are ubiquitously found in samples from IBD patients, however these workers failed to
demonstrate bands specific for M. paratuberculosis50.
Helicobacter species have also been implicated in IBD. These spiral shaped
organisms have been linked to IBD because of their association with gastric diseases
such as chronic gastritis, peptic ulcer, gastric adenocarcinoma and lymphoma; and
IBD demonstrates a chronic inflammation similar to gastritis caused by H. pylori67. It
has been recently shown that another Helicobacter species, H. hepaticus, can trigger
development of IBD in severe combined immunodeficient mice68. Furthermore,
17
clinical disease and histological lesions are more pronounced when H. hepaticus
infection is combined with the reconstitution with CD45RBhighCD4+ T cells and the
normal microflora as compared to mice just reconstituted with CD45RBhighCD4+ T
cells and the normal microflora in the absence of the H. hepaticus69. These
observations suggest that the combination of an abnormal immune response, normal
biota plus the presence of pathogenic bacteria, H. hepaticus could produce
inflammation similar to IBD. This is in contrast with the work performed by Sellon
and co-workers11 with IL-10 deficient mice. These workers noted that some of their
IL-10 deficient mice raised in a specific pathogen free (SPF) environment were
colonized by H. hepaticus as detected by PCR. To assess if intestinal inflammation
would develop in the absence of H. hepaticus, adult IL-10 mice were moved from
germ-free conditions to SPF conditions free of Helicobacter sp. and some germ-free
IL-10 mice were housed with bedding contaminated with Helicobacter-infected
stools. These groups of mice still exhibited inflammation and failed to become
positive for Helicobacter as determined by PCR. This suggests that Helicobacter
organisms were not essential for the pathogenesis of IBD in these mice. Thus, the role
of Helicobacter as the primary infectious agent in IBD still remains inconclusive.
Human IBD is observed to occur mostly in the terminal ileum, caecum and the colon.
These are areas of the gastrointestinal tract where there are a large numbers of
microorganisms and where there is less peristaltic movements and a slower transit
time which allow the mucosa prolonged contact with the microorganisms. This is
demonstrated when a stoma is temporarily created to divert faecal contents flow in
CD patients. It has been observed that sections of the bowel where faeces did not pass
through did not develop inflammation but when the stoma was reversed, inflammation
recurs70. This is also the case when an ileo-anal pouch is created in UC. A significant
proportion of patients developed pouchitis after a colectomy procedure71. This is
surprising because patients with UC do not develop inflammation in the small
intestine. These observations suggest that the microflora have pro-inflammatory
activities in susceptible hosts.
The most compelling evidence that the microbiota plays a critical role in the etiology
of IBD is that there is resolution of inflammation when antibiotics were administered
in experimental and human IBD9. Antibiotics are in fact one of the recommended
18
therapies to manage IBD, however, most controlled trials have shown effectivity in
CD patients but were not successful in cases of active UC or when used as a
maintenance therapy in UC patients72-74.
Despite the accumulating evidence that the intestinal microflora contribute to
intestinal inflammation, the mechanism by which the microflora induce and
perpetuate the inflammatory response is still inconclusive. Sartor (1998) summarised
the possible mechanisms by which lumenal microbial members could cause chronic
intestinal inflammation57 (Table 1.2). We proposed that the resident microflora could
either play a role in IBD through tissue invasion and damage, or by induction of
immunological reactions to microbial components.
1. 2. 3. Diversity of the Mucosa-Associated Bacteria in Bowel Inflammation
Mucosa associated bacteria could be of relevance in the disease process of bowel
inflammation as they are in close proximity or within the mucosa which is the
affected area in IBD. Moreover, it has been shown that there is a distinct difference
between the microbial community along the mucosa of the gastrointestinal tract and
those recovered from the faeces.
Earlier studies of the composition of the normal and diseased intestinal microflora
used culture dependent techniques and microbial isolates from the faeces. It has been
reported that there were normal levels of mucosa associated bacteria in CD253 while
decreased levels of total anaerobes including Gram negative anaerobes and
lactobacilli were observed in UC254-255. Other culture dependent studies revealed
higher levels of Bacteroides and enteric bacteria in IBD patients than in controls40, 255.
More recently molecular approaches such as oligonucleotide hybridization, real-time
PCR and fluorescent in situ hybridization have been used to deduce microbial
diversity in IBD. These methods reveal that both UC and CD are associated with high
levels of mucosal bacteria84, 256 and that there is a direct relationship between bacterial
concentration and severity of inflammation84.
19
However, there seems to be no difference in the composition of the mucosal
microflora compared to controls84. Desulfovibrios were found to be ubiquitous in the
faeces and mucosa tissue of healthy and UC patients257-258. Similar abundance and
diversity were also observed in both controls and IB patients when screened for E.
coli strains259. These studies contradict the study conducted by the group of Ott (2005)
wherein they showed a significant reduction in bacterial diversity in active IBD. They
demonstrated that mucosal inflammation is associated with the reduction of anaerobic
bacteria such as Bacteroides, Eubacterium and Lactobacillus260. Another group
(2005) reported that the prominent feature of biofilm in IBD patients, but not in other
intestinal diseases, is the presence of B. fragilis261. On the other hand, increased
bacterial counts for both anaerobes and aerobes were detected by another group and
that they have shown Bacteroides and E. coli were the most immunoreactive members
of the mucosal bacteria262.
The mechanisms by which the mucosa associated bacteria manifest their
pathogenicity in IBD have not been elucidated. It has been hypothesized that the
healthy mucosa has a cleaving function which prevents the close contact of the
luminal bacteria to the epithelium but this is compromised in IBD, thus, increasing the
association of the luminal bacteria with the mucus layer84.These may involve the
disruption of the mucosal layer and adherence or interaction with the putative
bacteria’s outer membrane. It has been shown that the sera of colitic patients reacted
most actively to the lipopolysachharide of Bacteroides263 while E. coli interacts
through bacterial adhesions264. Toxin or cytotoxic metabolic production from enteric
bacteria265 and desulfovibrios266 has also been thought to mediate the inflammatory
process and alter the protective function of the intestinal mucus in IBD. These
findings indicate that the aggressive inflammatory response in IBD might be induced
by common antigens of the bacterial population.
In summary, the intestinal microflora has a role in IBD. The mucosa associated
microorganisms maybe more closely related to the inflammatory process than the
faecal microflora. To date, conflicting reports are available as to which microbe or
group of microbes induce and perpetuate the immune response in IBD. It seems that
lactobacilli have been described as being reduced in several reports, whereas,
Bacteroides and enteric bacteria appear to increase in both animal and clinical IBD.
20
However, no consistent profile of the intestinal microflora in IBD has been
determined.
21
Table 1.2. Potential mechanisms by which microbial constituents could cause chronic
intestinal inflammation (adapted from Sartor, 1998).
1. Direct injury to epithelial cells
2. Enhanced mucosal permeability
3. Altered luminal metabolic activities i.e. production of SCFAs
4. Induction of antigen-specific immune responses
5. Auto-immune responses
22
1. 3. Mucosal Immunity and Bacteria in Bowel Inflammation
1. 3. 1. Gut Mucosal Barrier Function in Inflammation
The epithelium serves as the gatekeeper to protect the host and is considered to be one
of the physical barriers that protect the body from harmful effects of the environment.
It is a selectively permeable barrier that regulates the entry of digested nutrients and
solutes into the blood stream for immunological surveillance75, 76. Unfortunately, there
is an uncontrolled translocation of bacterial products into the mucosa observed in IBD
that alters the expression of genes in the epithelium. These genes are switched on
upon contact with the antigen receptors which leads to the expression of surface
molecule proteins such as class II major histocompatibility complexes, cytokines,
chemokines as well as antimicrobial compounds like mucins, lysozymes and
defensins from the epithelium that further destroys the integrity of the barrier77. This
increased mucosal permeability observed in IBD is known as “leaky gut”78. This
condition also compromises the structural components of the intestinal barrier such as
the water layer, mucus coat, mucosal surface hydrophobicity and the epithelial tight
junctions which are critical in the maintenance of intestinal tissue architecture and its
physiological functions78.
1. 3. 2. Mucosal Immune Response in Intestinal Inflammation
The gut is continually exposed to a many of dietary, microbial and self products that
may possess different degrees of antigenicity. The gastrointestinal immune system has
evolved to distinguish which, among these loads, are pathogenic and which are
considered “self”. At the same time the gastrointestinal tract (GIT) is required to
mount the appropriate immune response to destroy the former but be tolerant of the
latter. Thus, the gastrointestinal tract is always in the state of low grade inflammation
in the healthy host.
These putative antigens, even though barred by the epithelium, can still gain access to
host tissues through entry using one of two pathways. Antigens can be taken up by M
cells found overlying the Peyer’s patches or via a transcellular epithelial pathway.
Entry using the former route leads to the activation of antigen-specific lymphocytes.
23
These activated lymphocytes migrate to effector sites such as the lamina propria and
intraepithelial spaces. On the other hand, antigens entering using the transcellular
epithelial pathway are taken up by antigen-presenting cells (APC) in the lamina
propria and are then presented to the lamina propria T cells. These effector sites are
populated with a lot of antibody secreting B lymphocytes, T lymphocytes and
macrophages which are the immune cells responsible for up-regulating or down-
regulating the inflammatory response.
Secretory IgA (sIgA) is the major immunoglobulin isotype secreted by B cells in
normal lamina propria and other mucosal sites of the gastrointestinal tract79. It
contributes to immuno-exclusion of microbes and antigens by binding to them. In
addition, it does not activate the complement system which prevents development of
an excessive immune response against ubiquitous dietary and microbial antigens80.
However, colitic animals as well as patients with CD or UC exhibit impaired
tolerance to their own intestinal microbiota as indicated by the marked increase in the
counts of IgG producing lymphocytes in histological sections of their intestinal
mucosa81. This suggests that antibody production in IBD is altered as IgG neutralizes
the antigen, forms IgG-immunocomplexes that activate the complement and triggers
an inflammatory response in the host tissue.
Moreover, the cellular response in IBD is also seen to be hyperactive and is distinctly
different between the two forms of bowel diseases. The T lymphocytes upregulated in
CD are skewed towards T helper 1 (Th1) 1 wherein high concentrations of pro-
inflammatory cytokines such as interferon-gamma (IFN- ), TNF- , IL-1, IL-6, IL-12,
IL-18 are detected in the colonic mucosa. On the other hand, UC favours a Th2
response wherein lower levels of the so-called pro-inflammatory cytokines are
observed while Th2 cytokines such as IL-4, IL-5, IL-6 and IL-13 are markedly
elevated. The T lymphocytes profiles were found to be determinants of the nature of
the immune response in clinical IBD, however, there are instances in ulcerative colitis
when a mixed population of T cells are seen which makes it difficult to assess the
exact form of the disease. Nevertheless, the common impairment in the immune
profile of both forms, CD and UC, of bowel disease is the loss of tolerance towards
commensal bacteria of the resident microbiota57, 82-84. However, whether a different
(or the same) type of commensal bacteria is responsible for the unrestrained activation
24
of the intestinal immune system, and whether this bacteria is the one driving the
opposing T cell response and damaging the intestinal mucosa in CD and UC and of
the relapsing nature of both forms is yet to be determined.
1. 3. 3. T cell Responses in Inflammatory Bowel Diseases
As mentioned earlier, one of the desirable functions of the microflora is its ability to
modulate the immune response. It is responsible for generating immunocompetent
cells during development and maintaining the gut associated immune system85-87. At
birth, the gastrointestinal tract is devoid of any immunological functions and most of
the immune cells are in the naïve state. Microbial colonization begins right after birth
which happens upon contact with the maternal (vaginal) microbiota and from
surrounding environments28. The immune system and gut barrier then gradually
develops as a consequence of microbes establishing in the gut. The cellular immune
system of rodents, most animals and humans is biased towards a Th2 cytokine profile
during the neonatal period. As microbial colonization proceeds and exposure to food
products starts, the Th2 skewed immune response that is initially seen shifts towards a
more balanced Th1 and Th2 immune response88, 89.
Figure 1.4 illustrates the imbalance of inflammatory mediators in the presence of
mucosal inflammation. There is a delicate balance between the pro-inflammatory and
the anti-inflammatory cytokines in the gut, which in turn is regulated by the Th3 (or
regulatory) cytokines, IL-10 and TGF- . In a normal gut, the inflammatory response
is immediately down-regulated after the elimination of the pathogen but if the balance
is disturbed, chronic inflammation persists. When an antigen presenting cell (APC)
encounters an inducing antigen, it stimulates the mucosal lymphocytes to produce
IFN- and IL-2. IL-2 pushes the clonal expansion of naïve T cells and enhances the
functions of B and T cells, while, the IFN- triggers other surrounding APCs and
macrophages to secrete IL-12. The net result of this is the Th 1 cell differentiation and
activation in a self-sustaining cycle that consequently activates the Th1 cells to
produce more pro-inflammatory cytokines like IFN- , TNF- , IL-2, IL-12. The IFN-
also activates endothelial cells to increase endothelial cell adhesion molecule
expression which facilitates the recruitment of inflammatory cells into the mucosa.
IFN- further activates the macrophages to produce more pro-inflammatory cytokines
25
such as TNF- , IL-1, IL-6, IL-8, IL-12 and IL-18, reactive oxygen and nitrogen
species that leads to a severe tissue and immunological injury and clinical
manifestations of inflammation. The cytokine IL-10 performs a dual role in IBD. IL-
10 can both act as an inhibitor of the production of Th1 derived pro-inflammatory
cytokines and as a regulator of the T cell mucosal immune response. It has been
shown that low levels of IL-10 in UC patients contributes to the perpetuation of the
inflammatory changes90 and administration of IL-10 therapy in clinical trials have
suggested the potential of IL-10 in treating IBD91. Moreover, recent findings have
shown that IL-10 supports the development of the regulatory cytokine TGF- 92 and
that both cytokines further inhibit antigen presentation and subsequent pro-
inflammatory cytokine production.
One way of distinguishing the two forms of IBD is through the Th cell cytokine
profile. Cytotoxic T lymphocytes producing the cytokines IFN- , TNF- , IL-1, IL-6,
IL-8 and IL-12 are abundantly produced in the intestinal mucosa of CD whereas T-
suppressor lymphocytes producing the cytokines IL-4, IL-5, IL-11 and IL-13
predominantly mediate the inflammatory response in UC93. This holds true in the
context of disease classification but perhaps the ratio between the amounts of pro-
inflammatory and anti-inflammatory cytokines will provide the real account of the
intensity of inflammation in these diseases94. This is because manifestations of the
symptoms of CD and UC overlap in some patients and there is sometimes mixed
expression and production of the T cell lymphocytes95, 96.
The question still remains as to how the indigenous microflora drives the
inflammation seen in IBD. The identification of the new class of surface and
cytoplasmic receptors that bind to bacteria and bacterial products93, 97 paved the way
in understanding the mechanisms by which commensal bacteria activate, and how the
cytokines regulate the destructive cell-mediated immune response in bowel
inflammation. These pattern recognition receptors can be membrane bound or can be
found intracellularly. Membrane bound receptors are referred to as Toll-like receptor
(TLR) of which nine different types have been described (TLR 1-11). Each TLR
recognizes different bacterial products in the external environment while intracellular
receptors such NOD/CARD binds to intracellular bacterial products. After binding,
the transcription factor NF- B is activated through the ubiquitation and degradation of
26
I- B, an inhibitor that binds to and sequesters NF- B in the cytoplasm, by the I- B
kinases. The NF- B is then released and migrates to the nucleus where it switches on
multiple genes involved in intestinal inflammation. This then activates cytokine
production and the release of reactive oxygen species in monocytes, macrophages,
dendritic cells, mesencyhmal cells, epithelial and endothelial cells12, 98, 99. Other
transcription factors that are activated in IBD are the Smads and STATs, whereby,
the former influences the action of the regulatory cytokine TGF- and the latter
regulates immunity and inflammation associated with interleukins, interferons and
hemopoeitins, respectively95. Collectively, these pathways and pattern of cytokine
production may explain the absence of inflammation or the severity and sustained
inflammatory response in the gut mucosa when continually exposed to a variety of
indigenous bacteria.
27
Figure 1.4. T helper (Th) cytokine responses in inflammatory bowel diseases.
(T regulatory)
Dendritic
28
1. 4. Treatments in IBD
Different therapeutic strategies have been developed through the years to abrogate
IBD wherein the primary goals are to induce remission and prevent relapse. Current
available therapies of IBD include aminosalicylates, corticosteroids,
immunomodulators such as azathioprine and 6-mercaptopurine, methotrexate,
cyclosporine, biological agents, antibiotics and sometimes surgery is required.
Prescription of these therapies depends largely on the clinical goal and the patient’s
clinical status i.e. the extent and severity of disease, response to current and prior
medications and the presence of complications100, 101.
As reviewed by Lim and colleagues (2004), aminosalicylates are considered to be the
first-line therapy for mild to moderate UC and CD. They are more effective in
inducing and maintaining remission in UC while their use in CD is still controversial.
Corticosteroids, on the other hand, are the next therapy of choice when IBD patients
do not respond to aminosalicylates and are also prescribed to those with moderate to
severe forms of IBD. Corticosteroids are effective in suppressing active inflammation
but cannot maintain this effect and have a high relapse rate102. Immunomodulators
(immunosuppressants) are considered to be the choice for maintenance treatment
especially for steroid-dependent patients and for those whose IBD that was improved
using cyclosporine102. However, their effects are slow and they have potentially
serious side effects. Cyclosporine, on the other hand, had a rapid therapeutic effect on
severe UC that did not respond to conventional therapy but its use is limited to centres
experienced to monitor blood levels as its use is associated with increased
hypertension, altered nephorological and neurological functions, and opportunistic
infections100-102. Another drug that is used in CD but not UC is methotrexate100-102 that
has been shown to induce as well as maintain remission for as long as 40 weeks in
steroid-dependent CD patients but has serious side effects associated with its use such
as myelosupression, hepatotoxicity, teratogenic and abortigenic effects.
Aside from these mentioned conventional therapies in IBD, biological therapies are
being developed that focus on the suppression of the inflammatory process by
targeting the different mediators of inflammation (Table 1.3).
29
Table 1.3. Targets of biological therapy in IBD. As presented in “Emerging Biologic
Therapies in Inflammatory Bowel Disease” Vol 4 (2): 2004, Reviews in
Gastrointestinal Disorders.
Strategy Biologic Agents
Inhibitors of pro-inflammatory
Cytokines Anti-TNF therapies
Receptors IL-6R antibody
Transcription Factors Antisense NF- B
Anti-TNF therapies Infliximab, CDP571, CDP870,
etanercept, onercept, adalimumab,
RDP58
Anti-inflammatory cytokines IL-10, IL-11
Antileukocyte adhesion therapies
Anti- 4 integrin Natalizumab
Anti- 4 7 integrin MLN-02
Antisense ICAM-1 ISIS 2302
Inhibitors of Th1 polarization Anti-IL12, anti-IL-18 and IFN-
Inhibitors of T cell proliferation Anti-IL-2 receptor antibody (daclizumab,
basiliximab)
Inhibitors of T cell activation Anti-CD40L
Anti-CD4 therapy cM-T412, Max.16H5, BF-5
Anti-CD3 therapy Vasilizumab
Epithelial restitution and repair with
growth factors
EGF, KGF, growth hormone
Immunostimulation G-CSF (filgrastim), GM-CSF
(sargramostim)
Immunomodulators IFN- , IFN-
30
Most of the biological therapies available in clinical practice or those that are still
being evaluated are mostly recombinant peptides or proteins, partially or completely
humanized antibodies or nucleic acid based therapies103. An example of a biological
agent is the anti-TNF- therapy named Infliximab. TNF- is a pro-inflammatory
cytokine and an increased production of it was observed in CD4+ cells104,
mononuclear cells105 and cultures of biopsies from patients with CD106. Furthermore,
elevated levels of TNF- were detected in stools of CD patients107. Its mode of action
against CD is by suppressing the activity of TNF- in these samples. Infliximab is a
mouse-human chimeric monoclonal IgG1 antibody. Clinical studies have
demonstrated that it can dramatically improve active CD in two-thirds of the
participants after using a single dose108. Moreover, infliximab has also been shown to
be effective in the treatment of fistulae in CD patients100, 109-111. It is the only
biological agent approved by the U.S. Food and Drug Administration (FDA) for the
induction and maintenance of clinical remission in moderate to severe CD as well as
for the maintenance of fistulizing CD101. Infliximab has recently been found to be
beneficial in UC243.
Another approach being explored involves modulating the Th1 phenotype expression
and cytokine production using IL-10 therapy113. The clinical usefulness of IL-10
therapy, however, is limited because it must be administered frequently through
parental injections or rectal enemas114. These methods can ensure organ-specific
delivery of IL-10 but such procedures are inconvenient for many patients. Thus,
Steidler et al (2000) genetically engineered Lactococcus lactis to synthesize IL-10
and tested it first in IL-10 deficient mice. This strategy resulted in a high IL-10
concentration in the colon and there were significant improvements in the disease
activity of experimental colitis115. The advantages of this approach is that it allowed
the production of IL-10 in the intestinal lumen without the systemic exposure; and the
IL-10 is synthesized in lower, consistent doses which would have influenced the
therapeutic benefit seen in this procedure, as compared to when IL-10 is administered
systemically. This strategy employed by Steidler and colleagues should be attempted
in humans as parenteral IL-10 therapy, although safe and well tolerated, has not been
shown to result in significantly higher remission rates nor did it improve disease
activity indices compared to placebo treatment in a clinical setting91, 116, 117. Failure of
IL-10 to downregulate inflammatory responses in clinical studies could be due to the
31
low local concentrations of IL-10 in the intestine after systemic delivery. It could also
be that IL-10 alone is not sufficient to affect all the inflammatory mediators involved
in inflammation and that its immunostimulatory activities may be counterbalancing its
immunosuppressive properties118 or as indicated in animals studies, IL-10 is only
effective in preventing but not in reversing established disease63.
Antibiotics are also used in IBD because of the high levels of infectious
microorganisms found to be present in experimental and clinical samples of IBD119,
120. Metronidazole, ciprofloxacin and rifampicin are examples of antibiotics used in
IBD. Although these can ameliorate inflammation in IBD, the use of antibiotics still
poses a concern as they can weaken the intestinal microbiota especially since the
antigen responsible for bowel inflammation is still unidentified. Most of these
antibiotics are broad-spectrum and therefore they not only inhibit the growth of the
harmful microorganisms but may also eradicate many of the neutral and beneficial
members of the indigenous microflora. This could allow for the opportunistic
pathogens to become established in the gut and thereby give rise to other intestinal
conditions such as antibiotic associated diarrhea (AAD)121 or pseudomembranous
colitis122. Another problem that may arise with the continued use of antibiotics is that
a more resistant form of microorganims may develop in the gut microflora.
More recently, the group of Summers trialed the use of Trichuris suis, a parasitic
whipworm found in pigs, in patients with active CD123 and UC124. Trichuris suis is
known to influence the Th cell immune response towards a Th2 profile. Patients from
these studies were administered with 2,500 parasitic worms and these eggs were
allowed to hatch in the intestines. Afterwhich, a significant decrease in the number of
bowel movements were reported in these participants and these studies also showed
that these worms transiently established themselves in the gut as all were excreted
after a month. These studies supports the hygiene theory in IBD wherein the
overreaction of the immune response seen in IBD is due to an impaired immune
system because of a lack of exposure to microorganisms or parasites125, 126.
32
1. 5. Use of Functional Foods in IBD
Section 1.2.2 explored the role of the intestinal microflora in the pathogenesis of IBD
as well as discussing the differences in the microbial composition and concentration
of healthy and IBD patients. One can postulate that in an IBD gut there may be an
imbalance in the microflora with relative predominance of potentially pathogenic
enteric bacteria and an insufficient number of lactobacilli and bifidobacteria. Whether
this profile is the cause or a consequence of IBD is still unclear but it suggests that
altering the microbiota profile to a healthier state may lead to the amelioration of
intestinal inflammation. This strategy has already been demonstrated using antibiotics
but its use can have undesirable effects on intestinal health (Section 1.4). Another way
of manipulating the gut microbiota is through the use of functional foods. Functional
foods refer to any food or food ingredient that may provide health benefits beyond the
traditional nutrients it contains (Hasler, 1998). One type of functional food group
includes the food targeting the beneficial microbes: (a) live microorganisms
(probiotics); (b) the dietary fibres that enhance beneficial bacteria (prebiotics); and (c)
the combination of both probiotics and prebiotics (synbiotics).
1. 5. 1. Probiotics
Probiotics, as defined by Tannock et al, (2000) are “microbial cells which transit the
gastrointestinal tract and which in so doing, benefit the health of the host”127. The
majority of the probiotic microorganisms used are lactic acid bacteria, wherein,
Lactobacillus and Bifidobacterium are the commonly used strains. Lactic acid
bacteria are the preferred microorganisms used as probiotics because of their
longstanding application in the food industry and historical records of safety128 as well
as the fact they are normally found in the gastroinintestinal tracts of man and animals.
It is desirable that the probiotics to be administered to a host should originate from the
same species129. This is based on the principle that a probiotic isolated from one
species is less effective when used in another species. Moreover, an effective
probiotic should be able to survive transit along the gastrointestinal tract and
withstand the effects of bile acids130. The probiotics could be able to adhere onto the
mucosal surface and demonstrate a positive influence on the microecosystem.
33
Potential probiotics can exhibit their desirable effects by a number of modes of
actions (Table 1.4).
Several animal and clinical studies have demonstrated the protective role of probiotics
in IBD. Colitis in IL-10 deficient mice was resolved and lactobacilli numbers were
normalized after the rectal administration of 107 colony forming units (cfu).ml-1 of L.
reuteri51. Similar data have been described by Steidler and colleagues (2000), who
showed that continuous intragastric administration of L. lactis can reduce
inflammation in mice with colitis induced using dextran sodium sulfate. On the other
hand, it is theorized that a mixture of a large number of probiotic strains should be
most effective in IBD since a single strain is unlikely to colonize the gastrointestinal
tract or make significant modifications in the complex gastrointestinal environment.
Thus, Madsen et al, (2001) used a probiotic preparation consisting of 4 strains of
lactobacilli (L. casei, L. plantarum, L. acidophilus and L. delbruekii subsp
bulgaricus), 3 strains of bifidobacteria (B. longum, B. breve and B. infantis) and 1
strain of Streptococcus salivarus subsp thermophilus in IL-10 deficient mice and
showed significant improvement of colon inflammation131.
Oral bacteriotheraphy of 1010 cells of Lactobacillus GG twice daily in human IBD
patients resulted in a significant increase of specific IgA and shortened the duration of
diarrhea132. These indicate that Lactobacillus GG could enhance the gut immune
response and normalize gut permeability. Kruis and his colleagues initially (1997)
compared the effects of another probiotic, E. coli Nissle 1917 to the standard therapy
mesalazine in patients with inactive UC when given for 12 weeks133. The study did
not show any significant changes in disease activities or in relapse rates between the
two groups. Rambacken et al, (1999) tried the same probiotic but dosed E. coli Nissle
1917 for 1 year and assessed patients with active UC134. The UC patients received
their standard therapy until remission was achieved then they were randomized either
to receive, mesalazine, or probiotics. Results showed that 75% maintained remission
in the mesalzine group while 68% in the probiotic group and of these 74% of patients
on mesalazine relapsed while 67% of patients receiving E. coli relapsed. It was
concluded that E. coli Nissle 1917 is equivalent to mesalazine however it was not
clarified how E. coli Nissle 1917 induced remission when standard therapy was also
given. The same cocktail of probiotic bacteria (VSL#3) used by Madsen in a murine
34
model of IBD was also trialed by Gionchetti and his colleagues in 40 pouchitis
patients135. Twenty patients received 6g of the preparation daily for 12 months while
the control group (n=20) received the placebo. Microbiological analyses showed a
significant increase in lactobacilli and bifidobacteria populations 10 days after
administration of the probiotics, while faecal pH decreased significantly and persisted
throughout the treatment period. Moreover, only 15% in the probiotic group relapsed
compared to 100% in the placebo group. This latter study could be more effective for
two reasons (i) a high bacterial concentration and (ii) a mixture of probiotics that have
potential synergistic relationship to suppress worsening of the inflammation. Recent
researches have synthetised DNA or used DNA from VSL#3 and demonstrated
resolution of experimental colitis which indicates that non-viable probiotic bacteria
can be used in ameliorating colon inflammation136, 137. Table 1.4 summarizes the
probiotic strains that have been used in experimental and clinical IBD and the effect
these probiotics have exerted on bowel inflammation.
Although the findings on the use of probiotics in both animal and clinical research are
encouraging, clinical experts are still cautious in accepting probiotics as a routine
therapy in IBD because it is “only strong on rationale and preclinical data but still
weak on showing clinical efficacy”125. Therefore, larger controlled clinical trials are
required.
35
Table 1.4. Potential mode of actions of probiotics to reduce colon inflammation.
1. Increased acidity through production of lactic acid and SCFAs
2. Strengthening of intestinal cell barrier
3. Production of antimicrobials (example, bacteriocins)
4. Colonisation resistance
5. Stimulation of IgA antibody production or protective regulatory lymphocytes
6. Production of large quantities of intestinal mucus
36
Table 1.5. Efficacy of probiotic strains used in IBD. A= animal studies; H = clinical
studies.
Probiotic Strain Effect Reference
L. plantarum 299v Down-regulated TNF- production in
dextran sodium sulfate induced mice
A 139
Bifidobacterium
infantis
Inhibited B. vulagtus, a bacteroidal
species implicated in IBD, growth in a
culture system as well as in the
gnotobiotic murine model. It increased
numbers of Peyer’s patches and
suppressed antibody response raised by B.
vulgatus.
A 52
Lactobacillus GG Prevented recurrence of colitis in HLA-
B27 rats after antibiotic treatment.
Normalized microbiota balance and
decreased incidence of diarrhoea
A 140
H 132
Saccharomyces
boulardii
Small study showed 1 of 16 of CD
patients in probiotic group while 6 of 16
in the mesalazine group relapsed after 6
months of dosing.
H 141
E. coli Nissle 1917 Equivalent to mesalazine in preventing
relapse and maintaining remission; more
effective in human UC than CD
H 133, 134, 138
VSL# 3 Combination of 4 lactobacilli, 3
bifidobacteria and a streptococcus.
Suggested that it can be used for UC
patients that could not tolerate 5-ASA.
It can prevent pouchitis relapse after
colectomy in UC
H 135, 142
37
1. 5. 2. Prebiotics
Gibson and Roberfroid (1995) defined prebiotics as “non-digestible food ingredients
that beneficially affect the host by selectively stimulating the growth and/or activity of
one or more limited number of bacteria in the colon and thus improve health”143. This
would suggest that using such an approach is more advantageous than using
probiotics, since probiotic bacteria need to survive passage across the hostile
conditions of a healthy gastrointestinal tract and compete with the resident microflora
for nutrient and space. Prebiotics can easily transit the tract and target commensal
bacteria that are already specific in the host and possess effective colonization
properties by serving as a fermentable substrate for their growth and metabolic
activities144, 145. Most of the prebiotics available are carbohydrates which can
effectively be utilized by colonic saccharolytic bacteria which includes the protective
species of lactic acid bacteria but not the potentially harmful species as most of them
are proteolytic146. Examples of prebiotics are inulin, fructo-oligosaccharides, trans-
galacto-oligosaccharides, lactulose and resistant starches.
Prebiotics exhibit their beneficial effects in a number of ways. They selectively
stimulate the growth and/ or activities of health promoting microorganisms in the gut.
Bifidobacterium and Lactobacillus found in the indigenous gut are the major target of
prebiotics147-150. However, as found by these researchers, prebiotics can only increase
the number of these beneficial bacteria, especially bifidobacteria, if they are found in
low numbers but not when they are already present in high levels.
As a consequence of the proliferation of lactic acid bacteria, prebiotics mediate the re-
establishment of colonization resistance properties of these microorganisms. It has
been shown that the addition of mannose or palm kernel in the diet reduces the degree
of Salmonella colonization in the intestinal tract of broiler chicks151. A similar result
was observed, wherein, rats increased their resistance against S. enteriditis after they
were given a lactulose-calcium diet31. Pigs afflicted with diarrhea were observed to
have an altered microbiota and the oral electrolyte solution (OES) given to them only
replenished salt and water loss but failed to address the disturbances in the normal
densities and relative species abundance of the microbiota152. The addition of
fructooligosaccharides to the OES normalized the imbalance of the lactobacilli and
38
Enterobacteriaceae and, thus, accelerated the recovery from diarrhea152.
Supplementation of prebiotics could also be helpful in establishing a healthy flora in
bottle fed infants. Bottle fed infants are more susceptible to gastrointestinal infections
as their colons are predominantly colonized with potentially harmful bacteria which is
in contrast with breast fed babies whose colons are colonized with bifidobacteria28. It
has been reported that incorporation of lactulose in milk formula positively changes
the infant microflora to be similar to that of breast fed infants146, 153
Moreover, the physical structure of the carbohydrates may also block the
establishment of pathogenic microorganisms onto the gut epithelium and prevent
these microorganisms from causing disease154, 155. The epithelium has abundant
carbohydrate components to which pathogenic microorganisms attach themselves
through the use of surface receptors called adhesins. Thus, instead of the pathogens
binding onto the gut wall, they may recognize the carbohydrate structure of the
prebiotics as their target receptor154, 155.
Colonic microorganisms ferment the prebiotics, thereby, producing large amounts of
short chain fatty acids (SCFAs) such as acetic acid, formic acid, propionic acid,
butyric acid and lactic acid143, 146, 156. The presence of SCFAs in the gut subsequently
lowers the intestinal pH which is not ideal for the growth of many pathogens and
putrefactive bacteria since they prefer a neutral environment to thrive. Consequently,
SCFAs are inhibitory to their survival16, 26, 28. Absorption of these SCFAs stimulates
sodium and water resorption from the colonic lumen which can help in the
normalization of defaecation in constipation157. Moreover, SCFAs, especially
butyrate, are absorbed and serve as energy substrates by enterocytes and other
gastrointestinal cells and tissues. These SCFAs help in the development and
metabolism of colonic epithelial cells which are important in tissue regeneration158.
Limited studies are available on the role of prebiotics in IBD but those that are
available provide evidence of their benefit. The group of Jacobasch (1999) found that
low levels of SCFAs in the colons of trinitrobenzene induced colitic rats markedly
affected nutrient and water absorption of these animals. They fed the colitic rats with
the resistant starch from granular pea and this resulted in increased SCFAs levels and
decreased colonic tissue damage. In addition, supplementation of 400 mg.day-1 or 1%
39
inulin in drinking water of rats induced with colitis using dextran sulfate resulted in a
reduction in the inflammation of the distal colon159.
Inulin was also used in a small, randomized, double-blind, cross-over study in patients
with ileo-anal pouches160. These patients received a total daily dose of 24g of inulin
for 3 weeks and placebo for the next 3 weeks. This study showed that inflammation
was reduced in the mucosal lining of the pouches when the participants were
supplemented with inulin but not when they were administered with placebo,
suggestive that inulin influenced tissue repair in this patient group. In another small
study, 8.5g of lactosucrose syrup was given to 2 CD and 5 UC patients daily for 2
weeks161. It was reported that 4 of these patients had improvement in their bowel
movements after consuming the lactosucrose. Furthermore, bifidobacteria were
detected in all faecal samples at the end of the study course. Although a small study,
these results indicate that prebiotic supplementation has altered the balance of the
microbiota.
Clearly, more studies are required on the use of prebiotics to prove their safety and
effectiveness in the area of gastrointestinal inflammation.
1. 5. 2. 1. Role of resistant starch in bowel health and inflammation
Resistant starch (RS) is the prebiotic used in this study. RS is the starch fraction that
escapes digestion in the small intestine but is fermented in the large intestine by
colonic bacteria. Resistant starches are classified based on the source of the starch or
the resistance of their physical structure275. The three types of RS are RS1, RS2 and
RS3. RS1 are physically inaccessible to digestive enzymes and to be digested the
outer coating must be broken. Examples of RS1 starches are grains and seeds. RS2
has intact and raw starch granules such as those found in raw potatoes and green
bananas. These cannot be digested by amylases until gelatinized. RS3 are mainly
retrograded amylase or is crystalline in structure like in bread, cooked and cooled
potatoes and breakfast cereals. More recently, RS 4 is reported which are chemically
modified starches.
40
Resistant starch reportedly has similar physiological role similar to that of fiber.
Resistant starch has been shown to provide benefits such as an increased throughput
of the digestive tract and the production of desirable metabolites such as SCFAs277. In
a study conducted by the group of Cummings (1996), 12 human volunteers were
administered a glycemic diet containing either rapidly digested starch, slowly digested
starch, a non-starch polysaccharide, RS2 from green banana and RS 3 from
retrograded maize over a 15-day period. They showed that RS increased stool wet
weight and the excretion of faecal SCFAs. In a similar study, low (5g/day) and high
(49g/day) concentration RS diet were given to healthy human subjects over a four
week period279. This study demonstrated that a diet high in RS increased faecal
output, lowered faecal pH and significantly increased daily excretion of butyrate.
Thus, resistant starch has a significant impact on faecal bulk and butyrate production
which are the markers of colonic health.
An increase in faecal bulk is relevant because it prevents constipation and dilutes
potentially toxic compounds that might promote cancer cells280. RS has been shown to
be a better substrate for the production of butyrate compared to other nonstarch
polysaccharides281. This indicates that RS provides a good source of nutrients for
colonic bacteria and energy for proliferation of enterocytes and maintenance of
colonic health. Furthermore, the bowel microflora can be manipulated by providing
specific types of RS. It has been observed that bifidobacteria and lactobacilli levels
were raised after ingestion of high-amyose maize starch282. Brouns and colleagues
(2002) summarize the physiological properties and effects on colonic health on
resistant starches in Table 1.6
These properties of resistant starch listed in Table 1.6 suggest that the presence of RS
may help prevent inflammatory bowel diseases.
Feeding of RS from granular pea starch to TNBS colitic animals resulted in earlier
healing of the epithelium as compared to the group that received the non-starch
diet158. The regeneration of the colon coincided with the time of highest butyrate
production. Thus, it has been thought butyrate drives ATP production and then, the
ATP signals protein laminin to restore tissue integrity. The butyric effect of RS in
inflammation is further demonstrated in the study performed by Moreau and co-
41
workers (2003) where they compared the effects of fructo-oligosaccharides (FOS) and
RS in DSS colitis. They showed that both substrates produced butyrate but it was only
those rats that were fed RS recovered from colitis. They speculated that the protective
effect of RS could be due to the digestive pH reaching steady state rapidly compared
to the pH after FOS ingestion and that because RS produced more butyrate than FOS.
This highlights the fact that although substrates are capable to be fermented to
butyrate, they do not have similar effects in the amelioration of inflammation.
On the other hand, administration of high amylose starch has been shown to
significantly reduced endotoxin translocation to the liver of rats284. The mechanics of
this prevention could be due to the changes in the bowel microflora, stimulation of
secretory IgA and enhanced mucin production observed with ingestion of high
amylose starch. These may have promoted mucosal barrier function in the colon and
inhibit bacteria and/or endotoxin translocation.
Thus, there is a possible protective role of resistant starch in inflammatory bowel
diseases. It is postulated that resistant starch may protect against and treat IBD by:
modifying the colonic microflora to one less likely to produce toxic
metabolites and/or aggressive immune response;
yielding an environment in the colon less conducive to putative promoters
of inflammation;
causing stool bulking, thereby, decreasing the concentration of
inflammatory antigens;
increasing the rate of transit of colonic contents, thus, allowing less time
for inflammatory antigens to function.
42
Table 1.6 Functional potential of resistant starches (adapated from Brouns et al.2002)
Effects on intestinal flora and metabolism Completely fermented by intestinal flora Low levels of gas formation when fermented Elevates colonic butyrate levels more than non starch polysaccharides when fermented Reduces intestinal pH in dose dependent manners Selectively utilized by lactobacilli and bifidobacteria Promotes colonization of lactobacilli and bifidbacteria Reduces intestinal pathogen levels Reduces secondary bile acids Reduces faecal water toxicity
Effects on health, gut function and physiology Reduces symptoms of diarrhea (duration and fluid loss) Increases stool weight Mild laxative effect at higher intakes Reduces energy intake when substituted for normal starch in food Reduces insulin response compared to normal starch/carbohydrate Increase satiety response in the late post absorptive phase. Increases Ca and Mg absorption Stimulates immune system Reduces risk factors related to large bowel cancer
43
1. 5. 3. Synbiotics
A synbiotic is a combination of probiotics and prebiotics. It is defined as “a mixture
of a probiotic and prebiotic that beneficially affects the host by improving the survival
and implantation of live microbial dietary supplements in the gastrointestinal tract, by
selectively stimulating the growth and/or activating the metabolism of one or more of
a limited number of health promoting bacteria”143.
At present, only a single report was found that showed the effect of synbiotics in a
murine model162. Methotrexate was used to induce mucositis in the animals and the
mucositis animals were grouped according to the diet they received. One group of
mucositismice was dosed with lactobacilli while the other group received oat fibre in
addition to the lactobacilli. Severity of mucositis was reduced in both groups, but
improvement was significantly better in the animals fed the oat fibre-lactobacilli diet.
44
1. 6. Hypothesis
The antigen(s) that drives the chronic cell-mediated intestinal inflammation is yet to
be identified. Studies indicate that the indigenous microorganisms are central to the
development of inflammatory bowel diseases because: 1) genetically susceptible hosts
are found to be immunoreactive to their own commensal bacterial flora; and 2) the
potential pathogens are found in high levels while the putative beneficial species are
detected in low numbers in areas of the gastrointestinal tract where inflammation is
observed.
The conventional therapy used in IBD to inhibit the activities of these microorganisms
is through the use of antibiotics. Antibiotics are able to reduce severity of
inflammation but complications that may arise such as antibiotic associated diarrhea
(AAD) or pseudomembranous colitis with their continued usage as well as a higher
risk of resistant strains of microorganisms developing in the gut.
Thus, it is hypothesized that manipulating the gut microbes by increasing the
concentration and health promoting type of bacteria through the administration of
probiotic bacteria or prebiotic food additives can resolve intestinal inflammation in
colitic host. Anyone of a selection of probiotic Lactobacillus and Bifidobacterium
strains and/or the prebiotic high amylose maize resistant starch could be valuable in
reducing experimental murine colitis and clinical IBD. These probiotic strains were
previously shown to exhibit anti-microbial and positive immunomodulation properties
and as such may also reduce the likelihood of developing, or accelerate the recovery
time, in a condition with a dysregulated microbiota and immune response. On the
other hand, the prebiotic high amylose maize resistant starch may target inflammation
in IBD through its complex carbohydrate structure. Unlike other indigestible shorter
chain carbohydrates which are more rapidly utilized in the proximal colon, resistant
starch has longer chains of carbohydrate molecules and has been proposed to
influence microbial activity throughout the entire colon. Thus, the high amylose
maize resistant starch may provide more benefit to inflammation in the distal colon.
45
1. 7. Aims
The aim of work presented in this thesis was to study the effects of specific probiotic
strains (selection of Lactobacillus and Bifidobacterium strains) and the prebiotic, high
amylose maize resistant starch, on colitis and their potential to ameliorate colon
inflammation. Furthermore, the mechanisms of protection afforded by the probiotics
and the resistant starch in colon inflammation were examined.
The following approaches were utilized to realize the aims of this thesis:
I. Development of colon inflammation model in BALB/c mice using the
optimized trinitrbenzene sulfonic model of inflammatory bowel disease
(Chapter 2).
II. Determination of the lowest effective dose of the high amylose maize
resistant starch diet (Chapter 3) and the most effective probiotics (Chapter
4) for ameliorating murine colitis.
III. Correlation of the differences observed between colitic mice and colitic
mice that received the probiotics and prebiotic diet for the following
parameters:
Restoration of the microbiota balance (Chapter 3 and 4)
Stimulation of suppressive immune responses (Chapter 3 and 4)
Promotion of mucosal healing and mucosal barrier function as
indicated by:
a) short chain fatty acid production (Chapter 3)
b) bacterial translocation (Chapter 4)
c) histological profile (Chapter 3 and 4)
Presentation of disease activity indices (Chapter 3 and 4)
IV. Evaluation of the effects of Lactobacillus fermentum VRI 003 on the
faecal microbiota, cytokine production and symptoms associated with
Ulcerative Colitis (UC) when administered in conjunction with standard
therapy in UC patients undergoing remission (Chapter 5).
46
Chapter 2
Optimization of the trinitrobenzene sulfonic acid murine model of colitis
in BALB/c mice
2. 1. Introduction
Several murine models of intestinal inflammation have been developed over the
recent years and are used to assist researchers in understanding the pathogenesis of
inflammatory bowel diseases. These models are classified under four categories
according to how inflammation is induced in the animals. Chemically induced models
require an exogenous chemical compound such as trinitrobenzene sulfonic acid
(TNBS), dextran sodium sulfate (DSS) and acetic acid to be administered to animals2,
163, 164. There are also immunologically mediated models that adoptively transfer T
cells or bone marrow precursors into immunodeficient recipient mice2, 163, 164,
examples of which are the CD45RBhigh and bone marrow chimera transfer models
have been developed. Models wherein the genes necessary for the expression of pro-
inflammatory cytokines are knocked out or manipulated are also available2, 163, 164.
The majority of these models are gene deletions of cytokines such as IL-10, IL-12 and
T cell receptor (TCR) / . Finally, in the spontaneous models, inflammation develops
without any external manipulations2, 163, 164. Colitis in these models develops early on,
persists through the animal’s life cycle and resolves with age. Examples of this latter
model are the C3H-HeJBir and SAMP1/Yit mice.
Animal models provide a number of practical advantages in studying the pathogenic
mechanisms of inflammatory bowel disease compared to clinically based
investigations. The models allow the sequential analysis of the different stages of the
disease process, from prior to the onset of inflammation, during the peak of
inflammation until its resolution that is often difficult to follow in clinical subjects.
Experimental murine systems also allow the testing and comparison of the effects of
inflammatory mediators implicated in inflammatory bowel disease. Moreover, novel
therapies can be evaluated in a relatively inexpensive manner.
47
The TNBS murine model of colitis was employed in this study. This model is chosen
because it was as readily available while other IBD models were not. Furthermore, it
is one of the most widely used models wherein the irritant, 2, 4, 6 – trinitrobenzene
sulfonic acid, is administered with ethanol, as the barrier breaker, by luminal
instillation. The immunopathology in the TNBS model is characterized by the
infiltration of lamina propia with the autoaggressive CD4+ T cells2, 163, 246. It causes
disease through the excessive secretion of pro-inflammatory cytokines such as IL-12
and IFN- 166, 174, 246. Identification of the most prominent T cell profiles allows the
delineation of a particular kind of immune response to a particular histopathologic
reaction.
However, even if the TNBS model demonstrates an immune response similar to that
observed in human IBD, it still has its limitations. Firstly, inflammation does not
instantaneously develop unlike in human IBD. Inflammation in this model occurs
with exogenous manipulation. Moreover, experimental and/or genetic factors must be
present for inflammation to develop. Studies have shown that severity of
inflammation in the TNBS model is influenced by dosage and time of exposure to the
irritant48, 166,167, 174, 177, animal strain2, 177, 246 and the environment i.e animal housing
facility and microflora status of animals46.
Nevertheless, no animal models exist that exactly resemble human IBD. However,
when chosen appropriately, they can be used to explore immunopathogenic
mechanisms, test potential therapeutic alternatives and form hypotheses.
It was the objective of this study to optimize the dose of TNBS in BALB/c mice that
would induce inflammation similar to IBD as well as to characterize the immune
response and events associated with the inflammation observed.
48
2. 2. Materials and Methods
2. 2. 1. Animals
BALB/c mice were purchased from the Biological Resource Centre (BRC) Little Bay,
Sydney, Australia or from the Animal Resource Centre, Perth, Australia (ARC). All
mice were used between the ages of 6-8 weeks and had unlimited access to sterile
mouse food and water. They were kept under specific pathogen-free conditions in the
animal facility of the School of Biotechnology and Biomolecular Sciences of The
University New South Wales, Sydney, Australia. All experiments done on the animals
were approved by the Animal Care and Ethics Committee of the University of New
South Wales.
2. 2. 2. Induction of experimental colitis
The hapten reagent 2, 4, 6- trinitrobenzene sulphonic acid (TNBS; Sigma) was used
to induce colon inflammation in BALB/c mice by intra-rectal administration. The
concentration of ethanol and the dose of TNBS used in this study were optimized
prior to performing experiments on colitic mice using resistant starch (Chapter 3) and
probiotics (Chapter 4). Groups of mice (n=10) were exposed to varying
concentrations of ethanol, 50%, 45%, 30% and 20% as well as induced with a single
dose of TNBS at a concentration of 2.5, 2.0 and 1.5 mg in 100 µl ethanol. Before
trialing the TNBS doses, the best ethanol concentration to be used was determined
first by assessing the disease activity and histology of colon tissues of the animals
after 7 days. After this, the chosen ethanol concentration was used for the TNBS dose-
response experiment. Animals were subcutaneously anaesthetized with xylazine and
ketamine prior to the intrarectal administration of ethanol or TNBS mixture using a
sterile, stainless steel animal feeding tube. The animals were held in a vertical
position for 20 seconds while administering ethanol or TNBS to allow it to reach the
entire colon.
49
2. 2. 3. Assessment of severity of colon inflammation
The disease activity index (DAI) was assessed from the major clinical signs of colon
inflammation in mice and include weight loss, diarrhea, rectal bleeding and ruffling of
the fur coat. The final formula for DAI was the sum of the score given for weight loss,
diarrhea, rectal bleeding and ruffling of the fur coat.
Body weight loss was calculated by getting the difference between the percentage
weight change of the starting bodyweight and body weight on a particular day. The
appearance of diarrhea was defined as mucusy or loose consistency of faecal material
that was adherent to anal fur. The presence or absence of diarrhea was scored as either
1 or 0, respectively. A score of 2 was then given when a visible sign of blood was
observed during the diarrhea episode. On the other hand, ruffling of the fur coat of the
animal was scored as 1 and 0 if it remained smooth.
2. 2. 4. Histological analysis of colon tissues
Mice were sacrificed by CO2 asphyxiation 7 days after ethanol was administered or
colitis was induced using TNBS. The distal colon was quickly removed, opened
longitudinally and cleared of faecal contents using cold PBS. The colon tissue was
then fixed in 10% phosphate buffered formalin at room temperature overnight. The
tissues were then sliced into 5 mm pieces, dehydrated in ethanol, embedded in
paraffin wax, sectioned and stained with haemotoxylin and eosin (H & E) or Giemsa
stain. The section with the worst appearance of inflammation was assessed.
2. 2. 5. Organ culture conditions and cytokine assays
The colons were collected from control and TNBS-treated animals a week post-
induction of colitis. The colons were then cut into 3 mm pieces and cultured in RPMI
1640 medium supplemented with 10% heat-inactivated fetal calf serum, 5mM L-
glutamine and 100 U penicillin and 100 µg streptomycin at 37ºC in 5% CO2, 95% O2
for 24 hrs in the presence or absence of 5 µg/ml concanavalin A (ConA; Sigma). After
24 hrs, supernatants were harvested for cytokine assays. All tissue culture reagents
were purchased from Gibco unless otherwise stated.
50
Cytokine concentrations in the culture supernatants were measured by ELISA. In
brief, 96 well plates (NUNC) were coated with purified anti-cytokine capture
antibody and by incubation overnight at 4ºC. Subsequently, the plates were blocked
with 1% BSA in PBS/ 0.05% Tween 20 (PBS/T) and incubated at room temperature
for 90 mins. The standards were prepared and diluted two-fold in 1% BSA in PBS/T.
The standards and samples were then added to the wells and the plates were left to
incubate at room temperature for 90 minutes. Biotinylated anti-cytokine antibody
dissolved in 1% BSA-PBS/T was then added into the wells and was allowed to
incubate for another 90 minutes in room temperature. Streptavidin-horse radish
peroxidase (HRP) (Chemicon) was diluted 1/ 1000 in 1% BSA-PBS-T and allowed to
incubate in the wells at room temperature for another 30 minutes. The plates were
washed three times with PBS/T after each step until the addition of the substrate
3,3’,5,5’-tetramethyl benzidine (TMB; Sigma) and hydrogen peroxide into the plate.
Once substrate and hydrogen peroxide were added, colour reaction was allowed to
develop at room temperature. The reaction was stopped using H2SO4 and the plates
were read at a wavelength of 450 nm using BIORAD Microplate Manager Reader.
The antibodies used were rat anti-mouse IL-12 and IL-10 that were both obtained
from Pharmingen.
2. 2. 6. Determination of colonic bacterial concentration
After the animals were sacrificed, the colons were opened longitudinally and the
colonic pellets were collected, weighed and serially diluted ten-fold in half strength
Wilkins-Chalgren broth prepared by adding half the powder required per 1 litre of
sterile distilled water (1/2 WCB: 1 dH2O; Oxoid). From every dilution, 10 µl was
drop plated in triplicates onto Mac Conkey Agar (MAC; Oxoid), de Mann Rogosa
Sharpe Agar (MRS; Oxoid) and Wilkins-Chalgren agar supplemented with 5% horse
blood (WCAH; Oxoid) to enumerate enteric bacteria, total lactobacilli and total
anaerobes, respectively. The MAC agar plates were incubated aerobically for 24
hours at 37ºC while the MRS and WCAH agar plates were incubated in an anaerobic
chamber for 48 hours at 37ºC.
51
2. 2. 7. Statistical analyses
All data are expressed as mean ± SD. The statistical significance of the differences
was evaluated using t-test or ANOVA for normally distributed data and Mann-
Whitney U test or Wilcoxon Signed Rank test for non-parametric data. Statistical
significance was defined as p<0.05. All statistical analysis was performed using SPSS
for Windows version 11.5.
52
2. 3. Results
2. 3. 1. Comparison of colitis activity in BALB/c mice induced with 2.5 mg and
2.0 mg TNBS in 50% ethanol
The dose of 2.5 mg and 2.0 mg TNBS dissolved in 50% ethanol were initially
compared. These doses were used after consultation with other groups (Grimm,
personal communication) and from published studies that used the TNBS model of
colitis. It was found in this study that BALB/c mice induced with 2.5 mg TNBS and
2.0 mg TNBS in 50% ethanol resulted in a DAI of 15.29 and 13.23, respectively
(Table 2.1). The wasting effects of these doses proved to be lethal to the BALB/c
mouse strain as only 20-30% of animals survived after 5 days when the hapten
reagent was administered. Moreover, animals that had a total body weight loss of 13%
or more were culled as per the Animal Care and Ethics Committee (ACEC) guidelines
in conducting research on animals. Both of the groups experienced this amount of
weight loss just a day after TNBS was induced (Figure 2.1) BALB/c mice were also
exposed to 50% ethanol to determine if the ethanol had an effect on the activity of
colitis seen in BALB/c mice. DAI of the animals in the group that received 50%
ethanol alone was comparable with the recorded DAI and mortality of animals
receiving TNBS in 50% ethanol. Furthermore, injury was incurred using these
treatments that resulted in extensive inflammation of the colon tissue (Figure 2.2a to
2.2c) and appearance of granulomas in mice dosed with 2.5 mg TNBS in 50% ethanol
(Figure 2.2a). These results show the lethality of this concentration of ethanol. This
concentration was not used in subsequent experiments.
2. 3. 2. Effect of varying the ethanol concentration affects the intensity of disease
activity in BALB/c mice
A range of ethanol concentrations; 45%, 30%, 20% and 0%, were tested because 50%
ethanol alone was shown to be too damaging to the animals. The aim of this
experiment was to determine the ethanol concentration that would confer the minimal
injury to the animals prior to adding the TNBS reagent. The lowest DAI score was
obtained from mice
53
Table 2.1. Comparison of disease activity index (DAI) of BALB/c induced with 2.5
mg TNBS and 2.0 mg TNBS in 50% ethanol (n=10).
Group DAI on Final Day Survival (%)
2.5 mg TNBS in 50% ethanol 15.29 ± 6.37 3/10 (30%)
2.0 mg TNBS in 50% ethanol 13.23 ± 10.35 2/10 (20%)
50% ethanol 12.07 ± 8.73 5/10 (50%)
54
-10
-5
0
5
10
15
20
25
30
35
1 2 3 4 5
Days after colitis induction
Dise
ase
Act
ivity
Inde
x (D
AI)
Normal BALB/c mice 2.5 mg in 50% 2.0 mg in 50%ethanol 50% ethanol
Figure 2.1. Colitis activity in BALB/c mice (n=10) induced with 2.5 mg and 2.0 mg TNBS in 50% ethanol. Results are expressed as the mean DAI of surviving animals in each group ± SD.
55
(a)
(b)
(c)
Figure 2.2. Colon histology of BALB/c mice exposed to A) 2.5 mg TNBS in 50% ethanol, B) 2.0 mg TNBS in 50% ethanol and C) 50% ethanol. Colon tissues were stained with Geimsa and assessed at a magnification of 40X. Blocked arrow indicates appearance of granuloma.
56
dosed with 20% ethanol, however, the kinetics did not differ from those mice dosed
with 30% ethanol (Figure 2.3). Moreover, both 20% and 30% did not have dramatic
change in their DAI scores compared to the 0% ethanol concentration group as there
was only 1-2 score difference noted between these groups. These observations were
further confirmed by the histological profile of the colon tissues (Figure 2.4a-d). The
epithelium, goblet cells and crypts remained intact and there was minimal
lymphocytic infiltrations detected when 20% (Figure 2.4b) and 30% ethanol (Figure
2.4c) were administered. In fact, the tissue damage was very similar to that noted for
animals receiving 0% (Figure 2.4a). Conversely, intrarectal administration of 45%
ethanol produced consistent DAI kinetics that is different significantly from those
obtained using other ethanol concentrations and the control receiving 0% ethanol
(p<0.01). Body weight loss was less pronounced remaining within the acceptable
range not requiring sacrifice of the animals. Histology also revealed that the
epithelium barrier was broken (Figure 2.4d). No deaths were observed for any of the
concentrations of ethanol used. Based on these results, 45% ethanol was used to
dissolve TNBS in subsequent experiments.
2. 3. 3. Development of TNBS-induced colitis in BALB/c mice
Subsequent to determining the best ethanol concentration, the dose of TNBS was
optimized. Several parameters were investigated to confirm the presence of colon
inflammation in this murine model. The disease activity index was determined to
corroborate clinical signs of disease in the different concentrations used (Figure 2.5).
From the three doses, 2.5 mg TNBS resulted in significant colon inflammation
(p<0.01) on Days 2, 3, 4, 5 and 6 as manifested by greater weight loss, more frequent
diarrhea and rectal bleeding and rougher fur coat observed across time compared to
2.0 mg and 1.5 mg doses. On the other hand, the latter doses had signs of disease
similar to the onset, intensity and appearance of mice induced with 45% ethanol
alone. The 2.0 mg TNBS dosed mice had a slightly higher DAI compared to those
receiving 1.5 mg TNBS. The mortality rate also varied for the different concentrations
of TNBS used, from 10% when induced with 1.5 mg TNBS to 40% when induced
with either 2.0 mg or 2.5 mg TNBS.
57
-6
-3
0
3
6
9
12
15
0 1 2 3 4 5 6
Days after colitis induction
Dise
ase
Act
ivity
Inde
x (D
AI)
0% 20% 30% 45%
*
Figure 2.3. Disease activity index (DAI) of BALB/c mice a week after exposure to different concentrations of ethanol (n=10 per group). Results presented as mean DAI and error bars correspond to SD.* significantly different from other treatments at p<0.01.
58
Figure 2.4. Histopathological changes in H&E stained sections of the colon of BALB/c mice 7 days after exposure to different concentrations of ethanol. Colons of BALB/c mice induced with a) 0%, b) 20%, c) 30% and d) 45% ethanol.
BALB/c mice induced with 0% have regular crypts with mucin containing goblet cells that are found along the length of the crypt (40X). Similar changes were noted in BALB/c mice intra-rectally administered with 20% and 30% ethanol. Ethanol enema of 45% created a mild epithelial damage and ulceration.
(a) (b)
(c) (d)
59
-9-6-30369
12151821
0 1 2 3 4 5 6Days after colitis induction
Dise
ase
Act
ivity
Inde
x (D
AI)
1.5 mg TNBS 2.0 mg TNBS 2.5 mg TNBS 45% ethanol Normal
Figure 2.5. Disease activity index (DAI) of BALB/c mice (n=10) induced with colitis using different doses of TNBS in 45% ethanol for up to six days after induction.
Results are expressed as mean DAI, error bars are SD. Significantly (p<0.01) more severe colon pathology was observed in 2.5 mg compared to 2.0 and 1.5 mg doses of TNBS as determined by independent t-Test on Days 2, 3, 4, 5, 6 after colitis induction.
60
Histological studies revealed that mice that were induced with 2.5 mg TNBS (Figure
2.6c) had more severe inflammation of their large intestine compared to mice dosed
with intermediate and low doses of TNBS. The colitis observed in mice dosed with
2.5 mg TNBS in 45% ethanol was characterized by distortion of crypts, loss of goblet
cells and infiltration of neutrophils and mononuclear cells (Figure 2.6c), while,
inflammation seen in the other colitic groups (Figures 2.6a-b) appeared equivalent to
the ethanol-control group (Figure 2.6d), which was a mild form of colitis or no colitis
at all and there was no loss of goblet cells and no crypt abscesses were noted.
Macroscopic examination showed that the inflammation occurred throughout the
entire colon.
Cytokine production in the colons of TNBS-treated mice was also examined. This
was done by collecting the colonic tissue specimens 7 days after the induction of
colitis and cytokine profiles were compared among the groups. The colon tissues were
cultured for 24 hrs and culture supernatants were analyzed for concentration of one
nominated Th1 cytokine (IL-12), and one T regulatory cytokine (IL-10) by specific
ELISA. As shown in Figure 2.7, there was a significant increase (p<0.05) of IL-12
production a week after induction of colitis in 1.5 mg, 2.0 mg and 2.5 mg TNBS-
treated group compared to the ethanol-treated and normal groups. Furthermore, organ
cultures from colonic tissue of 2.5 mg TNBS-treated animals produced three times
higher levels of IL-12 than animals from colitic BALB/c mice (p<0.05). In contrast,
secretion of IL-10 from organ cultures of TNBS-treated mice was identical to the
amounts produced by ethanol-treated and normal control mice.
Based on the above results, 2.5 mg TNBS in 45% ethanol was used to induce colitis in
BALB/c mice for experiments performed in Chapters 3 and 4.
61
Figure 2.6. Histological features of TNBS-induced colitis in BALB/c mice receiving different doses of TNBS in 45% ethanol; a) 1.5 mg TNBS; b) 2.0 mg TNBS, c) 2.5 mg TNBS and d) 45% ethanol. Magnification was at 40X.
D
(b)
(c)
(a)
(d)
62
0
200
400
600
800
1000
1200
1400
1600
1.5 mg TNBS 2.0 mg TNBS 2.5 mg TNBS 45 % ethanol Normal
Cyt
okin
e C
once
ntra
tion
(pg.
ml-1
)
IL-10 IL-12
**
*
Figure 2.7. IL-12 and IL-10 production in organ cultures of colons from BALB/c mice induced with different concentrations of TNBS at 7 days post-induction of colitis.
Results are expressed as mean concentration (pg.ml-1) of cytokine ± SD. * significantly different from the ethanol-control and normal-control groups, p<0.05.
63
2. 3. 4. Colonic bacterial concentrations in BALB/c mice induced with TNBS
Concentrations of total lactobacilli, enterics and anaerobes, which are members of the
indigenous microflora considered to be involved in the pathogenesis of inflammatory
bowel diseases12, 57, 165 were measured in TNBS-treated versus ethanol-treated and
normal control (Figure 2.8a-c). The concentration of lactobacilli was significantly
reduced (p<0.05) in BALB/c mice induced with TNBS and ethanol a week post-
induction of colitis, relative to normal animals (Figure 2.8a). On the other hand,
counts of total Gram negative enteric bacteria significantly increased (p<0.05) in all
treated groups, with the 2.5mg TNBS-treated animals harbouring the highest number
of colonic enterics, compared to normal BALB/c mice (Figure 2.8b). No detectable
changes in the numbers of total anaerobes (Figure 2.8c) were observed in any of the
groups. Figure 2.9, on the other hand, shows that bacteria were found present on the
colonic epithelium during colitic development in BALB/c mice induced with TNBS.
64
Lactobacillus counts
0 2 4 6 8 10 12
Normal
45% ethanol
1.5 mg TNBS
2.0 mg TNBS
2.5 mg TNBS
Log colony forming units (CFU) per gram colon contents
p=0.05
Enteric counts
0 2 4 6 8 10 12
Normal
45% ethanol
1.5 mg TNBS
2.0 mg TNBS
2.5 mg TNBS
Log colony forming units (CFU) per gram colon contents
p=0.05
Total Anaerobe counts
0 2 4 6 8 10 12
Normal
45% ethanol
1.5 mg TNBS
2.0 mg TNBS
2.5 mg TNBS
Log colony forming units (CFU) per gram colon contents
Figure 2.8 Concentrations (Log CFU.g-1 of colon contents) of the bacterial groups involved in colon inflammation detected in colitic BALB/c mice (n=10). Results are expressed as mean bacterial concentrations ± SD.
Lactobacilli were significantly reduced (p<0.05) in TNBS-induced and ethanol-induced animals compared to normal control animals while enteric bacteria were significantly higher (p<0.05) in all TNBS-treated groups.
65
Figure 2.9. Bacteria detected on colitic tissue of BALB/c mice 3 days post-induction with 2.5 mg TNBS.
Colon sections were fixed, embedded on paraffin and section stained with H&E prior to microscopic assessment.
66
2. 4. Discussion
In these experiments, a murine model of intestinal inflammation induced by the
intracolonic injection of the hapten reagent TNBS is described. Significant findings of
this experiment include demonstration that the intensity of inflammation is dependent
on the dose of TNBS and concentration of the delivery vehicle (ethanol). Secondly,
inflammation is associated with a Th1 cytokine response and colonic lesions exhibited
an acute type of inflammation. Furthermore, TNBS-induced colitis is coupled with a
reduction in the number of potentially beneficial lactobacilli and proliferation of
Gram negative enteric bacteria in the colonic contents.
The trinitrobenzene sulfonic acid (TNBS) murine model is a chemically induced
model of inflammatory bowel disease. Tissue injury in this model is T cell mediated
as a result of covalent binding of TNP residues to both hapten and autologous proteins
such as the normal microflora47, 166, 167.
The relevance in the suitability of the TNBS-induced colitis model for studying the
effects of resistant starch (Chapter 3) and probiotics (Chapter 4) is that it can provide
a direct indication of any alterations in the colonic microflora and shifts in the
immune response by the resistant starch and probiotics. Faecal lactobacilli are
reported to be completely absent in TNBS model compared to the predominance of
lactobacilli in healthy animals247 while increased production of pro-inflammatory
cytokines is observed2, 163, 164, 174,177, 246. Resistant starch can act as a substrate for
intestinal microbes187-190 and provide energy for colonocytes regeneration while
probiotics can positively stimulate the immune response and produce antimicrobials
that could inhibit the destructive effects of colitis.
It is worth noting that the type and severity of inflammation seen in the TNBS model
is dependent on a number of factors such as dosage and time of exposure to the hapten
and the “barrier breaker”, genetic background of the animal and the status of the
environment. Thus, the intensity of the inflammation observed in one laboratory
cannot be inferred by other observers in other laboratories but has to be criticized
accordingly in terms of the conditions mentioned above.
67
Ethanol was used as delivery vehicle for TNBS in this experiment. Its role is to
facilitate the entry of TNBS from the colonic lumen into mucosal tissues by mediating
the breakdown of the epithelial barrier. It was found in this experiment that the
concentration of ethanol influences the mortality rate of animals. Fifty percent (50%)
ethanol was initially used because this concentration was used as in previous studies2,
48, 166, 169-171 but this proved to be too deleterious on survival as well as destruction of
the integrity of tissue architecture. Therefore, a range of doses was tried and 45%
ethanol was selected to be used in subsequent experiments. Other studies172, 173 also
used a lower concentrations of ethanol (40% and 45%, respectively) in their BALB/c
mice. Adjusting the ethanol concentration suggest that it can influence the clinical
course of the disease, therefore, it is necessary to ensure the right concentration to use
before performing an extensive colitis experiment.
It is also demonstrated in this study that a single intra-rectal administration of 2.5 mg
TNBS was sufficient to induce in BALB/c mice a chronic distal colitis that persisted
for a week. This result shows the advantage of using this experimental model of IBD
wherein inflammation rapidly and reliably develops in mice with a normal immune
system without the need of genetic manipulation of a major immune feature. The
variability in the TNBS dose between studies is a well recognized difficulty in this
model of IBD, presumably, the variability is related to differences in colonic flora in
different housing environments48, 166, 167, 174. Nevertheless, epithelial disruption
(Figures 2.6), bacterial translocation (Chapter 4) and infiltration of neutrophils (Figure
2.6) were observed when exposed to 2.5 mg TNBS. Moreover, these events were
associated with diarrhoea, rectal bleeding and weight loss which are the clinical
features that further underscore that TNBS-induced colitis mimics some important
features of human IBD.
One of the most intriguing findings reported here is that histology of the colonic
lesions of the animals with a single intracolonic challenge with 2.5 mg TNBS was
marked by ulceration, acute inflammatory infiltrates and regenerative changes while
analysis of cytokine production by the organ culture supernatants showed strikingly
elevated levels of the pro-inflammatory Th1 cytokine IL-12. The histological changes
observed parallel the acute phase of chronic ulcerative colitis175, 176, whereas, the
increased production of Th1 cytokine and the almost normal or reduced levels of IL-
68
10 secreted by the colons of TNBS-treated mice have resemblance to human Crohn’s
disease at the T cell cytokine level, as previously reported. Thus, these imply that the
pattern of inflammation or immune responses to intracolonic TNBS can induce either
Th1 or Th2 type of inflammation associated with distinct types of colitis177. These
could be due to differences in the mouse strain used, dose of TNBS and conditions in
the animal house facilities. These also reflects the situation in diagnosing clinical IBD
as there are cases which are difficult to categorise as either Crohn’s disease or
ulcerative colitis because of the presence of intermediate/ overlapping features. These
further indicate that the phenotype of disease can rarely shift from one to the other
during the course of the disease.
Moreover, it has also been suggested that luminal components such as the intestinal
microflora may also affect the local cytokine responses. An increase in the number of
enteric bacteria was detected in the study that is consistent with results from other
experimental colitis models as well as human ulcerative colitis and Crohn’s disease.
Vibrio cholerae, Escherichia coli, Helicobacter and Campylobacter spp., Salmonella
spp., Listeria monocytogenes and Streptococcus pneumoniae are some of the
enterobacteria found in these conditions. It is postulated that the lipopolysaccharides
(LPS) component of their cell wall and the endotoxins they produce are the factors
driving the abnormally aggressive Th1 immune response observed in colon
inflammation178. IL-12 significantly increased in colitic animals whereas low levels of
IL-10 were produced from the colons. This observation coincided with the
proliferation of enteric bacteria in the faeces of colitic mice. IL-12 is a cytokine that
biases the immune response towards a Th1 phenotype and it is believed to be driven
by the microorganisms and microbial products in colon inflammation. It also
synergistically acts with IL-18 in the induction of a strong Th1 mediated immune
response and it most notably amplifies the production of IFN- 179. On the other hand,
IL-10 is a regulatory cytokine that inhibits both antigen presentation and subsequent
upregulation of the pro-inflammatory response. However, IL-10 production in the
colitic animals used in the study was downregulated and there was no evidence of it
providing protection against the immunological effects of TNBS. It is thus postulated
that the high concentrations of enteric bacteria detected in BALB/c mice after
induction of colitis compels the immune response to produce a pro-inflammatory type
of response.
69
It is also noted in numerous studies that bowel inflammation is thought to be caused
by a loss of tolerance against indigenous bacteria of the intestinal tract48, 49, 180.
Garcia-Lafuente and colleagues (1998) further support the influence of bacteria in
colitis by demonstrating that inflammation induced by TNBS is more aggressive in
diverted colonic segments when colonized with bacteria, including Bacteroides, than
in segments exposed to antibiotics. Furthermore, normal tolerance that exists towards
indigenous microflora is broken in experimental colitis48. The specific
microorganisms responsible for perpetuating colon inflammation are yet to be
determined but one common observation of the microflora profile of this study and
other researches on experimental and clinical IBD is the reduction of potentially
protective genus, Lactobacillus. This suggests, then, that not all indigenous bacteria
are involved in the induction and perpetuation of inflammatory events in colitis and
some may afford protection.
In conclusion, this study showed that 2.5 mg TNBS in 45% ethanol generated an
acute type of inflammation, similar to Ulcerative Colitis, with a T cell response that is
skewed towards a Th1 pro-inflammatory type of response and which was associated
with a reduction of lactobacilli count and a concurrent increase in enterobacteria.
These results provide further evidence that symptoms of the two forms of IBD may
overlap which can lead to the difficulty in discriminating between them. Ulcerative
Colitis and Crohn’s Disease share many clinical and histopathological features and
there is no absolute differentiating marker. Thus, classifying bowel inflammation
according to the characteristics presented in Table 1.1may limit the enormity of the
disease. What is more significant to consider is that an aggressive inflammation
develops in the gastrointestinal tract which could be driven by a dysregulated T cell
immune response and the microflora and to regulate this immune response to be
comparable to healthy gut.
70
Chapter 3
Feeding of high amylose maize resistant starch diet to BALB/c mice with
experimentally induced colon inflammation
3. 1. Introduction
The barrier function of the epithelium is breached in intestinal inflammation thus
allowing the rapid translocation of microbial products possessing antigenic properties
into the mucosa89. This results in the accumulation of pro-inflammatory mediators
such as lymphocytes, chemokines, cytokines and reactive oxygen species in the
mucosa. These further contribute to the deterioration of the epithelial integrity. It is
believed that the exacerbated immune response seen in inflammatory bowel diseases
(IBD) is directed towards luminal microbes. Bacteroides vulgatus53, Enterococus
faecalis54, Mycobacterium paratuberculosis66, Helicobacter spp68, Salmonella
typhimurium181 and sulfate reducing bacteria55 have been found in high numbers in
samples from inflammatory bowel disease (IBD) patients, however these studies
showed contradictory results on the dominant microbe(s) present. Interestingly though
a common finding was that lactobacilli were present in low numbers.
Lower level of short chain fatty acids (SCFAs) have been detected in an inflamed
colon compared to those seen in healthy subjects182, 183. This is critical as SCFAs are
essential for several physiological activities in the bowel. SCFAs serve as a metabolic
fuel for regeneration of colonocytes and contribute to tissue repair184. The bacteria
contributing to the production of SCFAs are also involved in mucus production185,
lipogenesis186 and detoxification of mutagens and carcinogens187. These events
suggest the possibility that restoration of the gut integrity by modulation of the
immune response and physiological activities of the intestinal microbes can lead to
remission of colonic inflammation.
In recent years, there has been a growing interest in the use of indigestible
carbohydrates in the management of IBD because of their prebiotic effects153. These
carbohydrates can stimulate the growth of beneficial microbes in the colon and/or
influence the metabolic activities of colonic microbes that affect the physiological
71
functions of the host. Examples of these prebiotics are oligosaccharides, fructo-
oligsaccharides, inulin, rafitulose, germinated barley and resistant starches (RS). Of
significant interest in this study is examining the efficacy of RS in reducing the
development and severity of trinitrobenzene sulfonic acid (TNBS) induced colitis.
Resistant starch has been shown to produce the highest concentration of butyrate in
the colon compared to other dietary fibres148-150, 188, 189. This can facilitate wound
healing in the inflamed mucosa by promoting butyrate oxidation in the colonocytes.
Thus, elevating butyrate levels in the inflamed gut may be achieved by
supplementation with resistant starch as an alternative to giving butyrate as enemas.
The latter is poorly tolerated by patients because of the offensive odour (Grimm M.,
personal communication). Moreover, RS selectively promotes the growth of putative
beneficial Bifidobacterium that can prevent the colonization of disease causing
bacteria in the colon150. RS is also more slowly degraded in comparison to other
prebiotics and can reach the entire length of the colon and its associated bacteria190.
This ensures a steady supply of fermentable substrate used by the colonic microbes
thereby influencing the host physiological as well as microbial diversity and activity
in the colon.
Bulking and faster transit times have been suggested as two major protective
characteristics of resistant starch against colorectal diseases248-249. Resistant starch has
been shown to have modest effect on faecal bulking and tends to delay transit time278-
279. These characteristics of resistant starch are significant in the amelioration of
inflammatory bowel diseases because it indicates that starch is efficiently degraded by
bacteria yielding energy and carbon necessary for the synthesis and growth of both
the microflora and colonocytes. Subsequently, this results to diluting the
concentrations of phenols and ammonia, by products of fermentation in carbohydrate-
deficient environment, in the colon280. In addition to their bulking effect, resistant
starch can reduce transit time278. This is beneficial in IBD as this demonstrates that
resistant starch does not have a pronounced laxative effect, thereby, may modulate
contractile activity and water movements in the colon and eventually may decrease
incidence of diarrhea in IBD.
72
The aim of this study was to assess if two varieties of the high amylose maize
resistant starch diet, namely an unmodified and a modified form, given at a low and a
high concentration could attenuate TNBS-induced colitis in mice. It was hypothesized
that ingestion of high amylose maize resistant starch could prevent worsening of
colon inflammation by promoting SCFAs oxidation, restoring a colonic microbial
community resembling that seen in healthy animals, and modulating the cytokine
response in the colonic mucosa. Moreover, these two varieties of the high amylose
maize starch diet may have different effects because of the pattern of degradation
observed by others191, 192.
73
3. 2. Materials and Methods
3. 2. 1. Animals
Six to eight week old, female, specific pathogen free (SPF) BALB/c mice were used
in this study. The animals were allowed to adapt to the housing conditions of the
animal facility in the School of Biotechnology and Biomolecular Sciences, University
of New South Wales, Australia prior to use in all experiments. The animals were kept
on sawdust in plastic cages and were allowed access to sterile water and food ad
libitum. All work done has approval from, and was carried out in accordance with, the
guidelines of the Animal Care and Ethics Committee of the University of New South
Wales, Australia. Animals were either purchased at the Biological Resource Centre at
Little Bay, New South Wales or the Animal Resource Centre, Perth, Australia.
3. 2. 2. High amylose maize resistant starch diet
Three experimental diets enriched with high amylose maize resistant starch were
specifically prepared for this study (Table 3.1). The non-starch diet did not contain
any resistant starch but had an equivalent concentration of glucose and cellulose when
energy and fibre levels were considered. One resistant starch diet was enriched with
an unmodified starch, Culture ProTM, the other contained a modified resistant starch,
Hi-MaizeTM. Both high amylose maize resistant starches used in this study are
classified as RS Type 2. Hi-MaizeTM was produced by an additional heating process
using dry heat and was not retrograded. Seventy (70%) percent of the resistant starch
is amylase resistant276. Both RSs were kindly provided by Starch Australasia Pty Ltd.
Diets used in this study contained either 30% or 5% of resistant starch.
74
Table 3.1. Composition of high amylose maize resistant starch diet. Three diets, 0%, 5% and 30% were prepared and reflect the concentration of resistant starch used.
High Amylose Maize Resistant Starch Concentration (g.kg-1)Ingredient
No (0%)Resistant
Starch
Low (5%) Resistant Starch
High (30%) Resistant Starch
Sucrose 150.0 150.0 150.0
Glucose 280.0 245.0 70.0
Cellulose 120.0 105.0 30.0
Resistant Starch (unmodified or modified)
- 50.0 300.0
Wheat bran 100.0 100.0 100.0
Casein 200.0 200.0 200.0
Gelatine 20.0 20.0 20.0
Choline chloride 6.0 6.0 6.0
Methionine 4.5 4.5 4.5
Vitamin and mineral mix (Veterinary Biochemical Research Ltd., Mitagong, Australia)
6.0 6.0 6.0
Sunflower oil (ml) 25.0 25.0 25.0
Canola oil (ml) 25.0 25.0 25.0
75
3. 2. 3. Experimental design
Figure 3.1 presents the experimental design of this study. After the adaptation period,
the animals were grouped accordingly for each experiment in this study.
Experiment 1
The first experiment compared the effects of incorporating 30% unmodified high
amylose maize starch on the weight, survival rate, colonic microbial community and
cytokine expression profiles of colitic animals. The animals were randomly divided
into four groups (n=10 per group per sampling point). The first group was the healthy
control wherein they were not induced with TNBS. The second group was the ethanol
control group which was the control for the TNBS because it was dissolved in ethanol
prior to intrarectal dosage. The third and fourth groups were colitic animals that
received resistant starch free or the unmodified resistant starch diet, respectively.
Assessment of body weight loss and histological changes was carried out on Days 15,
18, 21 and 24; and the cytokine and microbial profiling was performed on Day 21 of
the study period.
Experiment 2
The second experiment compared the efficacy of the low and high concentration of
unmodified high amylose maize resistant starch in ameliorating TNBS induced colitis.
Each group in this second experiment consisted of 10 animals at each sampling time.
Inflamed colon was examined for any physical and immunological changes 1 and 7
days post-induction of colitis. In addition, the microbial profiles of the colonic
contents were assessed 7 days after inducing colitis with TNBS. The peak of
inflammation in this model was observed 7 days after intrarectal administration of
TNBS (Chapter 2).
Experiment 3
The final experiment of this chapter assessed two varieties of high amylose maize
resistant starch, unmodified and modified, administered at 30% and 5% concentration
and their influence on SCFA production and cytokine production in the colonic
mucosa of animals (n=8 per group) 7 days post-induction of colitis with the TNBS. In
76
total, seven groups were assessed in this experiment, namely, the healthy, ethanol,
colitic, coltic plus the two concentrations of unmodified RS and colitic plus the two
concentrations of modified RS.
The animals were fed with their respective diets for 14 days prior to the induction of
colitis and were maintained on this diet until the end of the experiment. Animals that
were in the healthy control and ethanol control groups were fed the normal mouse
feed
77
Figure 3.1. Experimental design to assess effects of high amylose maize resistant starch diet on colon inflammation induced by TNBS.
Three experiments were performed in this chapter: Experiment 1: Non-starch diet vs. unmodified high amylose maize resistant starch diet Experiment 2: Comparison of 0%, 5% and 30% unmodified high amylose maize starchExperiment 3: 5% and 30% of unmodified vs. modified high amylose maize starch
Severity of colon inflammation was assessed by evaluating clinical manifestation of disease, histology score of colon tissue, microbial profile, cytokine profile and SCFA production in colon contents of mice induced with colitis
High Amylose Maize Resistant Starch Feeding
Days 14 15 18 21 24
Induction of colon inflammation with 2.5mg TNBS in 45% ethanol ASSESSMENT TIME POINTS
78
(Gordon’s Mouse Feed, Sydney, Australia). Colitis was induced by intrarectal
instillation of 2.5 mg trinitrobenzene sulfonic acid (TNBS) (Sigma) dissolved in 45%
ethanol on Day 14. Disease activity was monitored during the course of the study by
assessment of weight loss, incidence of diarrhea, rectal bleeding and texture of fur
coat. Mortality and mobility of the animals were also noted. The animals were then
sacrificed by CO2 asphyxiation. The colon was quickly removed upon sacrifice, open
longitudinally and cleared of colonic contents using cold PBS. This distal colon
region was used for microbial analysis of the pelleted contents, the next section was
used for microscopic tissue assessment and the remainder was divided into three
pieces, one for the isolation of lamina propria cells, another for measuring cytokine
levels and another for RNA extraction.
3. 2. 4. Microscopic assessment of colonic tissue
The colon tissue samples were fixed in neutral buffered formalin, dehydrated and
embedded in paraffin. Sections were stained with haematoxylin and eosin and scored
blindly using the histological scale of Ameho and colleagues (1997)193. Briefly,
microscopic analysis was done on the whole tissue sample at a magnification of 10X,
20X and 40X. The area of the tissue section with the worst inflammation was scored.
A score of 0 was given if histological findings were identical to normal tissue; 1 if
there was a mild mucosal or submucosal inflammatory infiltration and edema but
muscularis mucosae was intact; 2 if the condition described previously involved
>50% of the tissue specimen; 3 when ulceration extends through the muscularis
propria, prominent infiltration of neutrophils and edema but without muscle necrosis;
4 when grade 3 changes involved >50% of the specimen; 5 when there was extensive
ulceration with coagulative necrosis characterized by numerous neutrophils and
mononuclear cells which extends into the muscularis propria; and 6 if grade 5 changes
involved >50% of specimen.
3. 2. 5. Isolation of lamina propria mononuclear cells (LPMCs).
The colon was sliced into 3-4 mm long pieces, placed in a flask containing Hank’s
Balanced Salt Solution (HBSS) (Gibco) with 2mM dithiotreitol (DTT) (Sigma) and
was stirred for 20 mins at 37ºC. The solution was replaced and the colon pieces
79
shaken with fresh HBSS containing 2 mM DTT and 0.01 M EDTA (Sigma) at 37ºC
for 20 mins. The colon was further digested with 1 mg.ml-1 collagenase and 1 mg.ml-1
dispase for 1 hr at 37ºC to generate LPMC suspensions. The LPMC suspensions were
then cultured at 1x106.ml-1 in RPMI 1640 (Gibco) supplemented with 10% heat-
inactivated fetal calf serum (Gibco), 5mM L-glutamine (Gibco) and 100 U penicillin
(Gibco) and 100 µg streptomycin (Gibco). After 3 days of incubation at 37ºC in an
atmosphere of 5% CO2, supernatants were collected and stored in -70ºC until use and
assayed by ELISA to measure cytokine production.
3. 2. 6. Determination of mucosal cytokine production in colon tissues
The colon tissue was rinsed in cold HBSS, cut into 3-4 mm squares and resuspended
in complete RPMI medium. Mucosal pieces were incubated at 37ºC in 5% CO2.
Supernatants were collected for measurement of IFN- and IL-4 after 48 hours and
stored at -70ºC prior to analysis. Cytokine secretion was measured using ELISA.
3. 2. 7. Cytokine assay by ELISA
Cytokine concentrations in the supernatants of LPMCs and colon mucosa were
measured by ELISA. Microtitre 96-well plates (NUNC) were coated with purified
anti-cytokine capture antibody and incubated overnight at 4ºC. Subsequently, the
plates were blocked with 1% BSA in PBS containing 0.05% Tween 20 (PBS/T) and
incubated at room temperature for 90 mins. Standards (Pharmingen) were prepared
and diluted two-fold in 1% BSA in PBS/T. Standards and samples were then added to
the wells and the plates were left to incubate at room temperature for 90 minutes.
Biotinylated anti-cytokine antibody dissolved in 1% BSA-PBS/T was then added into
the wells and was allowed to incubate for another 90 minutes at room temperature.
Streptavidin HRP (Chemicon) was diluted 1/ 1000 in 1% BSA-PBS/T and allowed to
incubate at room temperature for another 30 minutes. The plates were washed three
times with PBS/T after each step up until the addition of the substrate 3,3’,5,5’-
tetramethyl benzidine (TMB; Sigma) and hydrogen peroxide into the plate. Once
substrate and hydrogen peroxide were added, colour reaction was allowed to develop
at room temperature for 10 minutes. The reaction was stopped using 0.1M H2SO4 and
the plates were read at 450 nm using BIORAD Microplate Manager Reader. The
80
antibodies used were rat anti-mouse IL-4, IL-10 and IFN- which were all obtained
from Pharmingen.
3. 2. 8. RNA extraction and RT-PCR analyses of colonic lymphocytes
RNA was isolated from LPMCs suspensions from Section 3.2.5 or from tissue
samples using Tri Reagent (Sigma). Preparation and isolation of RNA from these
samples were performed as indicated in the technical bulletin of Tri Reagent provided
by Sigma. Briefly, lamina propria cells were isolated by centrifugation and lysed with
1ml Tri Reagent by repeated pipetting. In addition, colon sections were opened
longitudinally and cleared of contents and debris with Ca2+ and Mg2+ -free HBSS with
10 mM Hepes. The colon was then cut into 0.5 cm pieces and 50-100 mg tissue pieces
were transferred into 5 ml flat-bottomed tubes (Sarsdet) and homogenized with 1 ml
Tri Reagent. After lysis of the samples, the lysate was centrifuged at 4ºC and the
supernatant was collected into a fresh tube. The supernatant was further treated with
0.2 ml chloroform and centrifuged to collect the aqueous phase containing RNA. The
RNA was then precipitated with isopropanol, washed with 70% ethanol, rinsed with
absolute ethanol and air-dried in a Biosafety Hood. The RNA was resuspended in
diethyl-pyrocarbonate (DEPC)-treated water and kept at -70ºC until assayed by RT-
PCR.
Isolated RNA was transcribed to cDNA using MMLV transcriptase as described in
the Sigma protocol. PCR was performed on 2 µl cDNA from each RT-PCR cytokine
reaction. Cytokine primer sets were obtained from GeneWorks (Melbourne,
Australia). -actin primers were used as controls. Primers specific for murine IL-4
(352 bp), IL-10 (298 bp) and IFN- (288 bp) were used. The primer sets were as
follows:
IFN-
IFN- sense 5’ ATC TGG AGG AAC TGG CAA AAG GAC G 3’
IFN- antisense 3’CCT TAG GCT AGA TTC TGG TGA CAG C 3’
IL-4
IL-4 sense 5’ACC TTG CTG TCA CCC TGT TCT GC 3’
IL-4 antisense 5’GTT GTG AGC GTG GAC TCA TTC ACG 3’
81
IL-10
IL-10 sense 5’TGC AGG ACT TTA AGG GGT TAC TTG GGT T 3’
IL-10 antisense 5’GCT TCT ATG CAG TTG ATG AAG ATG TCA 3’
-actin
-actin sense 5’TGG AAT CCT GTG GCA TCC ATG AAA C 3’
-actin antisense 5’TAA AAC GCA GCT CAG TAA CAG TCC G 3’
The cDNA was amplified in a 50 µl PCR reaction mixture in a thermocycler with the
following conditions: 94ºC for 5 mins; 27 cycles of 94ºC for 1 min, 60ºC for 45 secs
and 72ºC for 2 mins; and a final extension of 72ºC for 7 mins. The PCR products were
electrophoresed in 1.5% agarose gel and stained with 1 µg ml-1 ethidium bromide.
Band sizes of PCR products were compared to a 1 kbp marker obtained from
Promega. Image analysis was performed on gels using BIORAD GelDoc to visualise
the intensity of each cytokine and -actin band.
3. 2. 9. Short chain fatty acid quantification
The concentrations of acetic acid, propionic acid and butyric acid were quantified
from colonic contents as previously described and using ethylbutyric acid (Sigma) as
the internal standard150. Briefly, colon pellets were diluted in Wilkins Charlgren broth
(Oxoid) (1:10), homogenized and frozen at -20ºC until they were used for analysis.
After thawing, aliquots of 100 µl from each sample were placed into clean, dry
microfuge tubes (Eppendorf) and 20 µl of 10 M sulphuric acid and 0.040 g of sodium
chloride were added. The mixture was extracted with 100 µl of diethyl ether,
centrifuged and the ether layer collected. The samples were allowed to settle at room
temperature for 10 mins and 2 µl was injected into the gas chromatograph (Perkin
Elmer).
3. 2. 10. Enumeration of Colonic Microorganisms Using Selective Media
Colonic pelleted contents were collected and serially diluted in ten fold steps in half
strength Wilkins Charlgen broth (WCB; Sigma).Details of how the half strength WCB
is prepared were presented in Chapter 2, Section 2.2.6. An aliquot was kept from the
first dilution to be used later for molecular analyses. Each dilution was drop plated on
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Nutrient Agar (NA; Sigma), MacConkey Agar CM7 (MAC; Sigma), Reinforced
Clostridial Agar (RCA; Sigma), Wilkins Charlgren agar (Sigma) + 0.05% Horse
blood (Sigma; WCA), Rogosa Agar (ROG; Sigma) and Raffinose Bifidobacteria
Medium (RB)194 to enumerate total aerobes, total enterics, spore-formers, Gram
negative anaerobes, lactobacilli and bifidobacteria, respectively. Colonies that formed
were reported as colony forming units (CFU) per gram of contents.
3. 2. 11. Extraction of Nucleic Acid from Colonic Pellet
The aliquots from the first dilution prepared from Section 3.2.10 were centrifuged at
12,000 x g for 3 mins. The resultant pellets were then placed in microfuge tubes
containing approximately 0.3 g zirconia silica beads 0.1 mm (Biospec, USA). Three
hundred microlitres (300 µl) of 2X lysis buffer pH 8.0 (200 mM Tris, 50mM EDTA, 2
mM sodium citrate, 10 mM CaCl2 and 200 mM NaCl) and 30 µl lysozyme (100
mg ml-1) (Sigma) were then added to each tube. The contents of the microfuge tube
were mixed by gentle inversion and then incubated for 40 mins at 37ºC. Subsequently,
10 µl of 20% SDS (Sigma) and 60 µl proteinase K (Sigma) were added to initiate lysis
by incubation at 50ºC for 30 mins. Cetyl Trimethyl Ammonium Bromide (CTAB) (80
µl of a 10% CTAB solution in 0.7 M NaCl) and 100 µl of 5M NaCl were then added
and the mixture incubated at 65ºC for 10 mins. To complete lysis and remove excess
protein, 200 µl of 10% SDS and 400 µl phenol: choloroform: isoamyl alcohol
(24:24:1) (Sigma) were added. The tube was then shaken for 30 secs at the maximum
setting using a bead beater (FastPrep FT120-BIO101 SAVANT). The sample was the
centrifuged for 10 mins at 12,000 x g and the aqueous layer was transferred to a
sterile microfuge tube. Nucleic acids were extracted twice with 400 µl of phenol:
choloroform: isoamyl alcohol (25:24:1) and subjected to ethanol precipitation by
adding 1/10 volume of 3 M sodium acetate pH 5.0 and two volumes of absolute
ethanol or one volume of isopropanol overnight at -20ºC (or -70ºC for an hour). The
DNA pellets were obtained by centrifugation for 15 mins at 12, 000 x g, washed with
70% ethanol and then air dried. The extracted DNA was dissolved in sterile distilled
water and the concentration was calculated from the absorbance measurements of the
solution at 260 and 320 nm taken using a spectrophotometer (Beckman, USA).
83
3. 2. 12. PCR amplification of colonic pellet DNA
PCR primers 341-GC (GC clamp CCT ACG GGA GGC AGC AG) and 534r (ATT
ACC GCG GCT GCT GG) were used to amplify the V3 region of eubacterial 16S
rDNA for DGGE analysis. A 40 nucleotide GC clamp (CGC CCG CCG CGC GCG
GCG GGC GGG GCG GGG GCC CGG GGG G) was incorporated onto the 5’ end of
the 341f primer to ensure the DNA remained partially double-stranded during DGGE.
PCR reactions were performed in a total volume of 50 µl containing 10X PCR buffer,
2 mM deoxyribonucleotides mix, 25 mM Magnesium chloride and 1 U Taq
polymerase F1 DNA polymerase (all reagents from Biotech International Limited,
Australia) using Hybaid PCR express thermal cycler. The PCR conditions applied
were 94ºC for 5 mins for initial denaturation , followed by 27 cycles of (94ºC x 5
mins) + (62ºC x 30 s) + (72ºC x 1 min) and final extension of 72ºC for 7 mins. The
PCR products were detected on 2% agarose gel in 1X TAE buffer and
electrophoresed at 75 V. The gel was stained with 1 µg/ ml ethidium bromide and
visualized under UV transillumination using BIORAD Gel Doc Imaging System.
3. 2. 13. Denaturing gradient gel electrophoresis (DGGE) analysis
DGGE was performed with the Dcode system (BIORAD) using the protocol as
described by Chi (2000). A total volume of 42 µl of faecal DNA PCR products and 10
µl of 6X dye were loaded into the gel with 20%-60% gradient (Experiment 1) or 30%-
70% gradient (Experiment 2). The PCR products were separated in the above
mentioned linear gradient at 200 V and 60ºC for 5 hrs. The gel was stained with 1
µg.ml-1 ethidium bromide and visualized under UV transillumination using BIORAD
Gel Doc Imaging System.
3. 2. 14. DNA sequencing analysis
Bands of interest were excised and recovered using the ‘crush and soak’ technique
described by Sambrook (1989) for sequencing195. DNA sequencing reactions were
performed using an ABI PrismTM Dye terminator Cycle Sequencing Ready reaction
Kit (Big Dye) with Amplitaq DNA polymerase (Perkin Elmer Germany) using 5 µl
84
DNA sample, 2 µl 341 f primer, 6 µl BigDye and 7 µl sterile distilled water. A
standard sequencing reaction of 1 min of initial denaturation; followed by 25 cycles of
96ºC for x 10 s, 50ºC x for 5 s and 60ºC for 4 mins was performed. Sequences were
determined using an Applied Biosystems 377 automated DNA sequence using
synthetic oligonucleotides (Applied Biosystems, Foster City CA). Sequences were
assembled using Auto Assembler package (Applied Biosystems). DNA homology
searches were performed online using the BLAST server maintained at the National
Centre for Biotechnology Information (NCBI), Bethesda MD, USA.
3. 2. 15. Statistical analyses
Comparisons were made using t-test for variables with a normal distribution and
otherwise using Mann-Whitney or Wilcoxon’s test. A one-way repeated measures
ANOVA was used to test differences across time. Chi-square was used for nominal
data. The results were then corrected for multiple comparison using the Tukey,
Bonferonni and Sidak tests. A p-value of p<0.05 was considered statistically
significant. Statistical analysis was performed using the SPSS 11.5 for Windows
program.
85
3. 3. Results
3. 3. 1. Assessment of disease activity of colitic mice fed with 30% unmodified
high amylose maize resistant starch diet
Mice were fed with 30% unmodified high amylose maize resistant starch diet for 2
weeks and were maintained on this diet until the end of the experiment. After 2 weeks
of consuming the resistant starch diet, TNBS was intrarectally administered to the
animals in order to generate colitis and the clinical outcomes of colitis measured over
10 days.
Animals with colitis lost weight throughout the experimental period while the group
that received intrarectal 45% ethanol transiently lost weight on the first day but
recovered quickly (Figure 3.2). A one-way repeated measures ANOVA was
conducted to compare the weight profiles of colitic animals fed the high amylose
maize diet at 1, 4, 7 and 10 days after the induction of colitis. Administration of the
30% unmodified high amylose maize resistant starch diet did not prevent weight loss
in mice dosed with TNBS. Colitic control mice significantly (p<0.01) continued to
lose more weight compared to healthy controls and ethanol controls across the
observation period. Body weight loss was greater in colitic animals given the 30%
unmodified resistant starch compared to the colitic group that received the starch free
diet (p<0.01) Healthy controls did not experience any weight loss during the
observation period. No groups experienced weight loss prior to the induction of
colitis. There was minimal increase in the weight of animals fed the resistant starch at
the time TNBS was administered but this was not different from the weights of
animals that received the non-starch diet.
The survival rate was monitored to support the weight profile results since the
reported body weight data can only include animals that survived the wasting effects
of TNBS colitis. It was also important to assess the effect of dietary supplementation
of resistant starch on the survival of colitic animals. As shown in Figure 3.3, 40% of
the mice that received the unmodified high amylose maize resistant starch diet died
within 10 days after TNBS administration, whereas, a significantly higher survival
rate (p<0.05) was observed in the group that received the resistant starch free diet.
86
02468
101214161820
0 1 4 7 10
Bod
y W
eigh
t Los
s (%
)
Healthy Ethanol Colitic + Non-Starch Colitic + 30% Resistant Starch
Figure 3.2. Weight loss of colitic mice fed 30% unmodified high amylose maize resistant starch diet or starch free diet. Observations presented 0, 1, 4, 7 and 10 days after induction of colitis with TNBS administered rectally.
Mice (n=10 per group and sampling time) were fed the high amylose maize resistant starch for 24 days and colitis was induced using 2.5 mg TNBS in 45% ethanol on Day 14. Body weight loss over the 10 days was significantly greater in colitis control mice and colitic mice fed the 30% resistant starch diet compared to ethanol control and healthy control mice (p<0.01).
87
0
20
40
60
80
100
120
0 1 4 7 10Time after induction of colitis (Days)
Surv
ival
(%)
Healthy EthanolColitic + Non-Starch Colitic + 30% Resistant Starch
*
Figure 3.3. Survival of colitic mice fed with 30% unmodified high amylose maize resistant starch diet over the 10 days after induction of colitis with TNBS.
Mice (n=10 per group and sampling time) were fed the high amylose maize resistant starch for the duration of the study and colitis was induced using 2.5 mg TNBS in 45% ethanol on Day 14 and animals were monitored for the remaining 10 days. Significantly * p<0.05 higher survival rate was observed in colitic group that received starch free diet.
88
3. 3. 2. Evaluation of colon pathology of colitic mice on 30% unmodified high
amylose maize resistant starch diet
The extent of mucosal tissue damage initiated by TNBS colitis and effect of 30%
unmodified high amylose maize resistant starch diet on the associated tissue injury in
this model of colitis were assessed microscopically using the histological scoring
system of Ameho and colleagues. The histological scores are presented in Figure 3.4
and representative colon sections from the various groups are shown in Figure 3.5a-d.
Pathology of the colon of healthy controls was normal (Figure 3.4 and 3.5a). Animals
in the ethanol control group had minimal colon damage with a total average
histological score of 0.9 across all time points, which were significantly different
(p<0.05) to the healthy control group (Figure 3.4). Ethanol was used to break the
barrier function of the epithelium to facilitate entry of TNBS into the mucosa. Mild
ulceration was observed on the colon section of the ethanol group (Figure 3.5b). Intra-
rectal infusion of TNBS resulted in a more extensive ulceration of the colon tissue
compared to ethanol controls and mild inflammatory cell infiltration (Figure 3.5c).
The histological score increased significantly on days 1, 4, 7 and 10 after colitis
induction compared to the healthy control group (p<0.01) and the ethanol control
group (p<0.05).
Colon tissues from animals that were administered TNBS and received the 30%
unmodified high amylose maize resistant starch exhibited more chronic inflammation
than the other experimental groups (Figure 3.5d). Tissue damage was characterized by
an extensive distortion of the crypts, neutrophils had infiltrated through the layers of
the tissue sample and a mononuclear cell infiltrate was present. Injury of the colon
tissue of resistant starch fed colitic mice worsened significantly 1, 4, 7 and 10
(p<0.01) days after colitis induction compared to untreated and ethanol controls; and
the score on days 7 and 10 was significantly different (p<0.05) to that seen in the
colitis control group.
89
0
2
4
6
1 4 7 10
Time after induction of colitis (Days)
Hist
olog
ical
Sco
re
Healthy Ethanol Colitic + Non-Starch Colitic + 30% Resistant Starch
¥, ¶ ¥, ¶ ¥, ¶,§¥, ¶,§
Figure 3.4. Colon histological scores of colitic mice on 30% unmodified high amylose maize resistant starch diet and starch free diet.
Mucosal tissue damage was quantified by the scoring system of Ameho and colleagues. Histological scores, ranked 0-6 where 6 = worst inflammation characterized by severe infiltration of neutrophils and mononuclear cells and necrosis extends through the muscularis mucosa; 0 = histology similar to normal colon tissue, present the average score of 10 mice per group per sampling point ± SD. Tissue injury in colitic mice fed the 30% unmodified high amylose maize resistant starch was significantly worse than healthy¥ and ethanol¶ controls (p<0.01) and the colitic group fed the starch free diet§ (p<0.05).
90
Figure 3.5. Representative colon sections from healthy control mice (a), ethanol control mice (b), colitic mice fed the resistant starch free diet (c) and the 30% unmodified high amylose maize resistant starch diet (d) 7 days after colitis induction.
Colon tissue samples were stained with hematoxylin and eosin (H&E) and examined at a magnification of 40X.
(a) (b)
(d)(c)
91
3. 3. 3. Effect of feeding 30% unmodified high amylose maize resistant starch
diet on cytokine gene expression in the lamina propria mononuclear cells
(LPMC) of colitic mice
Based on the disease activity profiles and colon injury scores, supplementation with
30% unmodified high amylose maize resistant starch seems to promote colitis instead
of abrogating the condition. The next step taken was to investigate the mechanism that
mediated colon inflammation in colitic mice when given the high concentration of
unmodified resistant starch diet by assessing the expression and production of pro-
inflammatory, anti-inflammatory and regulatory cytokines in the inflamed colon.
Increased IFN- RNA gene expression was observed in both colitic groups fed the
starch free and 30% unmodified resistant starch diet (Figure 3.6, Lanes 3 and 4)
compared to the untreated and ethanol controls (Figure 3.6, Lanes 1 and 2). Signals
for IL-4 expression were detected in all groups. Administration of 30% unmodified
high amylose maize resistant starch resulted in the suppression of IL-10 mRNA gene
expression in colitic mice (Figure 3.6, Lane 4).
3. 3. 4. Cytokine production from cultured lamina propria mononuclear cells
(LPMC) of colitic mice fed with 30% unmodified high amylose maize resistant
starch diet
Since IFN- , IL-4 and IL-10 mRNA expression in the colon of colitic mice was not
measured quantitatively in the above rt-PCR analyses, the protein synthesis of these
cytokines was assessed in a standard sandwich ELISA using murine anti-IFN- , IL-4
and IL-10 antibodies.
It has been described that the level of IFN- is elevated in the TNBS model of colitis2,
167 and that reducing the amount of IFN- brings about the resolution of colon
inflammation. In this study, significantly marked elevations of IFN- (p<0.01) were
detected in the LPMCs of TNBS-induced mice 7 days after colitis was induced and
92
Figure 3.6. Cytokine gene expression in lamina propria mononuclear cells (LPMCs) of colitic mice fed with 30% unmodified high amylose maize resistant starch diet.
RNA was extracted from 106 LPMCs on the 7th day of the colitic period and analyzed by RT-PCR using specific primers for each cytokine. Equivalent loading of each sample was determined by -actin message shown above. Lane 1: healthy control fed with normal murine feed, Lane 2: ethanol control fed with normal murine feed, Lane 3: colitic control fed with starch free diet and Lane 4: colitic animals fed with the 30% unmodified high amylose maize resistant starch diet.
1 2 3 4
- actin
IFN-
IL-4
IL-10
93
0
500
1000
1500
2000
2500
Healthy Ethanol Colitic + Non-Starch Colitic + 30% ResistantStarch
Con
cent
ratio
n (p
g/m
l) of
Cyt
okin
e
IFN- IL-4 IL-10
§
Figure 3.7. T cell cytokine production in the LPMCs from colitic mice fed with 30% unmodified high amylose maize resistant starch diet.
LPMCs were isolated from the colon 7 days after colitis was induced, cultured for 72 hrs and after which the supernatants were collected and assayed for IFN- , IL-4 and IL-10 by ELISA. Animals were divided into the following groups: healthy control and ethanol control groups received the normal murine feed diet; colitic animals received either the starch free diet or the 30% unmodified high amylose maize resistant starch diet. Results are expressed as mean concentrations (pg.ml-1) of each cytokine ± SD. One-way ANOVA was used to detect differences.
94
significantly increased two-fold (p<0.01) when mice were given the 30% unmodified
resistant starch diet (Figure 3.7). Levels of IFN- in starch fed colitic mice differed
significantly (p<0.01) from the untreated, ethanol and colitic control groups.
No statistical difference was detected in the production of the anti-inflammatory
cytokine, IL-4, in the colon of animals from the untreated, ethanol and colitic control
groups (Figure 3.7). However, colonic levels of IL-4 were significantly decreased
(p<0.01) in colitic mice that received the 30% unmodified high amylose maize
resistant starch diet. On the other hand, the immunosuppressive cytokine, IL-10 was
significantly upregulated in the colitic control animals that received the non-starch
diet (p<0.01) while its production was significantly reduced when colitic mice had the
30% unmodified high amylose maize resistant starch diet (p<0.01) compared to the
healthy control, ethanol control and colitic mice that received the non-starch diet.
3. 3. 5. Changes in the gut microbial populations of colitic mice on 30%
unmodified high amylose maize resistant starch diet
It has been shown previously that stress and disease can alter the composition or
metabolic activity of the gut microbial community. Several studies have also reported
that the intestinal microorganisms are involved in the pathogenesis of bowel
inflammation. However, whether the gut microbial flora has a primary role in the
initiation of inflammation in IBD or responsible for the perpetuation of the disease
remains to be elucidated. It was the objective of this experiment to correlate bacterial
load with the degree of inflammation seen in TNBS induced mice and to assess
whether a particular bacterial group was associated with TNBS-induced colitis
Incidence, viable counts and community shifts of certain bacterial types in the colon
contents of colitic mice on the resistant starch diet were determined using selective
culture media and molecular analysis with denaturing gradient gel electrophoresis
(DGGE). Bacterial profiles of colon contents were analysed on the 7th day of the
colitic period which is also the same time when the most severe physical damage
(Figure 3.4 and 3.5) and inflammatory responses (Figure 3.6 and 3.7) were observed
in the colon.
95
Development of colitis led to a significant change of the colonic flora. The total
number of enteric bacteria per gram significantly increased (p<0.01) by 1 log CFU in
colitic mice compared to the levels in untreated and ethanol controls (Figure 3.8a).
Feeding of 30% unmodified high amylose maize resistant starch to colitic mice
resulted in a minimal elevation of enterics numbers compared to healthy controls.
Enteric counts were significantly lower in the colitis animals that received the 30%
unmodified high amylose maize resistant starch than those that were fed the starch
free diet a log CFU (p<0.01).
No dramatic change in the viable counts of Gram negative anaerobes was detected in
the colonic contents of untreated, ethanol and colitic control mice (Figure 3.8b).
Counts of Gram negative anaerobes for these groups were maintained at about a level
of 108 CFU per gram while colitic mice that were administered the 30% high maize
diet had a lower level of Gram negative anaerobes. This, however, was not
statistically different from the other groups.
There was a reduction in the number of lactobacilli detected in colitic mice compared
to all the other groups (p<0.05, except ethanol group) (Figure 3.8c). Mice from the
ethanol group harboured lower lactobacillus counts in their colon contents compared
to untreated controls but the difference was not significant. Levels of lactobacilli in
colitic mice given the 30% unmodified resistant starch were significantly greater
(p<0.01) than the colitic group and the counts remained at levels similar to that in the
untreated control mice.
Bifidobacterium counts were also assessed using the selective RB medium.
Bifidobacteria colonies were only observed in the faecal contents of mice given the
30% unmodified high amylose maize resistant starch diet but not those animals on the
starch free or basal diet (Figure 3.8d). Only one bifidobacteria colony type was
detected on the RB medium. This colony was isolated and further confirmed by
microscopic examination for Bifidobacterium morphology, and by assaying for the
presence of the enzyme fructose-6-phosphate phosphokelotase and by PCR using
Bifidobacterium species specific primers (Table 3.2).
96
DNA was also extracted from pooled colonic pellets from each group and then
subjected to PCR using primers that amplify the variable V3 region of the eubacterial
16S rDNA for DGGE analysis. Denaturing gradient profiles of each group (Figure
3.9) indicate that the gastrointestinal microbial content changed during the
progression of inflammation. Distinct banding patterns were observed 1, 4, 7 and 10
days after colitis was induced using TNBS. Several bands appeared while others
fluctuated during the observation period. There were discernible differences in the
colonization profiles between the groups across the observation period. As seen in
Figure 3.9, there were 8 bands detected that were distributed in all groups. Band 1 was
only detected in the colitis mice given the starch diet but was not clearly discernible in
the other groups. Band 2 which was uniquely found in the ethanol control group 1 day
after colitis was induced was not detectable in the other sampling timepoints. Band 3
was common in all groups while Band 4 was found in the healthy control and ethanol
control groups only. Band 5 was found in all groups but exhibited faint banding
intensity in the group administered 30% unmodified high amylose maize resistant
starch. Bands 7 and 8 were largely detected in all groups during the observation
period.
In order to determine the changes that occurred within the groups in the colitic gut,
dominant bands from DGGE profiles were sequenced and compared to GenBank
database entries. DGGE analysis was repeated on faecal samples which were
collected 1 and 7 days after colitis induction using 30-60% gradient. These faecal
samples were selected for sequencing analysis as it is on these days that the animals
experienced the effects of intrarectal introduction of an external agent (1 day post-
induction) and peak inflammation in the TNBS model happens 7 days after the hapten
agent was administered. These most intense bands from each group are indicated on
Figure 3.10. These bands were sequenced and results compared with those in the
GenBank database. The results are presented in Table 3.3.
97
Figure 3.8. Viable counts of different bacterial types from colon contents of colitic mice fed with 30% unmodified high amylose maize resistant starch diet or starch free diet detected 7 days after colitis was induced.
Data are expressed as Log colony forming units (cfu) per gram colonic contents of 10 mice (mean ± SD). ¥ = significantly different from healthy control, ¶ = significantly different from ethanol control and § = significantly different from colitic mice + non-starch group.
a) Total Enterics
4
6
8
10
Healthy Ethanol Colitic +Non-
Starch
Colitic + 30%
ResistantStarch
Log
CFU
.g -1
of c
olon
con
tent
s¥ ¥
§
b) Gram negative Anaerobes
4
6
8
10
Healthy Ethanol Colitic +Non-Starch
Colitic + 30%
ResistantStarch
Log
CFU
.g-1 o
f co
lon
cont
ents
§
c) Lactobacilli
4
6
8
10
Healthy Ethanol Colitic +Non-
Starch
Colitic + 30%
ResistantStarch
Log
cfu
.g-1 o
f col
on c
onte
nts
¥, ¶
d) Bifidobacteria
4
6
8
10
Healthy Ethanol Colitic +Non-
Starch
Colitic + 30%
ResistantStarch
Log
cfu
.g-1 o
f col
on c
onte
nts
98
Figure 3.9. Bacterial community profiles of colonic contents from colitic mice on 30% unmodified high amylose maize resistant starch diet as determined by PCR-DGGE. Colon contents were collected 1, 4, 7 and 10 days after colitis induction with TNBS.
Lanes 1, 5, 9 and 13 corresponds to healthy controls that received mouse feed diet; Lanes 2, 6, 10 and 14 corresponds to ethanol control group that received mouse feed diet;Lanes 3, 7, 11 and 15 corresponds to colitic mice fed with starch free diet and Lanes 4, 8, 12 and 16 corresponds to colitic mice fed with 30% unmodified high amylose maize resistant starch diet
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Colitis Day 10 Colitis Day 7 Colitis Day 4 Colitis Day 1
2345
1
6
7
8
99
Table 3.2. Confirmation of the presence of bifidobacteria in the colonic contents of colitic mice on 30% unmodified high amylose maize resistant starch diet.
Bacteria Colony
Morphology
Fructose-6-
Fhosphate
Phosphokelotase
PCR
Blank - -
Negative Control
Salmonella typhimurium Gm (-), rod - -
Positive Control
Bifidobacterium animalis Gm (+), fusiform + +
Bifidobacterium lactis Gm (+), fusiform + +
Test Isolate
Group D-Colony Type 1 Gm (+), fusiform + +
100
Figure 3.10. Identified DGGE bands of DNA from colonic contents of healthy control mice fed with normal mouse diet (Lanes A1, A7), ethanol control mice fed with normal mouse diet (Lanes B1, B7), colitic mice fed with resistant starch free diet (Lanes C1, C7) and colitic mice fed the 30% unmodified high amylose maize resistant starch diet (Lanes D1, D7). Colonic contents were collected from 1st (Lanes A1, B1, C1, D1) and 7th day (Lanes A7, B7, C7, D7) of colitic period.
1
2
34
5
6
7
8
910
A1 B1 C1 D1 A7 B7 C7 D7
101
Table 3.3. Identification of isolates from DGGE profile of colitic mice on 30% unmodified high amylose resistant starch diet.
Band Species Identification Percentage
Similarity
Observed Change¶
1 Nitrogen utilizing uncultured bacterium 89 A1
2 Staphylococcus sp 92 A1
3 Lactobacillus grasserri 95 B7
4 Uncultured GI bacterium 89 A1,C1,A7,B7,D7
5 Uncultured GI bacterium 92 A1,C1,B7,C7,D7
6 Uncultured beta proteobacterium 90 B7
7 Uncultured bacterium that regulates fat
storage 94 D7
8 Bradyrhizobium sp 90 B1
9 Uncultured bacteria in the intestinal tract
of soil feeding bacteria 96 A1
10 Uncultured Bacteriodetes intestinal
bacterium 100 C1
¶ = indicates the lanes in which the band was detected in Figure 3.3.9. The letters A, B, C, and D refers to healthy control, ethanol control and colitis and starch diet, respectively. The number after the letter refers to 1 and 7 days after induction of colitis.
102
There were some dominant and common bands in all the groups, one of which
corresponded to a Lactobacillus species. Most of the bands that were sequenced,
however, displayed closest homology to uncultured gastrointestinal bacterium.
Alterations were consistent in all groups and bands emerged as well as disappeared
during the colitic period (Table 3.3 and Figure 3.10).
103
3. 3. 6. Effects of concentration of unmodified high amylose maize resistant
starch in the amelioration of colon inflammation
As shown in Sections 3.3.1 to 3.3.6 (Figures 3.2-3.7), supplementation of 30%
unmodified high amylose maize resistant starch diet did not improve the clinical
features nor reduce inflammation in TNBS colitis. A lower concentration of the
unmodified high amylose maize rich diet was then given to colitic mice to test
whether a concentration of 5% was more effective. A lower concentration was given
because it has previously been shown that high concentrations of indigestible
carbohydrates cause bulking effect that can cause discomfort and gas production. In
addition, the physical structure of the indigestible carbohydrate could be too coarse
thereby create friction as it moves along the gut thus further damaging the inflamed
colon.
Mice subjected to 5% unmodified high amylose maize resistant starch diet showed an
overall lower impact of the colonic damage induced by the TNBS compared with the
diet containing 30% of the unmodified resistant starch. Colitic mice that received the
5% unmodified diet had higher body weight as compared to the 30% treated group
and TNBS control group receiving the starch free diet (Figure 3.11). The weight
kinetics in the 5% high amylose maize group were similar to those of the healthy
control group while those that received the 30% unmodified resistant starch diet
continued to lose weight throughout the colitic period (p<0.05).
The weight profile was consistent with the histological scores of 0%, 30 and 5%
resistant starch treated colitic groups (Figure 3.12). The 5% unmodified resistant
starch diet resulted in a significantly lower damage score of the colon after TNBS
administration (p<0.05) compared to the score given for colitic animals receiving the
higher concentration or the starch free diet. Colon inflammation in the group fed the
5% starch diet only extended to the mucosal layer and there was little infiltration of
inflammatory cells, ulceration and minimal goblet cell depletion. Some of the tissue
sections revealed recovery of the epithelium structure. These results are in contrast to
the microscopic examination of the colon tissues from colitic mice on the 30%
unmodified resistant starch diet. Colon tissues of colitic animals that received the 30%
resistant starch had very severe edema and infiltration of neutrophils in the lamina
104
propria and submucosa which led to a loss of crypt architecture. No difference was
seen between the damage score of the 30% resistant starch fed colitic group compared
to the colitic control fed the starch free diet. However, significantly higher
histological scores were detected 7 days after colitis was induced (p<0.05) as
evaluated against the score on day 1 post-induction of colitis.
The assessment of mRNA expression of cytokines from colon cells (Figure 3.13)
revealed an increased expression of IFN- in the colitic group fed with the 30%
unmodified high amylose maize resistant starch in comparison to the colitic mice that
received the lower concentration of starch (Figure 3.13a; Lanes 4 and 5). On the other
hand, feeding with 5% unmodified high amylose maize diet resulted in increased
expression of IL-4 and IL-10 and reduced expression of IFN- as evidenced by the
intensity of the bands compared to the groups obtained from mice given the 0% and
30% concentration of unmodified high amylose maize resistant starch. The increased
inflammatory respone seen with the 30% concentration was also reflected in the ratio
between IL-4 and IFN- when cytokine production was measured and the profile was
observed to be comparable to that of the colitic group (Figure 3.13b). IL-10
production was not measured.
The colitic mice consuming the 5% and 30% unmodified resistant starch diet had
different bacterial profiles (Figures 3.14 and 3.15). Enumerating bacterial groups in
the colonic contents showed that mice on 30% unmodified resistant starch had higher
levels of lactobacilli in their colon contents than their 5% counterpart but the
difference did not reach statistical significance. Levels of lactobacilli in the 30%
resistant starch fed group were greater than those found in the colitis control group
(Figure 3.14a). Feeding of unmodified resistant starch regardless of concentration
promoted bifidobacterial growth. Bifidobacteria were not detected in colitic animals
on the resistant starch free diet (Figure 3.14b). Similar levels of Gram negative
anaerobes were excreted in all groups (Figure 3.14c).
105
60
70
80
90
100
110
120
0 1 3 7 10
Time after induction of colitis (Days)
Bod
y W
eigh
t (%
)
Healthy Ethanol 0% RS + Colitic 30% + Colitic 5% RS + Colitic
§
Figure 3.11. Changes in weight (%) during colitic period of colitic BALB/c mice (mean ± SE of 10 mice per group) treated with different concentrations of unmodified high amylose maize starch diet.
Mice were treated with the unmodified resistant starch diet or non-starch diet for 2 weeks prior to induction of colitis with TNBS. Administration of the unmodified high amylose resistant starch was continued for 10 days after colitis was induced. Animals receiving the 30% significantly lost more weight (p<0.05) during the observation period compared to those which received the 5% concentration or the starch free diet.
106
0
1
2
3
4
5
1 7
Time after colitis induction (Days)
Hist
olog
ical
Sco
re
Healthy Ethanol Colitic + 0% RS Colitic + 30% RS Colitic + 5% RS
Figure 3.12. Colonic damage scores of the colon from mice induced with TNBS colitis and fed different concentrations of unmodified high amylose maize resistant stach.
Results are expressed as means of colon histological scores ± SD of 10 mice per group. Colitic mice fed the 5% unmodified resistant starch have significantly less damage (p<0.05) in their colon tissue compared to colitic mice that received 0% and 30% concentration of the unmodified high amylose maize resistant starch.
107
Figure 3.13a. Gene expression of pro-inflammatory and anti-inflammatory cytokines determined by RT-PCR in the colonic tissues of mice with or without treatment of low and high concentration of unmodified high amylose maize resistant starch.
Lane 1: untreated control, Lane 2: ethanol control, Lane 3: colitic animal + starch free diet, Lane 4: colitic animal + 30% starch diet and Lane 5: colitic animal + 5% unmodified high amylose maize resistant starch diet.
-actin
IFN-
IL-10
IL-4
1 2 3 4 5
-0.1
-0.05
0
0.05
0.1
0.15
0.2
0.25
Healthy Ethanol Colitic 30% RS 5% RS
Animal groups
Rat
io IL
-4:I
FN-y
Figure 3.13b. Mean ratio mucosal cytokine secretion of IL-4 and IFN-in the colons of colitic mice (n=10) fed the low and high concentration of unmodified high amylose maize resistant starch (mean ± SD).
108
0% Resistant Starch 30% Resistant Starch 5% Resistant Starch
Figure 3.14. Effect of different concentrations of unmodified high amylose maize starch on colonic content counts of different types of bacteria.
Data are expressed as Log CFU.gram-1 colonic contents from10 mice (mean ± SD). Significantly higher levels (p<0.05§) of lactobacilli were detected in 30% starch fed colitic mice compared to the starch free diet. Bifidobacteria levels were significantly higher (p<0.01 ) in the 30% and 5% concentration. Detection limit for bifidobacteria is 1x102. Spore-formers were significantly detected in lesser number of animals ( )that received the 5% compared to 0% and 30% concentration.
Number of animals
d) Endospores
4
6
8
10
Log
CFU
/g o
f fae
ces
0123456789
Num
ber of Anim
als
c) Gram Negative Anaerobes
4
6
8
10
Log
CFU
/g o
f fae
ces
a) Lactobacilli
4
6
8
10Lo
g C
FU p
er g
ram
of f
aece
s
§
b) Bifidobacteria
4
6
8
10
Log
CFU
per
gra
m o
f fae
ces
109
The levels of bacterial endospores in the colon contents, were also enumerated
(Figure 3.14d). Endospores were detected in all colitic groups, however, the highest
levels were observed from colitic animals fed the 30% unmodified high amylose
maize resistant starch. The spore-forming bacteria were detected in lower numbers in
the colonic pellets of colitic mice on 5% unmodified starch diet as compared to their
0% and 30% counterparts. The number of affected animals varied significantly
(p<0.01). Endospores were found to persist in 8 of 10 colitic animals fed the 0% and
30% unmodified starch diet. Endospores were only detected in one mouse in the
group given the 5% unmodified resistant starch diet.
Levels of total faecal enterics were similar in all groups (Figure 3.15a). Interestingly,
giving different concentrations of unmodified high amylose maize resistant starch
diets to colitic mice yielded different colony types of enterics using the selective
medium Mac Conkey agar CM7 (MAC) (Figure 3.15b). Three colony types were
observed: the red, creamy colonies (coliforms); white, creamy colonies (non-lactose
fermenters); and the red, pinpoint colonies (enterococci). Colitic animals that did not
receive any resistant starch supplementation harboured all the three enteric colony
types but possess more of the non-lactose fermenters (p<0.01). Enteric colonies from
the colitic group receiving the 30% resistant starch were characterized as non-lactose
fermenters and coliforms. Although the presence of creamy white colonies are higher
in this group, coliforms were detected in significantly higher counts (p<0.05) in the
30% unmodified resistant starch fed group compared to the other colitic groups fed
the resistant starch diet. The group that received the 5% unmodified high amylose
maize starch excreted significantly (p<0.05) more of the enterococci.
110
4
6
8
10
12
Log
CFU
/g o
f fae
ces
(a)
4
6
8
10
12
Log
CFU
/g o
f fae
ces
Non-coliforms Coliforms Enterococci(b)
0% 5% 30%
§
§
Figure 3.15. Total enteric counts in the colonic contents of colitic mice fed different concentrations of unmodified high amylose maize resistant starch (a) and the enteric colony types detected in the colonic contents after treatment with unmodified resistant starch given at different concentrations (b).
Data are expressed as Log CFU.gram-1 colonic contents of 10 mice (mean ± SD). Significantly more non-lactose fermenters (p<0.05), coliforms (p<0.01) and enterococci (p<0.01) were detected in the colonic contents of mice fed with 0%, 5% and 30% unmodified high amylose maize resistant starch diet, respectively.
0% Resistant Starch 30% Resistant Starch 5% Resistant Starch
30% 5%0%
111
3. 3. 7. Supplementation of different concentrations of unmodified and modified
high amylose maize resistant starch diet to colitic mice
It was hypothesized that giving the same variety of resistant starch would generate a
similar faecal profile regardless of the amount fed to colitic mice. However, feeding
low and high concentration of unmodified resistant starch created a different bacterial
profile of colonic microorganisms as shown in Figures 3.14 - 3.15. Administration of
30% unmodified high amylose maize resistant starch caused an increase in non-
lactose fermenting enteric bacteria and coliforms numbers, persistence of endospores
and a disappearance of enterococci in colitic mice. The 5% concentration of
unmodified starch, on the other hand, elevated enteroccocus numbers whilst
maintaining coliforms, non-coliforms and endospores at low levels. This indicates
that there was a dose dependent variation in the fermentation kinetics of unmodified
resistant starch in colon inflammation, as bacterial profile reflects the fermentation
capacity of the substrate.
A modified type of the high amylose maize resistant starch was incorporated in the
diet to investigate fermentation patterns of high amylose maize resistant starch in
colitic mice as measured by SCFA production. Mucosal production of inflammatory
and immunosuppressive cytokines was also evaluated to determine whether
restoration of the integrity of the mucosal immunity is dependent on the type of
indigestible carbohydrate.
All the animals that were induced with TNBS exhibited body weight loss 7 days post-
induction of colitis (Figure 3.16). Colitic mice that were on starch free diet lost more
weight (p<0.05) compared to healthy-control animals. A greater reduction in weight
was observed in colitis animals that received the starch free diet, however, the weight
profile was not significantly different from the weights of colitic mice given different
concentrations and types of resistant starch diet. However, there were more deaths
observed in the colitic control group and those given the higher concentration of both
varieties of high amylose maize resistant starch compared to the colitic animals that
were administered the lower concentrations of the starches.
112
Cytokine profiles revealed that the 30% unmodified high amylose maize resistant
starch diet produced a ratio of IL-4:IFN- lower than obtained in the healthy control
mice and all the colitic groups that received the different types and concentration of
high maize starch (Figure 3.17). This suggests that the 30% unmodified high amylose
maize starch is upregulating the production of inflammatory cytokine, or inhibiting
the expression of anti-inflammatory cytokines, in this model of colitis. The 5%
modified form of the high amylose maize resistant starch exhibited a cytokine profile
closer to healthy levels. Moreover, the ratio of IL-4:IFN- showed that 5% modified
high maize starch produces significantly higher levels of the anti-inflammatory
cytokine (p<0.05) than the 30% and 5% unmodified high amylose maize starch. No
difference, however, was seen with the mice receiving the 30% modified starch diet.
Colitic animals given the unmodified and modified high amylose maize starch diet
had a non-significant increase in the levels of SCFA compared to the colitic mice fed
the starch free diet (Figure 3.18). Healthy mice that received normal mouse feed had
comparable levels of SCFA to colitic mice that received the high amylose maize
starch diet. No difference was also detected in the amount of individual fatty acids
produced when colitic mice were given the unmodified and modified high amylose
maize starch diet (Table 3.4).
113
-20
246
810
121416
1820
Healthy Ethanol Colitic 30%Unmodified
Starch
5%Unmodified
Starch
30%ModifiedStarch
5%Modified
Starch
Body Weight Loss(%)
Number of Survivors(n)
Figure 3.16. Body weight loss and survival of TNBS-induced colitic mice at the day of sacrifice.
The unmodified and modified high amylose maize resistant starches were administered for 14 days prior to colitis induction with TNBS and was continued for another 7 days.
114
Figure 3.17. Mean ratio mucosal cytokine secretion of IL-4 and IFN- in the colons of colitic mice (n=10) fed the of unmodified (UM) and modified (MD) high amylose maize resistant starch (mean ± SD). * significantly different from 30% and 5% UM high amylose maize starch diet at p<0.05.
5% MD30% MD5% UM30% UMColiticHealthy
95%
CI
Rat
io IL
-4:IF
N-g
amm
a
.2
.1
0.0
-.1
*
115
99.29.49.69.810
10.210.410.610.8
0% Starch 30% UM 5% UM 30% MD 5% MD HealthyNormal Diet
Tot
al S
CFA
Con
cent
ratio
n (u
m.g-1
dig
esta
wet
wei
ght)
Figure 3.18. Total short chain fatty acid (SCFA) concentration in the colon contents of colitic mice fed the 5% and 30% concentration of the unmodified (UM) and modified (MD) high amylose maize resistant starch diet and healthy mice fed the normal mouse diet.
Colonic contents were collected 7 days after induction of colitis. Values are expressed as mean±SD (n=8 per group).
116
Table 3.4. Acetate, butyrate and propionate levels 7 days after colitis induction in colitic mice fed two varieties of high amylose maize resistant starch and healthy mice fed the normal mouse diet. Concentration of each fatty acid is expressed mean (µm.g-1
of colon contents)±SD.
Healthy Colitic Non-RS Unmodified Modified Short Chain
Fatty Acid Normal Mouse Food 0% 30% 5% 30% 5%
Acetate (C2) 2.78±0.04 3.45±0.00 3.65±0.19 3.59±0.24 3.43±0.02 3.53±0.04
Butyrate (C4) 4.18±0.41 3.54±0.00 3.57±0.04 3.54±0.00 3.62±0.04 3.54±0.00
Propionate (C3) 3.17±0.07 3.01±0.00 3.01±0.07 3.01±0.00 3.01±0.00 3.01±0.00
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3. 4. Discussion
This study evaluated the effects of high amylose resistant starch on the symptoms of
experimental colitis induced using TNBS. Resolution of colon inflammation by high
amylose maize resistant starch was affected by the variety and the concentration
supplemented in the diet of colitic mice. This is supported by the following
observations:
5% concentration but not 30% of high amylose maize resistant starch included
in the diet afforded protection against TNBS colitis as indicated by lower
weight loss and histological scores of animals.
Feeding of low and high concentration of unmodified resistant starch
generated a different microbial profile in experimental colon inflammation
induced by TNBS.
Expression and production of pro- and anti-inflammatory was increased in
colitic animals administered 5% high amylose maize resistant starch.
Relative levels of individual SCFAs were both comparable with both
concentrations of resistant starch but some animals were lost in the
experiment, thus, it is difficult to draw conclusions.
3. 4. 1. Recovery from colon inflammation observed in low concentration high
amylose maize resistant starch
Colitic mice received a diet containing either 5% or 30% high amylose maize resistant
starch. Regardless of the form of the high amylose maize resistant starch, whether it is
unmodified or the modified type, it was shown that 5% concentration of high amylose
maize resistant starch delayed the progression of colitis but not the 30%
concentration, as evident by the lower weight loss (Figures 3.2, 3.11 and 3.16) higher
number of surviving animals (Figure 3.3), and less tissue damage (Figures 3.4 and
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3.12) observed in the colons of the mice. This supports the findings from two separate
experiments of Moreau and colleagues (2003) where they used 6% of retrograded
high amylose maize resistant starch in a dextran sulfate model of colitis196. They
showed that body weight was stable and that gross and microscopic injury scores
were lower in colitic animals fed the retrograded resistant starch. On the other hand, a
dietary supply of 15% granular pea starch was also effective in improving the clinical
features of TNBS colitis in rats158 while, dietary supplementation of 12% and 45%
galacto-oligosaccharides did not induce recovery from TNBS-induced damage. Other
indigestible carbohydrates such as germinated barley when included in the diet at a
concentration of 10%, were able to accelerate recovery from weight loss in a
spontaneous model of colitis157. Reduction of the clinical manifestations of colon
inflammation, however, was observed with 5% concentration of high amylose maize
starch and this was consistent with the outcome from studies giving 5% Plantago
ovata seeds to transgenic197 and chemically-induced198 colitic animals.
This difference in protection between the 5% and 30% concentrations of high amylose
maize resistant starch in TNBS model of colitis indicates that improvement in body
weight and colon architecture after resistant starch treatment is not due to the “shield-
like” effect of the starch from the damage of TNBS. If high amylose maize resistant
starch does block the TNBS from coming into contact with the colon epithelium, then,
the higher concentration diet would show colon pathology similar to untreated normal
colons and not a complete destruction of the mucosa. It is possible that the beneficial
effects of the 5% starch diet were lost with the 30% starch addition because not all of
the 30% could be degraded by the microbes and the residual undigested granulated
starch contributed to the detrimental effects.
Moreover, although similar individual SCFA concentration were observed on a per
gram digesta, it is recognized that resistant starch is a bulking agent. Thus, one can
postulate that higher total SCFA is anticipated. More bacterial growth may be
promoted with the higher RS concentration as more substrate becomes available. This
could be detrimental to the integrity of the mucosa as TNBS reduces surface
hydrophobicity of the colonic mucosa285 and thus becoming more permeable to
environmental factors. These bacteria and their products could possibly traverse and
initiate and perpetuate an aggressive immune response.
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3. 4. 2. Microbial profile of colonic contents of BALB/c mice induced with TNBS
colitis
It was speculated that giving the same variety of resistant starch would generate a
similar microbial profile, regardless of the amount fed to colitic mice. However,
feeding low and high concentration of unmodified resistant starch created a different
microbial profile of colonic microorganisms. Differences in the gastrointestinal
microbial profile were analysed by culturing onto selective medium (Figures 3.8, 3.14
and 3.15) and analyzing colonic contents using DGGE (Figures 3.9, 3.10 and Tables
3.2, 3.3).
The selective medium, RB, assisted in the differentiation of bifidobacteria colonies
from other lactose-fermenters in the colonic contents. Numbers of bifidobacteria were
elevated in colitic mice given both the 5% and 30% unmodified high amylose maize
resistant starch diet but their numbers were not increased when the starch free basal
diet (0% resistant starch) was administered (Figure 3.8d and 3.14b). Bifidobacterium
are generally regarded as having a positive effect on human health and different
research groups have demonstrated the elevation of bifidobacteria and lactobacilli by
resistant starch149, 150. Interestingly, elevation of beneficial bacteria did not ameliorate
colitis in the group that received the 30% unmodified high amylose maize starch. This
is consistent with the study using galacto-oligosaccharides in TNBS colitis wherein
elevation of bifidobacteria did not correlate with the resolution of colitis170. In
contrast, our study was able to demonstrate that there was an elevation of
bifidobacteria and at the same time restoration of colon integrity in colitic mice given
the lower concentration (5%) of high amylose maize resistant starch diet.
A slight increase in the levels of lactobacilli was detected in the starch fed groups but
this did not show any significant difference compared to the colitic control (Figures
3.8c and 3.14a). On the other hand, Gram negative anaerobes remained at a similar
level for all colitic groups given the different concentration of resistant starch (Figures
3.8b and 3.14d), whereas, endospores were detected in more colitic animals with 30%
dosage (Figure 3.14d). It has previously been shown that spore-formers such as
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Clostridium, and the Gram negative anaerobe, Bacteroides, can efficiently utilize high
amylose maize resistant starch granules147, 150. Moreover, these genera of bacteria can
be pathogenic and may produce toxins and have putrefactive activities. This is a
concern regarding the selectivity of resistant starch to just stimulate beneficial
microorganisms like Bifidobacterium or C. butyricum in inflammatory bowel diseases
because Bacteroides, Clostridium and Bacillus have all been implicated in the
pathogenesis of IBD and various gastrointestinal infections and could be enhanced by
the starch.
Colony types of enteric microorganisms also differed with the different concentrations
of resistant starch used in TNBS colitis (Figure 3.15b). Colitic animals that did not
receive any resistant starch supplementation harboured more of the non-lactose
fermenters in their colonic contents. Examples of non-coliforms include Salmonella
and Shigella. The colitic group receiving the 30% resistant starch, on the other hand,
had both the non-lactose fermenters and the coliform colonies detected in their faeces
in higher levels compared to the levels of enterococi. Examples of coliform
microorganisms are E. coli and Pseudomonas. The group which received the 5%
unmodified high amylose maize starch excreted significantly more of the enterococci
but maintained the coliforms and non-coliforms in low levels compared to the 30%
concentration and starch free diet. Aerobic, enteric bacteria have been implicated in
initiation and perpetuation of IBD as they were the most immunoreactive against the
sera of C3H/HeJBir mice induced to have colitis40, particularly E. coli, Proteus
mirabilis and S. typhimurium. Furthermore, there was an increase in the number of
aerobic bacteria that adhered and translocated into the tissue of IBD mice51 and that
were detected in clinical samples50. Coliforms, non-coliforms and enterococci belong
to Enterobacteriaceae group. Enterobacteriaceae compose a group of closely related
intestinal bacteria. Most of the species in this group are members of the normal
intestinal microflora, such as E. coli and Enetrobacter. Other members of the groups
are pathogens inducing severe infectious diarrhea such as the non-coliforms,
Salmonella and Shigella; and coliforms such as that of E. coli; namely the
enteropathogenic (EPEC), enteroinvasive (EIEC), enterotoxigenic (ETEC) and
eterohemorraghic (EHEC) strains286. These E. coli strains are medically significant as
these cause urinary tract infections, meningitis and gastroenteritis. Coliforms are also
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abundant in the environment. They are also used as indicators for water quality and
contamination.
The enhanced counts of Bifidobacterium seen in the resistant starch treated group may
contribute to the attenuation of colitis. However, assessing the population numbers of
lactobacilli, Gram negative anerobes, endospores and enterics suggests that it is not
only the total level of lactobacilli and bifidobacteria that is critical in protecting the
animals against the development of IBD but the presence and abundance of the other
members of the gut microbiota. These results also show that enteric bacteria
especially the lactose fermenters are detected in high numbers in TNBS colitis, but
whether enteric bacteria are responsible for intestinal inflammation or whether the
profile observed is a result of inflammation warrants further investigation.
It is recognized that not all microbes in the intestine can be cultured, thus, DGGE
analysis was performed to show microbial community changes and to identify distinct
banding patterns that appear during the course of colon inflammation. DGGE profiles
(Figures 3.9, 3.10 and Tables 3.3) showed that high amylose maize starch fed colitic
mice had many bands that were also found in the controls and hence many microbes
are unchanged (Figures 3.9, 3.10 and Tables 3.3). This indicates that gut
microorganisms are stable. There were bands that appeared that were only detected in
the healthy and ethanol controls but not in colitic animals or colitic animals fed the
resistant starch. The sequences of these bands were consistent with those reported for
Staphylococcus sp., L. grasserri, a nitrogen utilizing bacterium and an uncultured
bacteria from the intestinal tract. These bacteria contribute to the microflora of normal
mice that were lost in colitic mice. New bands emerged when colitis was induced and
when the high amylose maize starch diet was administered. The emerging band in the
colitic group displayed 100% sequence homology with that of an uncultured
Bacteroidetes intestinal bacterium. Bacteriodetes is a common commensal mucosa-
associated bacterial group. Bacteroidetes has been isolated in samples exposed to
faecal contamination and have been shown to cause gastrointestinal illnesses199-201.
Bacteroidetes has also been used as a novel indicator in testing the quality of water
and food samples201. On the other hand, 30% unmodified starch promoted the growth
of an intestinal microbe that has been reported to regulate fat storage in the gut of
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colitic mice. This indicates that microrganims can affect metabolic activities through
dietary intervention.
3. 4. 3. Pro-inflammatory and anti-inflammatory cytokine production in colitic
mice supplemented with high amylose maize starch diet
An increased expression (Figures 3.6 and 3.13) and production (Figures 3.7and 3.17)
of the pro-inflammatory (Th1) cytokine, IFN- , was seen in the LPMCs and from
colon tissue cultures of colitic mice on the 30% high amylose maize starch diet. The
previously reported immunoreactivity of the inflamed colon towards enteric bacterial
antigens and their products may account for the upregulation of the production of
IFN- in colitic mice fed the 30% starch diet. The pro-inflammatory activities of IFN-
in the gut may increase permeability of the mucosa allowing for the rapid uptake of
bacterial products such as endotoxins, peptidoglycan polysaccharides and
lipopolysaccharides into the mucosa. As a consequence, macrophages and antigen
presenting cells would be stimulated and this subsequently would enhance production
of more Th1 cytokines and recruitment of neutrophils into the mucosa.
The present study also showed that supplementing the diet with a minimal
concentration of indigestible carbohydrate (0% resistant starch) and a lower
concentration (5%) of high amylose maize resistant starch were able to lessen the
severity of colon inflammation, and markedly reduced the synthesis of IFN- and
simultaneously upregulated the production of anti-inflammatory cytokine (Th2), IL-4,
and T regulatory cytokine, IL-10. It was demonstrated that lower concentrations of
indigestible carbohydrates brought the ratio of Th1 and Th2 cytokine immune
response close to levels seen in healthy mice (Figures 3.7 and 3.17). Moreover, all
colitic animals were able to express both IFN- and IL-4 regardless of the dietary
treatment administered but it is only in association with IL-10 synthesis that the diet
was effective in reducing inflammation associated with TNBS colitis (Figures 3.7 and
3.13). It has been speculated that to have an overall impact in reducing intestinal
inflammation, the amounts of Th1 and Th2 cytokines should be more or less
balanced. Considerable evidence is available that shows that Th1 cytokine responses
and Th2 cytokine responses cross regulate each other. IL-4 and IL-10 are two anti-
inflammatory cytokines that enhance immune defence mechanisms by preventing
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destruction of intestinal mucosa and physiologically maintain the low grade
inflammation as seen in normal gut. IL-4 and IL-10 can also suppress production of
multiple inflammatory cytokines by macrophages, T-cells, dendritic cells and natural
killer cells. This may be one of the mechanisms involved in the anti-inflammatory
effect observed after dietary supplementation of 5% unmodified high amylose maize
resistant starch.
3. 4. 4. Effect of high amylose maize resistant starch on short chain fatty acid
production in TNBS-induced colitis
No significant differences were observed in the relative concentration of individual
SCFAs produced by the colitic mice given the different high amylose maize starch
diet (Figure 3.18). However, looking at the individual components that make up the
total SCFAs measured showed that the 0% starch diet, 5% unmodified, 30% and 5%
modified high amylose maize diets produced more butyrate in the colonic contents
than acetate and propionate, whilst, the 30% unmodified high amylose maize starch
diet produced more acetate than butyrate or propionate (Table 3.4).
Impaired colonic SCFA production is seen in clinical and experimental colitis75, 182.
SCFA is a principal source of energy in the colonic epithelium and modulates
enterocyte differentiation, proliferation and restitution158. It is also believed that
SCFAs have immunoregulatory effect on intestinal epithelial cells and other mucosal
cell populations202, 203. SCFA, especially butyrate, modulates expression of pro-
inflammatory cytokines by altering histone acetylation of DNA of colonic epithelium
or suppressing NF- B activation204, 205. It has been reported previously that inclusion
of high amylose maize starch in the diet of healthy mice150 and humans206 resulted in
a significant increase in the level of butyrate detected. Moreover, accumulation of this
SCFA in the gut can lower intestinal pH that can inhibit the invasion of pathogenic
microorganisms.
The potentiation of a pro-inflammatory response seen in colitic mice fed the 30%
unmodified can be explained by the ability of SCFA to collaborate with bacterial LPS
and synergistically induce production of pro-inflammatory cytokines and their
mediators203, 207, 208. As seen from Figures 3.8 and 3.15a-b, 30% unmodified high
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amylose maize starch diet stimulated the growth of Gram negative aerobic bacteria
which possess lipoploysaccharides on their cell wall. These may have modulated
immune function in colitic mice and augmented production of IFN- and increased
the extravasation of neutrophil in the colonic mucosa.
The exacerbation of the inflammatory response in the 30% unmodified starch may
also be attributed to the amount of acetic acid produced. Although acetic acid is part
of the total SCFAs and should be beneficial in resolving inflammation, acetic acid is
also used as an agent to induce inflammation in murine models of inflammatory
bowel diseases2, 209 Thus, the amount at which it is being produced by the 30%
unmodified resistant starch may be detrimental for the recovery from TNBS induced
inflammation. The protection from the lower concentration of high amylose maize
resistant starch diets, on the other hand, is in accordance with previous studies which
showed that prevention of colitis is associated with an increase in SCFA production
and a reduction of pro-inflammatory IFN- . These results indicate that the amount and
type of short chain fatty acid present in the bowel can be manipulated by providing
specific types of resistant starch to stimulate particular bacterial profiles and immune
responses.
In conclusion, dietary supplementation of 5% concentration of high amylose maize
resistant starch prevented the development of inflammation in the TNBS model of
colitis. This beneficial effect was associated with an abrogation of the expression and
synthesis of pro-inflammatory cytokine, IFN- , and an upregulation of anti-
inflammatory and T regulatory cytokines, IL-4 and IL-10, respectively. Moreover, 5%
unmodified high amylose maize resistant starch diet elevated the growth of
bifidobacteria while suppressing the levels of putatively pathogenic endospores and
Gram negative aerobic bacteria in colitic mice. This beneficial effect enhanced
production of SCFA which facilitated the restoration of intestinal barrier, inhibited
proliferation of enteric microorganisms and synergistically modulated immune
responses through downregulation of pro-inflammatory cytokines.
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Chapter 4
Effects of selected probiotic Bifidobacterium and Lactobacillus strains on the
TNBS-colitis murine model
4. 1. Introduction
The cause/s of inflammatory bowel diseases (IBD) have not been identified, however,
the initiation and perpetuation of the immune response in IBD seems to involve type1
T helper (Th1) cells as their cytokine products are found in increased amounts in the
inflamed colonic mucosa80, 210, 211. Th1 pro-inflammatory cytokines include interferon
(IFN)- and tumour necrosis factor (TNF)- which are characteristically elevated in
Crohn’s Disease (CD). Evidence also exists that the other form of IBD, ulcerative
colitis (UC), exhibits a mixed T cell immune response. Both forms of IBD undergo
similar pathway of inflammatory processes which are characterized by excessive
production of pro-inflammatory cytokines and free-radicals in the mucosa95, 96, 212.
This observed increase in pro-inflammatory cytokines in IBD led to the interest in
developing therapies that neutralized the activities of Th1-associated cytokines. Novel
therapies being trialed including anti-TNF therapy (infliximab), anti-IFN
antibodies213 and anti-inflammatory cytokine IL-10 therapy. Intravenous infusion of
infliximab resulted in significant improvement of disease outcome measures in
murine colitis models and human CD and rheumatoid arthritis (RA) patients214.
Furthermore, monoclonal IL-10 treatment inhibited Th1 cytokine production and
ameliorated inflammation in experimental colitis in animals215.
Unfortunately, mild to serious adverse events are associated with the use of anti-TNF-
therapy and these include headache, drug reactions and increased susceptibility to
mycobacterial infection, as well as possible malignancy214. Some workers failed to
show that IL-10 therapy was effective in clinical inflammation116, 117 with minimal
improvement on the clinical activity index of CD patients and no effect on remission.
Moreover, higher doses of IL-10 stimulated IFN- production, thus, worsening
intestinal inflammation118.
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Several studies have examined the effects of probiotics on murine colitis because of
the potential to immunomodulate, regulate intestinal microbes and because of their
safty profile216-218. Steidler et al (2000) fed L. lactis genetically engineered to secrete
IL-10 which was able to abrogate chronic colitis in DSS induced mice as well as
prevent the onset of colitis in IL-10 deficient mice. Moreover, Lactobacillus GG
treatment changed the course of inflammation in IL-10 deficient mice and modulated
the intestinal flora219. Feeding a lysed solution of Escherichia coli proved to be also
efficacious in ameliorating murine colitis, indicating that not only living bacteria but
also soluble bacterial products can modulate colitis220. In a human intestinal post-
surgical inflammatory condition known as pouchitis, Gionechetti and co-workers
(2000) were able to successfully show that a combination of eight Lactobacillus,
Bifidobacterium and Streptococcus species can prevent flare-ups in these patients.
This combination of strains has subsequently been used in TNBS induced murine
colitis to examine the involvement of regulatory IL-10 in the resolution of colitis221.
Thus, the use for specific probiotics in immunotherapy is attractive, however it is
recognized that strains of bacteria differ in their immunomodulating capacity and
therefore probably in their capacity to reduce colitis.
This study examined several strains of Bifidobacterium and Lactobacillus in an
experimental murine colitis model with dosage prior to and after the induction of
colitis. The profile of cytokines in the inflamed intestine, and the indigenous colonic
microbes have been studied and correlated with histopathological changes, disease
activity indices and gut wall integrity.
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4. 2. Materials and Methods
4. 2. 1. Bacteria and culture conditions
Bifidobacterium and Lactobacillus used in the study included: Bifidobacterium
animalis LAVRI™-B1 (DSM, Moorebank), B. lactis LAVRI™-B2 (DSM,
Moorebank), Lactobacillus acidophilus LAVRI™-A1 (DSM, Moorebank), L.
amylovorus FII 546400 and L. fermentum VRI 003 (VRI BioMedical and stocked in
the University of New South Wales Culture Collection). Glycerol stocks of the
Bifidobacterium and Lactobacillus strains were grown in Tryptone Peptone Yeast
(TPY; recipe can be found in the Appendix) and de Mann Rogosa Sharpe (MRS;
Oxoid) broths, respectively, at 37ºC in an anaerobic condition. A standard curve of
OD versus viable counts expressed as colony forming units (CFU) was generated for
each strain by measuring absorbance at 600 nm. The viable cell counts were
determined by plating serial dilutions onto MRS or TPY agar plates and were
expressed as colony forming units (CFU.ml-1). Bacteria were washed twice with
0.01M Phosphate Buffered Saline (PBS; pH 7.2) and adjusted to the required
concentration of viable cells using OD values and standard curve.
4. 2. 2. Induction of colitis
Female specific pathogen free (SPF) BALB/c mice aged 6-8 weeks were obtained
from the Animal Resource Centre (ARC), Perth, Australia. Animals were kept in the
School of Biotechnology and Biomolecular Science Animal Facility of the University
of New South Wales, Sydney. The mice were housed on sawdust in plastic cages and
kept in conditions of constant temperature (20ºC) and humidity with a 12 hour light-
dark period. They were fed ad libitum a mouse feed (Gordon’s Animal Feed,
Australia) and allowed unlimited access to autoclaved water. All work conducted on
the mice had approval of the University of New South Wales Animal Care and Ethics
Committee. Animals were allowed to adapt to animal house conditions prior to the
start of the experiments.
Animals were anaesthetized with xylazine and ketamine prior to the induction of
experimental colitis. Colon inflammation was induced by intrarectal administration of
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2.5 mg 2,4,6-trinitrobenzene sulfonic acid (TNBS; Sigma) in 45% ethanol for colitic-
treated groups and 45% ethanol for ethanol control group. A total injection volume of
100µl was instilled into the colon of each mouse. The animals were held in a vertical
position for 20 seconds while administering the TNBS to allow it to reach the entire
colon.
4. 2. 3. Experimental designs
Three studies were performed in this chapter and are outlined in Figure 4.1.
Experimental design A compared the ability of several strains of probiotics to prevent
the development of colitis. B. animalis, B. lactis, L. acidophilus, L. amylovorus and L.
fermentum were fed every other day for a week prior to the induction of colitis on Day
7 until the animals were sacrificed 7 days after induction of colitis using TNBS.
Animals (n=10) were dosed with 200 µl containing 2x109 CFU of the probiotic
bacteria each dosing day while the healthy-control, ethanol-control and colitic-control
groups received equivalent volume of 0.01M PBS.
Experimental design B determined the most effective dose of L. fermentum using
1x109, 1x108 and 1x107 CFU per dose (n=9 mice per dosage level). A total of 200 µl
of L. fermentum were dosed while the control groups received the equivalent amount
of 0.01M PBS. Feeding regime is identical with Experiment A.
Experimental design C tested the effect of L. fermentum on established colitis.
Animals (n=8 per group) were induced with colitis and 6 hrs after were dosed with
1x108 CFU per dose per day of L. fermentum. Feeding of L. fermentum was continued
each day thereafter for 7 days.
All the animals from all experiments were sacrificed 7 days after TNBS
administration and their colon tissues collected as soon as possible and examined for
tissue damage, and expression of pro- and anti-inflammatory cytokines. Spleens were
also harvested to determine translocation of bacteria and colon contents collected for
enumeration of concentrations of bacteria.
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Experimental Designs A and B
Figure 4.1. Experiment design of Chapter 4
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sacrifice mice
Colitis Induction with TNBS
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sacrifice mice
Experimental Design C
Colitis Induction with TNBS
Days
Days
= Probiotic dosing
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4. 2. 4. Generation of lamina propria mononuclear cells (LPMCs)
The colon contents and debris were removed and the colon was then sliced into 5 mm
long pieces, placed in Hank’s Balanced Salt Solution (HBSS; Gibco) with 2mM
dithiotreitol (DTT; Sigma) with stirring for 20 mins at 37ºC. The solution was
replaced with fresh HBSS, 2mM DTT and 0.01M EDTA (Sigma) and the tube shaken
at 37ºC for 20 mins. The colon was further digested by addition of 1mg.ml-1
collagenase (Roche) and 1 mg.ml-1 dispase (Roche) and incubated for 1 hr at 37ºC.
The LPMC suspensions were harvested by centrifugation and then cultured at
1x106.ml-1 in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated
fetal calf serum, 5mM L-glutamine and 100 U penicillin and 100µg streptomycin.
Replicate wells stimulated with 1 µg.ml-1 Concanavalin A (ConA; Sigma) served as
positive controls. After 3 days of incubation at 37ºC in an atmosphere of 5% CO2,
supernatants were collected and stored in -70ºC until use.
4. 2. 5. Cytokine assay
Cytokine concentrations in the supernatant of LPMC were measured by ELISA. In
brief, 96-well plates (NUNC) were coated with purified anti-cytokine capture
antibody and incubated overnight at 4ºC. Subsequently, the plates were blocked with
1% BSA in PBS/0.05% Tween 20 (PBS/T) and incubated at room temperature for 90
minutes. Standards were prepared and diluted two-fold in 1% BSA in PBS/T. The
standards and samples were then added to the wells and the plates were left to
incubate at room temperature for 90 minutes. Biotinylated anti-cytokine antibody
dissolved in 1% BSA-PBS/T was then added into the wells and allowed to incubate
for an hour in room temperature. Streptavidin HRP (Chemicon) was diluted 1/1000 in
1%BSA-PBS/T and allowed to incubate at room temperature for another 30 minutes.
The plates were washed three times with PBS/T after each step until the addition of
the substrate 3,3’,5,5’-tetramethyl benzidine (TMB; Sigma) and hydrogen peroxide
into the plate. Once substrate and hydrogen peroxide were added, the colour reaction
was allowed to develop at room temperature. The reaction was stopped using 1M
H2SO4 and the plates were read at 450 nm using a BIORAD Microplate Manager
Reader. The antibodies used were rat anti-mouse IL-4, IL-10 and IFN- which were
all obtained from Pharmingen.
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4. 2. 6. Assessment of colon inflammation.
Colon tissues were examined histologically to determine the extent of colitis induced
by TNBS compared to inflammation in colitic animals fed with probiotics and to the
colons of healthy-control and ethanol-control groups. The distal colon was quickly
removed upon sacrifice, opened longitudinally and cleared of faecal contents using
cold PBS. The colon tissue was fixed in 10% phosphate buffered formalin at room
temperature overnight. The tissues were then sliced into 5 mm pieces, dehydrated in
ethanol, embedded in paraffin wax, sectioned and stained with haemotoxylin and
eosin. The slides were scored blindly using the histological grading system described
in Chapter 3.
4. 2. 7. Bacterial translocation from colon
Bacterial translocation into the spleen as a result of TNBS colitis was monitored. The
spleens were aseptically collected, weighed and homogenised in sterile cold PBS. Ten
fold serial dilutions were prepared in half strength Wilkins Chalgren broth (WC;
Oxoid) and suspensions were cultured onto MacConkey CM7 Agar (MAC; Oxoid),
MRS agar and Wilkins Chalgren Agar (WCA; Oxoid) with 5% horse blood (Oxoid)
for detection of enteric bacteria, Lactic Acid Bacteria (LAB) and total anaerobes,
respectively. All plates were incubated in an anaerobic chamber at 37ºC for 48 hours,
except MAC plates which were incubated aerobically at 37ºC. The colonies that grew
were counted and quantified as colony forming units per gram of tissue (CFU.g-1).
4. 2. 8. Enumeration of colonic bacteria.
After the animals were sacrificed, the colon tissues were opened longitudinally and
the pellets were collected, weighed and serially diluted ten-fold in half strength
Wilkins-Chalgren broth (Oxoid). From every dilution, 10 µl aliquots were drop plated
in triplicates onto Mac Conkey Agar CM7 (MAC; Oxoid), de Mann Rogosa Sharpe
Agar (MRS; Oxoid), Wilkins Chalgren Agar (WCA; Oxoid) with 5% horse blood
(Oxoid) and Yeast Extract Peptone Dextrose Agar (YEPD) (D-glucose, 20g;
Bactopeptone, 20g; Yeast Extract, 10g; Agar, 20g per litre) to enumerate
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enterobacteria, total lactobacilli, total anaerobes and yeast type microorganisms,
respectively. The MAC agar plates were incubated aerobically for 24 hours at 37ºC
while the MRS and WCA agar plates were incubated in an anaerobic chamber for 48
hours at 37ºC. YEPD plates were incubated at 30ºC for 48 hours.
4. 2. 9. Statistical analyses
The SPSS 11.5 for Windows statistical package was used for the analyses of data. All
data were expressed as mean ± SD and evaluated using Mann-Whitney or Wilcoxon
test for non-parametric data and t-test for normally distributed data to test difference
between two groups and Chi-square test for categorical data. The results were then
corrected for multiple comparisons using the Tukey, Bonferonni and Sidak tests
Values of p<0.05 were considered statistically significant.
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4. 3. Results
4. 3. 1. Protection provided by probiotic strains against TNBS induced murine
colitis
The effectiveness of various probiotic strains in amelioration of TNBS induced
murine colitis was investigated. Disease severity was evaluated by monitoring body
weight profile and survival of animals. Table 4.1 shows that B. animalis, B. lactis, and
L. amylovorus did not prevent the wasting effects of TNBS colitis. The weight and the
number of surviving colitic animals that received these probiotics did not improve.
All animals induced with TNBS colitis developed watery diarrhea and suffered a
reduction of mobility and body weight. These conditions worsened in all the treatment
groups and colitic control groups except in colitic animals dosed with L. acidophilus
and L. fermentum. Both L. acidophilus and L. fermentum had body weight loss lower
than and significantly different (p<0.05) from colitic mice on the day of sacrifice.
Notably, fewer deaths were seen in animals fed with L. fermentum and in L.
acidophilus fed colitic mice. Thus, the effects of L. acidophilus and L. fermentum on
TNBS colitis were further assessed by looking at the expression of inflammatory
cytokines in the colon (Figure 4.2) and effects on colon tissue damage (Figure 4.3).
Expression of interferon gamma (IFN- ) and interleukin 10 (IL-10) and the ratio of
the two for L. acidophilus and L. fermentum dosed mice was compared to that for
colitic control, ethanol control mice as well as healthy mice (Figure 4.2). It was
assumed that a ratio similar to healthy animals was more ideal than that of colitic
animals. It is established in the TNBS model of colon inflammation that there is an
overproduction of pro-inflammatory cytokines, such as IFN- , and that inflammation
may be abrogated by anti-inflammatory cytokines such as IL-10. The “physiological”
inflammation of the normal intestinal environment is associated with an optimal ratio
of IFN- production to IL-10 production. The colon cells of L. fermentum fed mice
increased the level of anti-inflammatory IL-10 and simultaneously decreased the
concentration of pro-inflammatory IFN- but did not reach statistical significance
when compared with the colitic-control group and L. acidophilus group. Nevertheless,
134
this indicates that L. fermentum can shift cytokine response of TNBS colitis from a
type 1 helper response to one with an anti-inflammatory profile.
Microscopic examination of inflamed tissue revealed dense neutrophil infiltration,
ulceration of the epithelium and distortion of the crypts in colitic mice and less severe
pathology in L. acidophilus and L. fermentum groups (Figure 4.3). Significance
(p<0.01) in tissue damage, however, was only seen in L. fermentum when compared
to the colitic-control group. No colonic histological abnormalities were identified in
the healthy group. Based on the above results, L. fermentum was used in subsequent
experiments.
135
Table 4.1. Body weight profile and survival of TNBS-induced colitic mice at the day of sacrifice. Bifidobacterium and Lactobacillus were administered for 7 days prior to colitis induction with TNBS. Values are significantly different from colitic control group at p<0.05*
Group Body Weight Profile
(%)
Survival
Loss Gain
Healthy Control 3.00* 10/10
Ethanol Control 4.42 7/10
Colitic Control 11.94 5/10
L. acidophilus 4.40* 9/10
L. amylovorus 14.04 4/10
L. fermentum 2.00* 9/10
B. animalis 8.28 5/10
B. lactis 14.76¥ 0/10 ¥ Body weight reported was taken 5 days after induction of colitis. Animals from this group had to be sacrificed before finishing the experiment as per Animal Research Ethics Committee guidelines on animal handling.
136
GROUP
LfLaColiticEthanolHealthy
95%
CI
Rat
io o
f IL-
10:IF
N-g
amm
a
.4
.3
.2
.1
0.0
-.1
Figure 4.2. Ratio of IL-10:IFN- cytokines in LPMCs of L. acidophilus and L.fermentum fed mice 7 days after induction of colitis. Results are expressed as mean ratio ± SD in 10 mice. La = L. acidophilus; Lf = L. fermentum.
137
0
0.5
1
1.5
2
2.5
3
3.5
4
Healthy Ethanol Colitic La Lf
Hist
olog
ical
Sco
re
¶
§,
¶
Figure 4.3. Histological scores of colon tissue of colitic animals fed with L.acidophilus and L. fermentum In all animals, except healthy-control and ethanol-control, colitis was induced with TNBS. Values represent mean±SD. Significance (p<0.01) was observed between colitic-control group and L. fermentum fed colitic mice. La = L. acidophilus and Lf = L. fermentum.
¶ = significantly different from ethanol-control group at p<0.01 § = significantly different from colitic-control group at p<0.01 * = significantly different from the La-treated group at p<0.05
138
4. 3. 2. Dose response effects of L. fermentum in colon inflammation
A dose response study was then performed to determine the effective dose of L.
fermentum VRI 003 required to protect mice from the inflammation caused by TNBS.
The concentrations of bacteria that were orally administered were 1x109, 1x108 and
1x107 CFU.dose-1 with mice receiving daily doses for 7 days prior to TNBS dosage. It
was observed that different doses of L. fermentum affected the weight kinetics (Figure
4.4). Body weight gain was significantly suppressed (p<0.01) in the colitic group in
contrast to the healthy group. Administration of L. fermentum at a daily dose of 1x109
CFU (p<0.01) significantly attenuated the TNBS effect on body weight, whereas, a
non-significant trend was observed with 1x108 CFU. There was no difference
observed in the body weight kinetics among the groups that received the dose of
1x107 CFU compared to the colitic-control group. There was no mortality seen in the
group given the 1x108 CFU of L. fermentum while 1 out of 9 mice died in the groups
given the 1x109 and 1x107 CFU per day of L. fermentum. Fewer animals survived in
the colitic group (Figure 4.4).
The concentration of L. fermentum also influenced how it modulated the pattern of the
Th2/Th1 balance in TNBS-colitic mice. This model of colon inflammation is
associated with increased concentrations of Th1 cytokine, IFN- , and decreased
concentration of Th2 cytokine, IL-4. Figure 4.5 presents the dose-response of L.
fermentum given in different concentrations on cytokine expression in the colon of
colitic animals. Administration of 1x108 CFU per day of L. fermentum modulated
production of Th2/Th1 cytokines to be comparable to the levels expressed by healthy
group. These results suggest that L. fermentum given at a dose of 1x108 CFU per
mouse per day can restore IL-4 production, thereby, inhibiting the pro-Th1 cytokine
milieu seen in the TNBS model of colon inflammation.
Another relevant observation is that L. fermentum lowered the incidence of
translocating bacteria from the lumen to the spleen. Colon inflammation results in an
increased intestinal permeability which facilitates the translocation of bacteria from
the colonic lumen to the systemic organs. This increased uptake of luminal contents
can worsen the inflammation as well as cause systemic sepsis. As presented in Table
4.2, L. fermentum given at a dose of of 1x109, 1x108 and 1x107 CFU significantly
139
inhibited (p<0.01) bacterial translocation to the spleen compared to the colitic-control
group as translocation was detected in more animals in the colitic-control group.
These doses of L. fermentum effectively reduced the number of Gram negative enteric
bacteria that translocated into the spleen. No translocating anaerobic bacteria and
Lactic Acid Bacteria (LAB) were detected in the spleens of any of the groups.
Concentrations of different types of bacteria collected from colonic contents were also
determined. Concentrations of Gram negative aerobic bacteria were significantly
lower (p<0.01) in the L. fermentum treated groups compared to the ethanol-control
and colitic-control groups, whereas, counts of enterics in the colitic animals that
received different doses of L. fermentum were comparable to the healthy-control
group (Figure 4.6a). Interestingly, yeast type (Candida species) microorganisms
(Figure 4.6b) were significantly detected 2 logs lower (p<0.01) in mice given 1x109
CFU per day and 1 log lower (p<0.01) in the group that received 1x108 and 1x107
CFU per day compared to the ethanol control and colitic-control animals. Candida
counts in the L. fermentum treated groups were comparable to the healthy control
group. On the other hand, there was a significant decrease (p<0.05) observed in the
counts of potentially beneficial species, Lactobacillus, in the ethanol-control and
colitic-control groups which is in contrast to the colitic animals that received L.
fermentum (Figure 4.6c). All 3 doses of L. fermentum maintained the levels of
lactobacilli in the colonic contents of colitic mice comparable to the healthy animals.
No significant changes were detected in the numbers of total anaerobe in any groups
(Figure 4.6d).
140
Figure 4.4 Body weight loss and survival of colitic animals (n=9) 7 days after induction of colitis with TNBS.
Animals were administered with 1x107, 1x108 and 1x109 CFU of L. fermentum every other day for two weeks. Colitis was induced using TNBS a week after L. fermentumdosage commenced. L. fermentum dose of 1x109* is significantly different from colitic group at p<0.01. All groups are statistically different from the healthy-control group (p<0.01).
GROUP
HealthyEthanolColitic1e71e81e9
95%
CI
Bod
y W
eigh
t Los
s
30
20
10
0
-10
***
8/9 9/9 8/9 6/9 7/9 9/9Number of Survivors
141
0
0.5
1
1.5
2
2.5
1.00E+09 1.00E+08 1.00E+07 Colitic Ethanol Healthy
Rat
io IL
-4:I
FN-g
amm
a ¶¶
¶
Figure 4.5 Ratio of IL-4:IFN- cytokines in the LPMCs of animals fed with different concentrations of L. fermentum VRI 0037 days after the induction of colitis with TNBS.
Animals were administered with 1x107, 1x108 and 1x109 CFU of L. fermentum every other day for two weeks. Colitis was induced using TNBS a week after L. fermentumdosage commenced. Results are expressed as mean ratio±SD in 9 mice.
142
Table 4.2. Levels of enteric bacteria in the spleens of mice after dosage with either 1x109, 1x108 or 1x107 CFU of L. fermentum or PBS (colitic, ethanol, healthy controls) every other day for 2 weeks. After 1 week colitis was induced using TNBS in all groups except ethanol and healthy controls.
Groups Bacterial count
in spleena
(Log CFU.g-1)
Number of animals with
detectable enteric bacteria
(n=9)
1x109 2.86 2
1x108 2.75 1
1x107 3.60 1
Colitic 3.04 7¥
Ethanol ND 0
Healthy ND 0
ND = not detected. Detection limit =102.gram-1
a = mean value of spleen with detectable enteric bacteria ¥ = significantly different from all 3 dosage levels of L. fermentum VRI 003 (p<0.01)
143
Figure 4.6. Concentrations of colonic microorganisms detected 7 days after TNBS induction of colitis in colitic animals (n=9) administered with a range of oral doses of L. fermentum.
The animals were given every second day a dose of 1x109 (Dose 1), 1x108 (Dose 2) and 1x107 (Dose 3) CFU of L. fermentum for 14 days prior to the induction of colitis with TNBS and continued until the end of the study. Counts of bacteria from colitic mice fed with L. fermentum are statistically different from the counts detected in the ethanol-control and colitic-control groups. * significantly different from colitic-control animals, p<0.05.
a) Enterics
0 2 4 6 8 10 12
Dose 1
Dose 2
Dose 3
Colitic
Ethanol
Healthy
Log CFU.g-1 colon contents
0.01
*
b) Yeast type species
0 2 4 6 8 10 12
Dose 1
Dose 2
Dose 3
Colitic
Ethanol
Healthy
Log CFU.g-1 colon contents
0.01
*
c) Lactobacilli
0 2 4 6 8 10 12
Dose 1
Dose 2
Dose 3
Colitic
Ethanol
Healthy
Log CFU.g-1 colon contents
0.05
*
d) Total anaerobes
0 2 4 6 8 10 12
Dose 1
Dose 2
Dose 3
Colitic
Ethanol
Healthy
Log CFU.g-1 colon contents
144
4. 3. 3. Administration of L. fermentum after induction of experimental colitis
using TNBS
Since the 1x108 CFU daily dose generated higher survival, promoted Th2 cytokine
expression and lowered incidence of translocating bacteria into the spleens of colitic
animals, this dose was used to assess whether L. fermentum could ameliorate an
established colitis. Colitic animals fed therapeutically with L. fermentum after
induction of colitis had a significantly improved (p<0.05) body weight compared to
control colitic mice. Feeding of L. fermentum to healthy mice had no effect since body
weights were maintained in healthy animals (Figure 4.7).
The therapeutic mode of L. fermentum treatment significantly inhibited (p<0.05) the
generation of pro-inflammatory cytokines in the colon as compared with the TNBS
colitic control (Figure 4.8) Administration of the same dose to healthy animals
produced a cytokine profile comparable to untreated healthy animals. Feeding of L.
fermentum did not markedly shift the cytokine profile of healthy animals.
145
-6
-4
-2
0
2
4
6
8
10
12B
ody
Wei
ght L
oss (
%)
Lf + Colitic Colitic Ethanol Healthy Lf + Healthy
Figure 4.7. Body weight profile of animals (n=8) therapeutically fed with a daily dose of 1x108 CFU of L. fermentum.
L. fermentum was dosed to the animals after induction of colitis using TNBS, and to healthy, normal BALB/c mice. Values are significantly different compared to colitic-control mice ** at p<0.05.
** **
**
146
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Lf + Colitic Colitic Healthy Lf + Healthy
Rat
io IL
-10:
IFN
-gam
ma
** ****
Figure 4.8. Ratio of IL-10:IFN- cytokines in LPMCs of colitic mice given L.fermentum or healthy animals given L. fermentum following the induction of colitis with TNBS.
Mean values were significant at p<0.05** between the colitic groups fed with L.fermentum and colitic-control group. Healthy-control group is also statistically different to the colitic-control group. Lf = L. fermentum.
147
4. 4. Discussion
In this chapter, the capacity of probiotics to attenuate colon inflammation whether
administered prior to and/ or after induction of murine colitis was examined. It was
demonstrated that:
Dosage of L. fermentum improved the clinical features of colonic
inflammation induced by TNBS.
L. fermentum altered the immune dysregulation engendered in the colonic
tissue as exhibited by the simultaneous decrease in the levels of pro-
inflammatory cytokines and production of anti-inflammatory and regulatory
cytokines; and lessened histological damage of the colonic tissues.
Different probiotic strains have different capacities to protect BALB/c mice
against chemically-induced colitis.
A greater incidence of translocating enteric bacteria is detected in the spleens
of colitic mice compared to L. fermentum fed colitic mice.
Concentrations of Gram negative enteric bacteria and yeast type
microorganisms were significantly increased while significant reduction in
lactobacilli numbers was detected and total anaerobes remained unchanged in
the colonic contents of colitic mice. Feeding of L. fermentum modified the
microbial profile comparable to healthy control microflora.
The selection of probiotics strains used in this study is based on previous in vitro and
in vivo studies that showed their ability to adhere to human epithelial cells, to inhibit
pathogenic bacterial growth, to stimulate the immune response and to hydrolyze
carbohydrates in the case of L. amylovorus. Based on the body weight loss profile
and number of survivors (Table 4.1), L. acidophilus and L. fermentum exhibited
greater recovery from weight loss and resulted in better survival rate compared to B.
animalis, B. lactis and L. amylovorus. Viability of Bifidobacterium and Lactobacillus
148
was measured after probiotic dosing to colitic mice by recovering them from the
faeces. Low numbers of Bifidobacterium were detected which is indicative of
Bifidobacterium not surviving transit in the gastrointestinal tract. This may explain
why there was no protective effect seen when Bifidobacterium was given to colitic
mice. This result suggests that not all probiotic strains can protect the animals from
the wasting effects of TNBS colitis. Although a combination of probiotics has already
been shown to have a therapeutic effect in clinical and experimental IBD221, the mode
of action of each of the strains in the mixture has not been reported. As shown in this
chapter, each strain has a distinctive effect on the course of colon inflammation.
This is further demonstrated when the effects of L. acidophilus and L. fermentum on
cytokine production in the LPMCs (Figure 4.2) and on the microscopic examination
of tissue sections (Figure 4.3) were assessed. Both L. acidophilus and L. fermentum
were able to increase the level of anti-inflammatory IL-10 and simultaneously
decrease the concentration of pro-inflammatory IFN- , indicative that both
Lactobacillus strains can shift cytokine response of TNBS colitis from a type 1 helper
response to an anti-inflammatory profile. However, L. fermentum was able to produce
a ratio of IL-10:IFN- closer to that of a healthy-control animal than was obtained in
L. acidophilus dosed mice. This shows that some probiotic strains may promote the
production of pro-inflammatory cytokines while others may trigger the release of
immunosuppressive cytokines in inflammation. This difference may be useful in
determining which probiotic strains are to be administered in cell-mediated diseases
and in conditions where there is an overproduction of Th2 cytokines, such as parasitic
diseases and allergies. However, this strict separation as to what type of cytokines a
probiotic can trigger may be an over-simplification of the complexity of immune
homeostasis. IL-10 may have a regulatory role in reversing severity of intestinal
inflammation but the application of high doses of IL-10 stimulates IFN-
production118, thus, potentially worsening intestinal inflammation. A more desirable
outcome is to change the chronic, recurrent type of inflammation in colitis to a
“physiological” inflammation as seen in the normal intestinal environment which is
associated with an optimal ratio of IFN- production to IL-10 production.
This cytokine profile is consistent with the histological profile exhibited by L.
fermentum and L. acidophilus fed colitic groups. Mice fed L. fermentum showed less
149
colonic tissue damage as characterized by the less dense neutrophil infiltration and
more intact crypt architecture and the presence of more goblet cells as compared to
mice dosed with L. acidophilus which presented more severe pathology. This result
further confirms that production of large amounts of IFN- in the colon is associated
with physical injury of the colon in bowel inflammation and that IL-10 may prevent
tissue destruction. If integrity of the colon tissue is intact, goblet cells distributed
along the gastrointestinal tract produce large amounts of mucus that contain mucins
that can be utilized by bacteria as carbon and nitrogen sources. Mucus on the intact
epithelial surface is colonized by indigenous microbes, including Lactobacillus,
throughout the different regions of the gastrointestinal tract. Loss of goblet cells that
produce the mucus is observed in colon inflammation. Thus, the physical protection
from the mucus plus the probiotic effects from L. fermentum may account for the
protection afforded against colon inflammation observed in BALB/c mice.
A dose response study using L. fermentum was performed to determine the effect of
dose on the capacity of L. fermentum to ameliorate colon inflammation. L. fermentum
has previously been shown to adhere onto the gut wall efficiently and can colonise the
gastrointestinal tract of mice for over 24 hr and hence one can speculate that a lower
dose may be also effective. Other workers have shown an effect of a probiotic mix of
many strains, VSL#3, in experimental colitis using 1011 CFU per daily dose222 and a
daily dose of 1010 CFU using a single strain, Lactobacillus GG140 while a much lower
dose of 106 CFU of Bifidobacterium sp and L. plantarum 299v in the dextran sodium
sulphate model of colitis139, 222. In this chapter, the lower dose range, 107; 108 and 109
CFU doses, of L. fermentum fed every other day for 14 days (details in Section 4.2.3
and illustration in Figure 4.1) was trialed.
Oral doses of 107; 108 and 109 CFU of L. fermentum were able to differentially affect
Th1 and Th2 cytokine responses in TNBS-induced colitic mice. The shift in cytokine
responses was dose-dependent. The ratio of IL-4:IFN- was comparable in healthy-
control mice and mice given 107 and 108 CFU per dose. Interestingly, the dose of 109
CFU had a cytokine profile closer to the colitic-control group but this group still
exhibited a milder form of inflammation and lower number of mortality compared to
colitic-control group which indicates that 109 CFU dose of L. fermentum still
suppresses the effects of TNBS colitis. A possible explanation for the resistance of
150
this group is the increased production of IL-4 which counteracts the Th1 response
seen in TNBS colitis. However, this dose simultaneously produced high levels of
IFN- and the amount of anti-inflammatory IL-4 may not be enough to offer
maximum protection at 109 CFU. On the other hand, 107 CFU dose seemed to have an
equivalent effect as the 109 CFU dose on the course of inflammation of colitis
however considerable variation was observed in the amounts of IL-4 and IFN-
produced in the colon. Consequently, it is difficult to predict if the 107 CFU dose
could create the desired balance between Th1 and Th2 cells. Moreover, of the three
doses tested, the colitic group of animals exposed to 107 CFU L. fermentum had the
highest body weight loss which indicates less protection by this dose. L. fermentum
given at a dose of 108 CFU prevented colon inflammation as revealed by the greater
weight recovery at the peak of the disease in TNBS colitis, in addition, there were no
deaths reported in mice given 108 CFU per dose. Furthermore, the 108 CFU of L.
fermentum shows a preferential stimulation of IL-4, which brought the pro-
inflammatory and anti-inflammatory cytokine balance to a healthier profile (Figure
4.5). These results then indicate that the 109 CFU of L. fermentum dose is best in
preventing body weight loss associated with TNBS colitis while 108 CFU dose is best
in modulating cytokine balance closer to healthy profile and that the 107 CFU dose of
L. fermentum did not exhibit protection. The reason for these observations could be
that the in vivo effects of IL-4 is one of both anti-inflammatory (cell-mediated
diseases) and pro-inflammatory (allergic diseases) and the responses are dependent on
the local concentrations of anti-inflammatory cytokines, and types of antigens present
within the intestinal milieu and the activation state of the immune cells in the mucosa.
It is evident that physical injury of the colon is more pronounced and extensive in
colitic animals compared to L. fermentum treated animals. This destruction to the
integrity of the gut can also account for the increased permeability in intestinal
inflammation. This is evident with the greater incidence of translocation of Gram
negative, aerobic bacteria from the colonic lumen to the spleen of colitic animals
(Table 4.2) and that this is ameliorated by L. fermentum in parallel with the anti-
inflammatory effect. Interestingly, not all bacterial types examined were able to
translocate into spleen. Gram negative, enteric bacteria were detected in the spleens
but not anaerobic bacteria or LAB. It is speculated that anaerobes may not have been
detected because of low numbers, below the limits of detection. It could also be that
151
the oxidation-reduction potential of the gut environment during inflammation was
higher than normal and hence facilitated the survival and growth of the enteric
bacteria26. The enteric bacteria may have been the inducing factor that stimulated
cytokine production of the colonic epithelial cells as a response to their translocation,
thus resulting in the infiltration of neutrophils and pathogenic T lymphocytes that
further damage the integrity of the epithelium. Alternatively, the translocation
phenomenon may simply reflect the mucosal damage already in place and
minimization of this by the use of L. fermentum, thereby, reduces bacterial
translocation.
Feeding of the different doses of L. fermentum also resulted in the modification of the
colonic microflora, but the effect was not dose dependent. Concentrations of Gram
negative enteric bacteria and yeast type microorganisms were significantly increased
while a significant reduction in lactobacillus numbers was detected and total
anaerobes remained unchanged in the colonic contents of colitic mice. L. fermentum
modified this microbial profile comparable to healthy control microflora (Figure 4.6).
Although the antigen responsible for chronic colon inflammation is still unknown, the
microbial profile observed may harbour the antigen that activates the immune system.
In addition, the concentration of these microorganisms may be detrimental to an
immunocompromised host but not an immunocompetent host. It is a possibility that
one of the mechanisms employed by L. fermentum to reduce or prevent inflammatory
insult is by altering the microbiota and thereby modulating disease severity in TNBS
colitis.
Thereafter, 108 CFU per dose of L. fermentum was administered after the induction of
colitis. This mode of treatment enhanced production of IL-10 and downregulated IFN-
in the local sites of inflammation (Figure 4.8). This mechanism of IL-10 production
is inherently attractive as IL-10 is currently administered in human IBD via the
systemic route. This method of IL-10 administration presents a number of limitations:
it has to be given in high doses because systemic administration of IL-10 does not
allow for the efficient delivery of the cytokine in the intestinal mucosa; and the serum
half-life of IL-10 is only 1.1-2.6 hours. Moreover, high doses of IL-10 have a
tendency to stimulate production of IFN- . Administration of IL-10 via the oral route
will destroy the cytokine as it cannot withstand the acidity of the stomach. Steidler
152
and colleagues (2000) genetically engineered Lactococcus lactis as an alternative
method to deliver IL-10 to the local site of inflammation as well as lower the dose of
IL-10 required and reduce adverse effects. They showed this using the dextran sodium
sulphate and IL-10 knock-out mouse models of colitis. They allowed for colitis to
develop in the former model and colitis to develop spontaneously for 8 weeks in the
latter model prior to giving the IL-10 secreting L. lactis. They showed that there was
50% reduction in the severity of colitis in the dextran sodium sulphate model and L.
lactis prevented the onset of colitis in IL-10 mice. However, using genetically
modified organisms in a clinical setting raises safety and ethical concerns. Moreover,
L .lactis has to be re-engineered again as human IL-10 is different from murine IL-10.
L .lactis is still needed to be tested if it can withstand human bile and stomach acids.
The TNBS model mimics the inflammation observed in IBD by generating a T cell
profile biased to produce an inflammatory response. Cytokines from each T helper
cell subclass were quantified in this study. It was observed that colitic animals
expressed more IL-12223 and IFN- in their colons, consistent with previous
observations48, 166. IL-12 is a Th1 cytokine and it is known to be stimulated by
bacteria and bacterial products95, 96. IFN- is also a pro-inflammatory cytokine and its
production is influenced by the presence of IL-12 in the environment79, 96. Both
cytokines have an important pathogenic role in TNBS colitis as they promote the
infiltration of polymorphonuclear cells into the colonic tissues and potentiate the Th1
immune response. IL-4 and IL-10 are the two other cytokines which were assayed and
lie in the opposite spectrum of the Th1 and Th2 balance. The anti-inflammatory
cytokines, IL-4 and IL-10, were monitored. These cytokines function by inhibiting the
migration and growth of polymorphonuclear cells into the colonic tissue as well as
inducing tolerance to bacterial antigens indigenous to the host95, 114. It was shown in
this study that L. fermentum can activate the production of IL-4 and IL-10 in colitic
mice and simultaneously downregulate the production of IFN- , which was associated
with reduction of migration of inflammatory cells into the colonic mucosa. These
results suggest that L. fermentum suppresses TNBS colitis through the preferential
stimulation of anti-inflammatory and regulatory cytokines which inhibits expression
of immunostimulatory immune responses. Thus, L. fermentum can confer protection
against colon inflammation through its ability to modulate cytokine balance towards a
“healthier” state.
153
The TNBS model of colonic inflammation employed in this study results in a physical
and immunological injury to the colon. Administration of L. fermentum at a dose of
1x108 CFU every other day for 2 weeks was able to improve clinical features and
immune response of colitic animals. This study offers insight into the potential use of
L. fermentum in preventing colonic mucosal inflammation.
154
Chapter 5
Effects of Lactobacillus fermentum VRI 003 on the symptoms, cytokine immune
responses and faecal bacterial profile of UC patients in remission
5. 1. Introduction
Ulcerative Colitis (UC) is a chronic relapsing disease which involves an uncontrolled
immune response to luminal antigens. It affects the area of the gastrointestinal tract
with high bacterial population namely the colon and rectum. It has been suggested
that the intestinal microflora are involved in the pathogenesis of inflammatory bowel
diseases (IBD). The primary goal in the management of inflammatory bowel diseases
is to prevent relapse of the condition and thereby maintain state of remission.
Antibacterial agents have been used to control the symptoms of IBD and beneficial
effects have been noted119, 120. The use of antibiotics is limited since a microbial agent
responsible for IBD is yet to be identified and long term antibiotics usage every time
there is a flare-up can result in adverse events such as antibiotic associated diarrhea or
increased resistance of the microbes to the antimicrobial agent itself.
Aminosalicylates, immunosuppressants and corticosteroids have been recommended
but while they induce and/or maintain remission, their use is associated with side
effects 100, 101.
Since the indigenous microbes of the colon are implicated in UC, there is potential for
beneficial microbes, referred to as probiotics, to influence the colonic microbes and
thereby colon inflammation. The approach has been explored using animal models
(Chapter 4) and results are encouraging using single strains of lactobacilli and
mixtures of various strains. A probiotic mixture of bifidobacteria, lactobacilli and
streptococci has been shown to have therapeutic benefits in the prevention137 and
maintenance of remission135 in chronic pouchitis. Pouchitis is an ulcerative colitis-like
condition affecting the surgical neo-rectum in patients with UC who have undergone
total colectomy. Murine studies in Chapter 4 showed that a single strain of
Lactobacillus, namely L. fermentum VRI 003, could ameliorate colon inflammation
by changing the nature of the immune response and modulating the intestinal
155
microflora. The present study was undertaken to evaluate the effects of L. fermentum
VRI 003 on UC patients that were in remission and maintained on their usual
medications.
156
5. 2. Materials and Methods
5. 2. 1. Participants
Twelve patients with UC in remission were recruited during August to November
2003 from the outpatient clinic of the gastroenterology group of the Townsville
Hospital, Townsville, Queensland, Australia or by public advertising through the
Australian Crohn’s and Colitis Association (ACCA). Remission was confirmed
through the overall assessment of the attending gastroenterologist and the evaluation
of the clinical activity index (CAI) for UC (CAI 4), endoscopic index (EI) (EI
4)287 and no histological signs of acute inflammation. Participants were included in
the study if they have experienced remission for a duration of no longer than 12
months. They were excluded from the study if they had active UC, Crohn’s Disease
(CD), infectious colitis, chronic liver disease or significant co-morbidities such as
advanced neoplasia, severe allergic, renal or pulmonary disease, on-going antibiotic
usage, known intolerance to lactose or had consumed probiotics within the previous
six months prior to trial entry. All patients gave informed consent prior to
participation in the study.
5. 2. 2. Study Design
This was a randomized, double-blinded, placebo-controlled, cross-over study that
assessed the activity of L. fermentum VRI 003 in UC patients in remission whilst
given together with their standard medication over 12 months.
Sample size was calculated using the power calculation program from
hedwig.mgh.harvard.edu. A total of 39 patients were needed for a probability of 99%
that the study will detect a treatment difference at a two-sided 5% significance level,
if the true difference between the two was 5 units. This was based on the assumption
that the within-patient standard deviation of the response variable was 5.
The patients were randomized in a double-blind, cross-over manner to receive either
6 months of placebo followed by 6 months of active treatment (group A) or 6 months
of active treatment followed by 6 months of placebo (group B). Randomization was
157
developed and maintained by the study pharmacist (HR) with all investigators and
patients remaining blinded to the details. Participants were allotted an identification
number and dispensed the first month’s supply of the investigational treatment after
baseline information was obtained. Baseline assessment was done on Day 0 whereby
participant’s demographic characteristics, medical history and medications were
recorded.
Participants took either the active gelatin capsules that contained a minimum of
1.5x109 colony forming units (CFU) per capsule of freeze-dried L. fermentum VRI
300 (active treatment) mixed with microcrystalline cellulose or the placebo gelatin
capsules containing microcrystalline cellulose. Each participant was asked to take two
capsules in the morning and two capsules in the evening with food each day for 6
months. The UC participants continued treatment with their standard medication
during the study period. All patients of both groups were instructed not to take any
probiotics during the study period. Investigational capsules were placed in medication
bottles which were dispensed every month to the participants. These used bottles were
then collected back the following month.
Participants filled out a daily questionnaire for 12 months to ensure consistent
reporting. They were also required to visit the attending gastroenterologist at the end
of every month to assess their clinical status and standard therapy. At the end of each
month, the questionnaires and medication bottles were taken by a research nurse for
assessment of compliance. Serum and faecal samples were collected at 0, 3, 6, 9 and
12 months of the study period for cytokine and microbiological analyses, respectively.
Cytokine and microbiological analyses were performed in the Probiotics Lab, School
of Biomolecular Sciences, University of New South Wales, Sydney, Australia. Figure
5.1 illustrates the study design and the parameters assessed at each scheduled visit.
Dr Stephen Fairley from Townsville, Queensland was the recruiting clinician for this
study. Thus, the study protocol was submitted for evaluation to and subsequently was
approved by The Townsville Health Service District Institutional Ethics Committee
(14/03).
158
5. 2. 3. Evaluation of symptom scores
The primary endpoint was considered to be a reduction in the incidence and severity
of exacerbation of the symptoms, namely frequency of bowel movements, abdominal
pain and general well being, as measured using a visual analogue scale. It is
anticipated that because of the nature of the disease, symptoms will worsen, even
when patients are maintained on their standard medications. Severity of each
condition was measured on a scale of 1-10 (1:lowest, 10:highest). Other parameters
that were monitored using the patient’s daily questionnaire include change in
medication, appearance of blood or mucus in the stools, occurrence of an adverse
event and other relevant events (such as hospital admission). Participants were asked
to fill out the questionnaire daily for the whole duration of the study period. In the
questionnaire, the participants were asked whether, in comparison with the previous
month, there had been any changes in the frequency of bowel motions or abdominal
pain, or if there was blood or mucus in the stools. They were asked to indicate
whether they evaluated these symptoms during the last intervention period to be
generally better, unchanged or worse. It was considered an exacerbation if the
frequency of bowel actions increased, or if abdominal pain or general well-being
worsened.
5. 2. 4. Measurement of cytokine levels in the serum
Serum was collected from each participant prior to commencement of treatment (0
month) and then at 3, 6, 9 and 12 months of the study period. The serum sample was
collected in a serum tube and was kept frozen in -80°C until transported to the
Probiotics Lab for cytokine analysis. The serum samples were kept in dry ice during
transport. The serum was thawed and aliquots were transferred in microfuge tubes
(Eppendorf) and were kept at -80ºC until they were assayed.
Cytokine levels were assayed using IFN- and IL-10 (Pharmingen) antibodies and the
ELISA methodology. One hundred microlitres (100 µl) of serum was added to a well
of a microtitre plate (Nunc) coated with 1 µg.ml-1 antibodies to IFN- or IL-10. After
incubation for 90 mins at room temperature, the microtitre plate were washed three
times with Phosphate Buffer Saline containing Tween 80 (Sigma) (PBS/T) and
159
blocked with 200 µl of 3% Bovine Serum Albumin (BSA; Sigma) in PBS/T for 60
mins at room temperature. After this, the plates were then washed again three times
with PBS/T and 100 µl of 0.5 µg.ml-1 biotinylated antibody to IFN- or IL-10 was
added. Streptavidin HRP (Chemicon) was diluted 1/ 2000 in 1% BSA-PBS/T and
allowed to incubate at room temperature for another 30 minutes The plates were
washed again 3X with PBS/T until the addition of the substrate 3,3’,5,5’-tetramethyl
benzidine (TMB; Sigma) and hydrogen peroxide into the wells. Once substrate and
hydrogen peroxide were added, colour reaction was allowed to develop at room
temperature for 10-20 minutes. The reaction was stopped using 1M H2SO4 and the
plates were read at 450 nm using BIORAD Microplate Manager Reader. A cytokine
standard (Pharmingen) for IFN- or IL-10 was included on each plate to allow
quantitation of the concentration of IFN- or IL-10 in the serum.
5. 2. 5. Enumeration of faecal bacteria from UC patients
Faecal samples were collected immediately following recruitment (0 month), 3, 6, 9
and 12 months of the study period. Participants were asked to collect stool into a
sample container and to then tightly seal this faecal container. The faecal samples
were stored at -50ºC until transported in dry ice to the Probotics Lab. These were then
kept in -50ºC until analysis.
The faecal samples were processed by weighing 1 g of the sample in a pre-weighed
50 ml falcon tube (Sarsdet) and mixing it with 9 mls in half-strength Wilkin-Chalgren
broth (1/2 WCB; Oxoid) prepared by diluting WCB in distilled water. The tube was
then mixed for 2 mins using a tube mixer at the high speed setting or until the faecal
sample formed a homogeneous mixture. An aliquot (1 ml) from the first dilution was
heat treated at 100ºC for 10 minutes for the enumeration of endospores. After this,
ten-fold serial dilutions were performed in half strength WCB. Ten microlitres (10 µl)
was taken from each dilution step and drop plated in triplicate onto MacConkey Agar
CM7 (MAC; Oxoid) for the cultivation of total enterics, Rogosa Agar (ROG; Oxoid)
for culture of total lactobacilli, and Reinforced Clostridia Agar (RCA; Oxoid) for
endospores. MAC plates were incubated aerobically at 37ºC for 24 hours while ROG,
WCA and RCA plates were incubated in an anaerobic chamber at 37ºC for 48 hours.
The colonies that grew were expressed and quantified as colony forming units per
160
gram of faecal sample (CFU.g-1). The 3rd and 6th month bacterial counts were
averaged and compared between the two treatment groups.
5. 2. 6. Statistical analyses.
All data were audited prior to their analysis using SPSS version 11.5 for Windows.
Descriptive statistics were used to summarise baseline characteristics and Chi-square
to evaluate significance of categorical information. Linear regression was performed
on each patient’s visual analogue score for each parameter to establish the trend of the
effect of the treatment and to get the “line of best fit” on the parameter. The slope of
each line for each parameter and treatment were then compared. A test for normality
was employed to determine distribution of data points and the statistical tests to be
used. Analysis of the effect of treatment was carried out by looking at the differences
for each period. Independent t-tests and paired sample t-tests were used for normally
distributed data while Mann-Whitney U test and Wilcoxon Signed Rank test were
performed for non-parametric data. Log rank statistical analysis was used to
determine the cumulative exacerbation rates of bowel motions. Results were reported
as two tailed p-values and assumed statistical significance if p<0.05.
161
5. 3. Results
5. 3. 1. Study population characteristics and progress in the study
There was a slow recruitment rate experienced in the beginning of the study and
because of the time constraints on this PhD project, only data from 12 participants
who were able to complete the clinical course one year after the August-December
2003 recruitment are presented in this chapter. This entire study is still in progress and
an additional 28 participants have since been recruited. It is anticipated that this study
will be completed at the end of the first half of 2005.
Table 5.1 shows the baseline demographic characteristics of study participants. A
greater proportion of UC patients that participated in this study are from the male
population. More than half of the participants have been afflicted with colitis for more
than 5 years and have been suffering for a range of 10 to 35 years. These long-
suffering UC patients are the ones who have the longest smoking history among the
participants. It has been reported that smoking is associated with a higher likelihood
of developing late-onset UC224. Whereas, other studies showed that smoking can
decrease risk of developing colon inflammation and could have favourable effect on
disease progression244. This study, however, lacks sufficient numbers or a UC-
negative control group to look at the influence of smoking. Eighty three percent
(83%) of the participants do not have a family history of inflammatory bowel
diseases. Most of the UC patients recruited in the study are from a Caucasian
background (p<0.01) which is consistent with the demographics of the Townsville
area. However, it may not be a representative of all other centres in Australia. The
prescribed medications to the 12 participants were corticosteroids, aminosalicylates or
a combined therapy of corticosteroids and immunosuppressant or aminosalicylates
plus the immunosuppressant. Half (50%) of the study population was on
aminosalicylates and immune system suppressors during the study period.
All 12 participants completed the study. Protocol violations were assessed before
breaking the randomization code. Deviations from the protocol include: usage of
antibiotics during the study period (1 patient from Group A and 2 patients from Group
B), consumption of a probiotic product during the 5th month of the active treatment
162
and was continued until the 3rd month of the placebo arm of the study course (1
patient in Group B); a week of not taking the investigational medicines due to failure
of their delivery to trial site on time (all patients from both groups). These deviations
were accepted and data from all patients were included in the data assessment. A
detailed description of the progress and compliance of each participant is presented in
the case report (Appendix).
163
Table 5.1. Baseline characteristics of participants (n=12). Values are expressed as frequencies, except for age.
Characteristics Group A
(placebo then
active)
N=6
Group B
(active then placebo)
N=6
p-value
Age (mean age in years) 53.6 46.7 NS
Gender
Male 3 6
Female 3 0
NS
Duration of UC
5 years 2 2
> 5 years 4 4
NS
Smoking History
yes 4 4
no 2 2
NS
Family History
yes 1 1
no 5 5
NS
Ethnicity
Caucasian 5 6
Others 1 0
0.001
Medications
Aminosalicylates 2 0
Cortecosteroids 0 1
Aminosalicylates and
Immunosuppressant
3 2
Aminosalicylates and
Immunosuppressant
1 3
NS
NS = no significant difference
164
5. 3. 2. Participants’ subjective evaluations of their symptoms
Patients scored their symptoms using a visual analogue scale. Figure 5.2 reveals a
more severe bowel action score in 9/12 (75%) of UC patients when administered the
placebo treatment resulting in a significant difference between the two treatments
(p=0.001). L. fermentum VRI 003 use was associated with a lower frequency of bowel
movements in 50% of the 12 UC participants, while, 25% of the patients maintained
their bowel habits and 25% experienced an increase in bowel motions during the
active treatment. A comparison of the time for symptoms to exacerbate in L.
fermentum VRI 003 and placebo treatments was then assessed by Kaplan-Meier
curves (Figure 5.3a & b) as both treatments were able to worsen bowel frequency in
UC. The median time to increase the frequency of bowel actions when placebo
treatment was administered was 2 months while it was 4 months when L. fermentum
VRI 003 was given. Furthermore, Log rank statistical analysis shows that L.
fermentum VRI 003 significantly reduced the frequency of bowel movements in
comparison with the placebo treatment at p=0.0037. Moreover, blood or mucus
appeared together with the bowel motions in 5 out of 12 participants when
administered the placebo treatment but it was seen in only 2 participants on L.
fermentum VRI 003 treatment.
Six patients (50%) felt that their abdominal pain subsided during L. fermentum VRI
003 treatment and 7 out of 12 patients (58.3%) reported the pain to be more
pronounced during placebo treatment (p=0.004) (Figure 5.1). As further revealed in
Figure 5.2, 50% of the participants that received L. fermentum VRI 003 and 33.3% of
the participants administered the placebo scored that there was no change in the
intensity of their abdominal pain. However, no significant difference (p=0.814) was
observed between L. fermentum VRI 003 and placebo treatment in reducing the
severity of abdominal pain (Figure 5.4) although the mean value was lower during
active treatment.
As rated by the participants (Figure 5.2), L. fermentum VRI 003 was significantly
better than placebo (p-value=0.007) in improving and maintaining the general well
being of UC patients in remission when compared to the placebo treatment. None of
12
Diet also has a major influence on the composition of the microflora and the ability of
the host to resist infection. Infants that are breast fed have predominantly high
bifidobacteria counts with low numbers of enteric bacteria, clostridia and Bacteroides
in their faeces, while infants that are formula fed have higher levels of enterics,
clostridia and Bacteroides. This difference in the microbial profile of breast and
formula fed infants is significant as the latter are more susceptible to pediatric
infections and allergies28. It should also be noted that the relationship between
formula feeding and level of enterics, Clostridia and Bacteroides may not necessarily
be causative but may reflect the array of anti-infectious factor profile in the breast
milk compared with formula. Breast milk contains maternal immunocompetent cells,
immunoglobulins, immune reactive peptides, anti-infectious oligosaccharides, growth
factors, cytokines, lysozyme, lactoferrin and complement components236, 237 which
may be involved in the selection of the intestinal microflora by providing protection
against microorganisms coming from the maternal environment and bringing in
microbes needed in the development of the immune system238-242. On the other hand,
a diet with a high fat to protein ratio is claimed to support the growth and metabolic
activities of putrefactive gut microorganisms that generates carcinogens in the gut that
may be responsible for the development of colon cancer29. On the other hand,
consumption of a high fibre diet leads to an increase in higher level of SCFA
production which is detrimental to many enteric pathogens26, 30-32.
Numerous metabolic activities of the indigenous gut microflora benefit the host. It has
been demonstrated that the combined activities of the microbes and the host can
eliminate or minimize toxic effects of substances that are potential carcinogens. Rat
studies have shown that administration of L. casei, L. bulgaricus, Streptococcus
thermophilus and some Bifidobacterium strains were able to reduce DNA and cell
damage in gastric and colonic mucosa cells33. Indigenous lactic acid bacteria may also
effectively assimilate cholesterol from the system and precipitate bile acids to prevent
their transformation to secondary toxic forms34, 35.
The intestinal microflora contributes immunostimulation which protects the host.
Exposure to the microflora initiates the development of the immune system, the
induction of tolerance to themselves as well as the establishment of the intestinal
ecosystem. Secretory IgA antibodies are responsible for the immunoexclusion of
165
the participants reported that their general well being improved while on placebo
treatment. Comparing the slopes of the daily general well being scores during L.
fermentum VRI 003 and placebo treatment, it can be seen that L. fermentum VRI 003
statistically improved (p-value=0.014) the general well being of UC patients when
taken together with standard therapy (Figure 5.5).
167
0
20
40
60
80
100
120
Active Placebo Active Placebo Active Placebo
Num
ber
of P
atie
nts (
%)
Bowel Actions Abdominal Pain General Well Being
25 %
50 %
25 %
75 %
50 %
50 %
8.3 %
58.3 %
33.3 % 50 %
50 % 41.7 %
58.3 %
25 %
Sustained relief of symptoms Increased severity of symptoms Reduction of severity of symptoms
p=0.001 p=0.004 p=0.007
Figure 5.2. Proportion of UC patients that experienced an increase in symptoms, sustained relief of symptoms and experienced reduction of symptoms during active treatment with L. fermentum or placebo treatment (n=12). P value refers to the difference between the incidence of the symptoms in 12 patients between the active and placebo treatments.
168
Monthly Evaluation of Placebo Patient1 2 3 4 5 6
1 2 3 4 5 6 7 8 9
10 11 12
Monthly Evaluation of Active Patient
1 2 3 4 5 6 1 2 3 4 5 6 7 8 9
10 11 12
Figure 5.3a. Frequency of exacerbation of bowel actions in individual patients of the placebo treatment and L. fermentum treatment. Number of bowel actions was evaluated by the patient. A shaded box represents an increase in bowel actions compared to the previous month.
169
Time (months)
76543210-1
Cum
ulat
ive
Exac
erba
tion
Rat
e
1.2
1.0
.8
.6
.4
.2
0.0
-.2
Figure 5.3b. Cumulative exacerbation rates in the two groups (log rank statistical analysis, p=0.0037). At each monthly appointment of patient, frequency of bowel motions were evaluated in comparison with one month earlier. Hatched lines show the month of exacerbation of diarrhea in UC patients in remission.
PlaceboTreatment
L. fermentum Treatment
170
GROUP
ACTIVEPLACEBO
ABD
OM
INAL
PAI
N
.02
.01
0.00
-.01
-.02
10
Figure 5.4. Influence of L. fermentum and placebo treatment on the severity score of the abdominal pain symptoms in 12 UC patients in remission.
Results expressed as the mean slope of daily scores for each treatment dosed over 6 months. Circles with numbers are outliers detected for each treatment.
171
1212N =
ACTIVEPLACEBO
GEN
ERAL
WEL
L BE
ING
.01
0.00
-.01
-.02
-.03
23
4
Figure 5.5. Influence of L. fermentum and placebo treatment on the general well being score of 12 UC patients in remission (n=12). L. fermentum significantly improved general well being at p=0.014.
Results are expressed as the mean slope of the plot of daily scores for each treatment dosed over 6 months. Circles with numbers were outliers detected for each treatment while the asterix indicated an extreme point that extended more than 3 box lengths from the edge of the box.
172
5. 3. 3. Effects of L. fermentum VRI 003 on serum cytokine production of UC
patients in remission
No difference was demonstrated between the serum IL-10 levels of patients in the
active and placebo treatments (Figure 5.6). On the other hand, comparable levels of
IFN- were detected in the serum of both treatments (Figure 5.7).
Consequently, results were grouped according to the classification of the medicines
the patients were taking as it is recognized that these therapies have different mode of
actions on the immune system, and this may influence the effects of the probiotic
treatment. Medicines that were used include aminosalicylates only (n=2),
corticosteroids only (n=1), aminosalicylates and immunosuppressants (n=6) and
corticosteroids and immunosuppressants (n=3). However, while different treatments
(corticosteroids vs. salicylates vs. immunosuppressants) might have different effects
on cytokine levels, the numbers of patients in each therapeutic group are too small to
perform meaningful comparisons.
173
0
100
200
300
400
500
600
0 3 6
Time (Months)
IL-1
0 Se
rum
Con
cent
ratio
n (p
g.m
l-1)
PlaceboActive
Figure 5.6. Serum IL-10 concentration of UC patients in remission prior to and after 3 and 6 months dosage of L. fermentum and placebo treatments taken concurrently with standard medication. Results are expressed as mean concentration pg.ml-1 of IL-10 ± SD.
174
0
100
200
300
400
500
600
0 3 6
Time (Months)
IFN
-y S
erum
Con
cent
ratio
n(p
g.m
l-1)
PlaceboActive
Figure 5.7. Serum IFN- concentration of UC patients in remission prior to and after 3 and 6 months dosage of L. fermentum and placebo treatments taken concurrently with standard medication. Results are expressed as mean concentration pg.ml-1 of IFN- ± SD.
175
5. 3. 4. Bacterial analysis of faeces of UC patients in remission prior to and after
treatment with L. fermentum VRI 003
Although slightly higher levels of total enterics were detected at baseline and after
placebo treatment compared to L. fermentum VRI 003 treatment, no differences were
seen between the two treatment groups (Figure 5.8). However, the relative incidence
of the different types of enteric bacteria detected on the selective medium (MAC) for
total enterics differed significantly (p<0.05) between the active and placebo
treatments (Figure 5.9). Enterococci were detected in 58.3% of UC participants during
the active treatment whilst only 33.3% of faecal samples from placebo treatment had
detectable enterococci. The prevalence of coliforms was higher (50%) during the
placebo treatment as compared to L. fermentum VRI 003 treatment with coliforms
being detected in 33.3% of the faecal samples. Non-lactose fermenters were only
detected in 1 patient during the active treatment and were not detected during placebo
treatment. Detection limit was at 100 per gram.
Higher lactobacillus counts were detected after dosage of placebo and L. fermentum
VRI 003 (Figure 5.10) as compared to the baseline counts of faeces from UC patients
in remission. However, the increased faecal lactobacilli counts observed during the
placebo and active treatments did not reach statistical significance. In addition,
endospores were only detected at a mean count of 3.5 log CFU in 6 out of 12
participants when placebo treatment was administered and were not detected during L.
fermentum VRI 003 treatment (Table 5.2).
176
Total Enterics
0
1
2
3
4
5
6
7
Baseline Placebo Active
Log
CFU
.g-1
of fa
eces
Figure 5.8. Levels of total enterics detected in the faeces of UC patients in remission during placebo and L. fermentum treatments. Results are expressed as mean log CFU per gram of faeces ± SD (n=12).
177
0
20
40
60
80
Placebo Active
Prop
ortio
n of
Pat
ient
s (%
)
Enterococci Coliforms Non-lactose fermenters
p=0.034p=0.021
Figure 5.9. Frequency of detection of the various types of enteric bacteria detected in the faeces of UC patients in remission (n=12).
Enterococci were significantly detected in more patients (p<0.05) than coliforms and non-lactose fermenters during L. fermentum VRI 003 treatment while incidence of coliforms were higher compared to other enteric bacteria types during placebo treatment ( 0.05).
178
Lactobacilli
0
1
2
3
4
5
6
7
8
Baseline Placebo Active
Log
CFU
.g-1
of f
aece
sp>0.05
Figure 5.10. Levels of total lactobacilli detected in the faeces of UC patients in remission during placebo and L. fermentum treatments. Results are expressed as mean log CFU per gram of faeces ± SD (n=12).
179
Table 5.2. Concentrations (CFU g-1) of endospores detected in the faeces of UC patients in remission during placebo and L. fermentum treatment. Results are expressed as mean concentration ± SD.
Treatment Endospores(Log CFU.g-1 of faeces)
Number of positive patients (n)
Placebo 3.45 ± 0.21 6/12
L. fermentum VRI 003 ND 0/12
ND = not detected at lowest dilution used at 10-1
180
5. 4. Discussion
Therapies developed for inflammatory bowel diseases target control of inflammation,
nutritional deficiencies and symptoms including diarrhea, rectal bleeding and
abdominal pain100-102. Aminosalicylates are given singly or in combination with
antibiotics or steroids to successfully ameliorate or lessen the disease activities of UC.
However, current therapies are not without adverse side effects. Patients with UC
intermittently experience an increase in the frequency of bowel movements, and of
bleeding despite the long-term continuous use of their standard medication100-102.
These may be associated with alterations in the profile of the colon microflora
because of the on-going use of antibiotics, steroids and/or immuno-therapies9, 74.
Alternatively, exacerbations of disease may be a direct result of, or result in
alterations in bacterial profile. Consequently, probiotic dosage may manipulate the
microflora and thereby contribute to a relief of symptoms.
The study was designed to evaluate the effects of oral administration of L. fermentum
VRI 003 on four symptoms: diarrhoea, abdominal pain and general well being; in UC
patients who were in remission when dosed with L. fermentum VRI 003. A cross-over
study was used to minimize any between-subject variation. UC patients in remission
were recruited in the study. L. fermentum VRI 003 or placebo was administered in
addition to the patient’s standard medication during the study course which ran for a
total of 12 months. Symptoms were recorded by the subjects using a visual analogue
scale for frequency of bowel movements, severity of abdominal pain and general well
being. The L. fermentum VRI 003 dosage significantly reduced the frequency of
bowel movements (p=0.001) and abdominal pain (p=0.004), and improved the general
well being score (p=0.007) of more UC patients (Figure 5.2). Moreover, the
exacerbation of diarrhea took a longer period of time to occur again with
administration of L. fermentum VRI 003 (Figure 5.3.a-b) and bleeding was observed
in fewer participants dosed with L. fermentum VRI 003 compared to placebo. These
findings suggest that L. fermentum VRI 003 exerts beneficial effects on some of the
major symptoms associated with UC.
The 12 participants in this study used three classes of medicines for their colon
inflammation: aminosalicylates, corticosteroids and immunosuppressive agents.
181
Aminosalicylates, such as mesalazine and sulfasalazine, are recommended for use as
maintenance therapy. The mode of anti-inflammatory action is its concerted effects on
leukotrienes, cytokines and oxygen radicals138. Corticosteroids are hormones
produced naturally by adrenal glands which have anti-inflammatory actions100-102.
Prednisolone is the commonly used synthetic corticosteroid and is found to be most
effective in active disease100-102. Corticosteroids are given in large doses initially but
can then be tapered to a low maintenance dose or withdrawn completely when the
condition is brought under control. Although this class of drugs induces immediate
suppression of inflammation, corticosteroids can cause serious side effects, including
susceptibility to microbial infection as these agents reduce the normal response of the
immune system to microorganisms100-102. Immunosuppressive agents such as
azathioprine are usually given together with aminosalicylates or corticosteroids to
enhance their efficacy in reducing inflammation. Azathioprine exerts its anti-
inflammatory action by interfering with the production of sensitized B and T cell
clones, inhibiting the production of blood-forming cells in the bone marrow as well as
inhibiting the production of white blood cells100-102.
All of these medicines down-regulate the overly active immune response in intestinal
inflammation but have different mechanisms of action and as such may have different
effects when combined with L. fermentum VRI 003. Therefore, the results of cytokine
profile were examined across all subjects and divided according to the class of
medication used. No differences were seen in the cytokine profiles of patients during
placebo or active treatments, however, the low numbers could have contributed to this
effect and analysis of the full data at the end of the clinical study will provide more
information on this important variable. The cytokine endpoints have not helped in
understanding the mechanism. However, it is speculated that since dosage of
probiotics gave beneficial effects, this may give an indirect effect of the probiotics on
the indigenous microflora of inflamed intestine rather than a direct effect on the
immune system.
Microbiological examination of the faeces was also performed to determine the
microbial profile during remission in UC as previous reports have suggested that
exacerbation of symptoms in inflammatory bowel disease may be associated with
microbial infection225. Results showed that levels of total enteric bacteria were similar
182
in both treatment groups and similar to the counts obtained prior to the start of the
study (Figure 5.8). However, different types of enteric bacteria were detected when
placebo or L. fermentum VRI 003 was administered. UC patients given the former
treatment harboured more of the coliform type of enterics while entercocci were
detected in more patients during oral dosage of L. fermentum VRI 003 (Figure 5.9).
Coliforms such as E. coli and Pseudomonas are potentially pathogenic enteric
microorganisms when present in high numbers and thses species may be associated
with intestinal inflammation55, 74. Enteroccocci are lactic acid bacteria which are
potentially beneficial microorganisms226 but the E. faecalis strain has been detected in
mucosa of CD patients227 and was shown to induce inflammation in mice54.
Lactobacilli (Figure 5.10) and endospore levels (Table 5.2) varied as a result of L.
fermentum VRI 003 dosage. Higher levels of faecal lactobacilli were detected during
placebo and L. fermentum VRI 003 treatments compared to baseline levels. The
higher levels of lactobacilli in the active treatment may indicate the survival of orally
administered lactobacilli, however, there the increase in the placebo treatment cannot
be explained. The cellulose component of the placebo formulation may be acting as a
prebiotic which can enhance the growth and activities of colonic microorganisms,
however, the levels used were considerably less than used for other prebiotics.
Endospores were detected during placebo treatment but not when the UC patients
were dosed with the L. fermentum VRI 003. It has been shown previously that spore-
formers, especially the toxin A-producing Clostridium difficile strain, are the most
prevalent microbes detected in the faeces of inflammatory bowel disease patients that
were experiencing a relapse225, 228, 229. Consequently, the beneficial effects on the
frequency of bowel movements noted in Figures 5.2 - 5.3 could be linked to the lower
levels of endospores during L. fermentum dosage.
This is the first study to use the probiotic strain L. fermentum VRI 003 in combination
with the patient’s standard medication in UC. Most of the studies on probiotic usage
in UC have utilized the parallel design rather than a cross-over design as used here
and have compared therapeutic efficacy of the probiotics with mesalazine. The E coli
Nissle 1917 was shown to have beneficial effects in UC133, 134. The group of Kruis
(2004) conducted a longer, homeogenous, multicentre study recruiting only patients
183
whose UC was in remission. This study demonstrated the efficacy of E coli Nissle
1917 in maintaining remission in colitis patients and at the same time it was able to
show that these effects were equivalent to mesalazine. Although an extensive safety
data are available on this strain, the use of E coli Nissle 1917 as a probiotic in
inflammatory bowel diseases needs further investigation. This is because of the role
the enteric bacteria, such as E coli can play in the pathogenesis of bowel
inflammation230.
Lactic acid bacteria strains that might be used in inflammatory bowel diseases include
Lactobacillus and Bifidobacterium. Lactobacillus GG has been tested against clinical
Crohn’s disease (CD). It failed to maintain remission in moderate to active CD cases
when used as a maintenance therapy in surgical-induced231 and antibiotic-induced232
remission. Daily administration of milk supplemented with 1x1010 cfu of
Bifidobacterium breve, B. bifidum and L. acidophilus YIT 0168 (n=11) for 12 months
to patients with mild to moderate UC resulted in a reduction in the severity of
symptoms associated with colitis and prevented relapse in this group of patients
compared to a parallel placebo control group of 10 subjects233. Oral administration of
another combination which consisted of eight different lactic acid bacteria, VSL# 3,
lead to both prophylactic137 and therapeutic benefits in chronic pouchitis following
colectomy and ileo-anal anastomosis135.
The current study demonstrates the benefits of administration of a single strain of
lactic acid bacterium on symptoms and well being of UC patients in remission. In this
study, the participants received a total daily dose of 6 x109 CFU of L. fermentum VRI
003 per day. Other workers have used up to 1010 CFU per day140 and it is interesting
to speculate whether a lower dose of L. fermentum VRI 003 could result to enhanced
benefits.
From the faecal microbial profiles one can postulate that the beneficial effects may be
associated with a decrease of endospores and coliforms. Laboratory studies have
shown that L. fermentum VRI 003 can inhibit growth of spore-forming and enteric
bacteria234 and decrease translocation across the intestinal the intestinal mucosa235.
Consequently these characteristics of L. fermentum VRI 003 could contribute to the
observed outcomes in UC patients during L. fermentum VRI 003 dosage. It is
184
encouraging that side effects associated with L. fermentum VRI 003 were minimal,
however, it is acknowledged that the power of this study was not sufficient to detect
significant side effects. L. fermentum VRI 003 may in the future be considered as an
appropriate routine maintenance therapy in colon inflammation.
The design of the study has several limitations. First, there was no wash-out period
which means that the group that received active first followed by the L. fermentum
VRI 003 may have been affected by probiotic treatment. However, the study shows
that placebo treatment given before or after the probiotic treatment had no major
impact noted at the end of 6 months. (Appendix II). A second limitation of the study
was its small population size. Thirdly, the L. fermentum VRI 003 total daily dose used
in this study, 4 capsules with 1.5x109 CFU per capsule, was arbitrarily chosen. This
may be lower than needed as other clinical studies showed efficacy at a total daily
dose of 1010 CFU of single strain probiotic bacterium. Results from Chapter 4 indicate
abrogation of murine colitis using a total daily dose range of 1x108 to 2x109 CFU of
L. fermentum VRI 003. However, caution is needed before extrapolating the effective
dose observed in mice to be used in humans as the mice received a much higher dose
in relation to their weight as compared to the dose received by the participants in this
study. Therefore, subsequent dose response studies in clinical inflammatory bowel
diseases are needed. Finally, the clinical benefits of L. fermentum VRI 003 on
symptoms associated with UC symptoms needs to be supported by endoscopic and
histological findings, before comments on the effects of L. fermentum VRI 003 on
relapse and remission of the condition can be made.
In conclusion, L. fermentum VRI 003 administered at a total daily dose of 6x109 CFU
resulted in beneficial effects on symptoms and well being of UC patients in remission
and is consistent with the findings in the murine model of colon inflammation in
Chapter 4.
185
Chapter 6
Concluding Discussion
The work presented in this thesis examined the effects of probiotics and prebiotics on
colitis. The mucosal cytokine response, colonic microbial profiles and symptoms in
both murine and human colon inflammation were assessed. Studies included
supplementing the diet with high amylose maize resistant starch as a prebiotic
(Chapter 3) and orally dosing probiotics, Bifidobacterium and Lactobacillus strains
(Chapter 4), to BALB/c mice with experimentally induced colitis. Intrarectal injection
of trinitrobenzene sulfonic acid (TNBS) was used to induce colitis. Furthermore, a
randomized, double-blinded, cross-over study was performed in UC patients whose
colitis was in remission in order to monitor the impact of dosing L. fermentum VRI
003 when administered together with their routine therapy.
Previous studies have provided evidence that the indigenous microorganisms are
central to the development of bowel inflammation because genetically susceptible
hosts are found to be immunoreactive to their own commensal bacterial flora and
because the potentially aggressive types of bacteria are found in high levels while
those putative beneficial bacteria are detected in low numbers in the inflamed gut.
Antibiotics can be, and are still, used to inhibit the activities of these microorganisms.
However, the antigen(s) that drives the chronic cell-mediated immune response in
intestinal inflammation is (are) yet to be identified and continued usage of antibiotics
to target an unidentified bacterial antigen may result in an increased risk that resistant
strains of microorganisms will emerge in the gut. Consequently, it was proposed that
the gut microflora in colon inflammation can be manipulated by using prebiotics and
probiotics.
The initial work in this study focused on optimizing the TNBS murine model of colon
inflammation in BALB/c mice (Chapter 2). A dose response study was performed to
determine the dose of ethanol and TNBS that would produce colitis in the BALB/c
strain of mice. Ethanol was used as vehicle for TNBS in this experiment. Its role is to
facilitate the entry of TNBS into the colonic lumen, thereby, mediating the breakdown
186
of the epithelial barrier leading to mucosal injury. It was found in this experiment that
the concentration of ethanol influences the mortality rate of colitic animals. It was
also demonstrated in this chapter that a single intrarectal administration of 2.5 mg
TNBS in 45% ethanol was sufficient to induce colitis in BALB/c mice.
Administration of 2.5 mg TNBS in 45% ethanol generated an acute type of
inflammation with a T cell response skewed towards a Th1 response and which was
associated with a reduction in Lactobacillus counts and a concurrent increase of
enteric bacteria. Chapter 2 provided useful insights into the pathogenesis of colon
inflammation and resulted in the establishment of a model for evaluating the effects of
prebiotics and probiotics on colitis.
From the studies in Chapter 3, it was shown that resolution of colonic inflammation
by high amylose maize resistant starch is affected by the variety and the concentration
of starch supplemented in the diet of mice. A 5% concentration, but not 30% of high
amylose maize resistant starch included in the diet delayed progression of TNBS
colitis as evidenced by lower weight loss and histological scores in animals from the
5% starch dosed group. This improvement in the disease activities of colitic mice with
the 5% high amylose maize resistant starch diet was associated with an abrogation of
the expression and synthesis of the pro-inflammatory cytokine, IFN- , and
upregulation of anti-inflammatory and T regulatory cytokines, IL-4 and IL-10,
respectively. The exacerbation of the inflammatory response in the 30% high amylose
maize starch concentration may be attributed to resistant starch being a bulking agent.
This could result to higher production of substrate that may promote bacterial growth.
As the mucosa is already compromised in colon inflammation, bacteria and their
products could possibly traverse easily into mucosa milieu and thus, may initiate and
perpetuate an aggressive immune response.
Moreover, 30% unmodified high amylose maize resistant starch diet increased levels
of spore-formers (Clostridium species) and potentially pathogenic Gram negative
aerobic bacteria in colitic mice as determined by culture method. Furthermore
Bacteriodetes was detected only in the colonic contents of colitic mice as determined
by DGGE analysis. The potentiation of a pro-inflammatory response seen in colitic
mice fed the 30% unmodified resistant starch may occur because the SCFA in
combination with bacterial LPS act synergistically to induce the production of pro-
187
inflammatory cytokines and their mediators as already reported by other group203, 207,
208. It could also be that these microorganisms, e.g. clostridia and Bacteroidetes,
produce toxins that induce the diarrhea associated with colitis201, 225, 228.
Both concentrations of unmodified high amylose maize resistant starch enhanced
levels of Bifidobacterium in colitic animals and one would anticipate that this may
contribute to the attenuation of colitis. However, assessing the population numbers of
lactobacilli, Gram negative anaerobes, spore-formers and enterics, it is most probable
that it is not only the total level of beneficial microorganisms that is critical in
protecting the animals against the development of IBD, but the presence and
abundance of the other members of the gut microflora. It was shown that enteric
bacteria especially the lactose fermenters and coliforms, were detected in high
numbers in the colons of TNBS induced colitic mice but whether enteric bacteria are
responsible for intestinal inflammation or whether the profile observed is a result of
inflammation warrants further investigation.
In Chapter 4, some Bifidobacterium and Lactobacillus strains were orally
administered to colitic mice to determine if these probiotics strains could ameliorate
murine colitis. It was demonstrated that different probiotic strains have different
capacities to protect BALB/c mice against chemically-induced, immune-mediated
colitis. B. animalis, B. lactis and L. amylovorus did not prevent the wasting effects of
TNBS colitis in these animals as indicated by greater weight loss and lower survival
rates; however, strains L. acidophilus and L. fermentum VRI 003 provided benefits.
Therefore, generic statements on the effects of probiotics on colitis cannot be made
since each specific strain will have a distinctive effect on the course of inflammation.
L. acidophilus and L. fermentum VRI 003 were further evaluated and both strains
were able to increase the level of regulatory cytokine IL-10 and simultaneously
decrease the concentration of pro-inflammatory IFN- , indicative that both
Lactobacillus strains can shift the cytokine response of TNBS colitis from a type 1
helper response to an anti-inflammatory profile. A more desirable outcome, however,
is to change the chronic, recurrent type of inflammation observed in colitis to a
“physiological” inflammation as seen in the normal intestinal environment which is
associated with an optimal ratio of IFN- production to IL-10 production. This is
188
because IL-10, if present in high levels, may worsen status of inflammation by
promoting IFN- production118. L. fermentum VRI 003 dosage to colitic mice restored
the ratio of IL-10:IFN- closer to that of a healthy-control animal and thus,
subsequent studies in this thesis focused on this strain.
Physical injury of the colon was more severe in colitic animals. This loss of gut
integrity contributed to the increased permeability in intestinal inflammation as
greater incidence of Gram negative aerobic bacteria translocated into the spleens of
colitic mice. Dosing L. fermentum VRI 003 prevented the increased uptake of Gram
negative aerobic bacteria into the spleen. The enteric bacteria which were also
detected in elevated levels in the colonic contents may have contributed to the
stimulation of the colonic epithelial cells leading to an inflammatory cytokine
production and inflammation. This would result in the infiltration of neutrophils and
pathogenic T lymphocytes that would further lead to the damage to the integrity of the
epithelium.
Most of the probiotic studies in inflammatory bowel diseases used high
concentrations of the probiotic strains, a daily dose 1010 to 1012 CFU of the probiotic
strain. This high dose may be needed because many strains survive poorly in the
gastrointestinal tract and cannot attach onto the mucosa. Non-adhering strains will be
washed out due to peristalsis. An experiment was performed to evaluate a lower dose
range, 107; 108 and 109 CFU, per day of L. fermentum VRI 003 in colitis induced mice
because the VRI 003 strain has previously been shown to adhere to the gut mucosa
and one could speculate that a lower dose may also be effective in ameliorating colon
inflammation. The doses of 108 and 109 CFU every other day for 2 weeks afforded
more protection against colon inflammation as shown by the greater weight recovery
and greater survival rate at the peak of the disease in TNBS colitis as compared to the
dose of 107 CFU dose. However, it was 108 CFU dose every other day for 2 weeks
which showed preferential stimulation of IL-4 and subsequently brought the pro-
inflammatory and anti-inflammatory cytokine balance to be more consistent with that
in healthy mice.
The dosage of 108 CFU per day of L. fermentum VRI 003 was then administered as
therapy after colitis induction with TNBS instead of using it prophylactically as
189
carried out in the study reported above. This study design was performed because it is
similar to human with established disease. This mode of dosage enhanced production
of IL-10 and downregulated IFN- , indicative of activation of anti-inflammatory
pathways. This activation at the sites of inflammation could be more beneficial than
the administration of IL-10 via the systemic route as recently trialled for IBD
patients114. Systemic administration of IL-10 has a number of limitations. It has to be
given in high doses as systemic administration does not allow for the efficient
delivery of the cytokine in the intestinal mucosa as the serum half-life of IL-10 is
between 1.1-2.6 hours and a lot would be cleared out of the system. Moreover, high
doses of IL-10 have a tendency to stimulate production of IFN- 118. Orally
administered IL-10 would be destroyed by the acidity of the stomach. Thus,
administration of L. fermentum VRI 003 could have benefits since it can influence the
nature of the cell-mediated immune response in colon inflammation by stimulating
production of anti-inflammatory cytokine, IL-4, and regulatory cytokine, IL-10;
restoring the balance between the pro-inflammatory cytokines and simultaneously
modifying the microbial profile to be more comparable to that of healthy control
animals.
The work in Chapter 4 can be extended to study the integrity of the intestinal mucosa
as well as the mechanisms of interaction of L. fermentum VRI 003 with the epithelial
cells of the inflamed tissue and their receptors. It would also be of interest to evaluate
how the inflammatory immune response is initiated in colitis and how L. fermentum
VRI 003 can modulate the induction as well as maintain a state of remission. It would
also be useful to conduct an experiment that will show the effects of L. fermentum
VRI 003 on active and recurrent colitis.
As shown in Chapter 4, L. fermentum VRI 003 prevented experimentally induced
colitis. L. fermentum VRI 003 was then administered to ulcerative colitis (UC)
patients who were in remission, to evaluate whether L. fermentum VRI 003 could
prevent the exacerbation of the symptoms. Although the study designs of the animal
model and the clinical study were different, benefits of the host were seen with L.
fermentum VRI 003 dosage both in disease induction in mice and maintenance of the
reduced symptom scores in UC patients in remission.
190
A daily dose of 6x109 CFU of L. fermentum VRI 003 for 6 months to UC patients
maintained in remission with medication, reduced severity and incidence of diarrhea,
rectal bleeding and abdominal pain as well as improved the general well being of the
patients. Effects on the pro-inflammatory cytokine IFN- were not seen, most
probably because of the effectiveness of the patients’ medications, while serum levels
of the immunosuppressive cytokine, IL-10, were either elevated or comparable levels
were achieved with the medications above. Consistent with the observations in
Chapters 3 and 4, concentrations of coliforms and endospores were lower and levels
of lactobacilli increased with L. fermentum VRI 003 administration to patients. The
higher levels of lactobacilli during L. fermentum VRI 003 treatment are indicative of
the survival of the orally administered lactobacilli in the colitic gut during standard
medication with aminosalicylates, corticosteroids and immunosuppressants.
The clinical study produced some very encouraging preliminary results that warrant
further investigation. Further studies should consider aspects such as dose response. It
may also be interesting to include a follow-up period in the clinical study design to
further assess the recovery of patients from the symptoms associated with colon
inflammation. A wash-out period is recommended to be included to prevent any carry
over effects from the treatment should a cross-over study be conducted again. It is
also suggested that patients are randomized according to the medication and the study
conducted based on one type of standard medication used. The effect of L. fermentum
VRI 003 on the different form and activity of colitis, i.e. mild, moderate, active and
relapsing, may be worth exploring. Finally, a comparison on the effects of L.
fermentum VRI 003 on UC and Crohn’s disease will be worth investigating as these
two forms of inflammatory bowel disease have different clinical presentations and
could be interesting to see how L. fermentum VRI 003 can affect the progression of
each disease form.
In conclusion, it has been shown that it can be possible to reduce the severity of colitis
in the TNBS murine model by using a prebiotic and probiotics, however, this is not a
general phenomenon for any prebiotics or probiotics. The mechanism is most
probably by altering the dominant intestinal microbes and thereby the immune
response of the patient. A relief of symptoms and a change in intestinal microbes was
seen in the patients with ulcerative colitis with dosage of a specific probiotic,
191
supporting the findings from the mouse studies. Further studies are needed to confirm
these benefits in UC patients and to identify the mechanisms involved.
192
References
1. Stenson WF. Inflammatory bowel disease. In: Yamada T, ed. Philadelphia: JB Lippincott Company, 1995.
2. Elson CO. Experimental models of inflammatory bowel disease. Gastroenterology 1995;109:1344-1367.
3. Palm O, Moum B, Jahnsen J, Gran JT. The prevalence and incidence of peripheral arthritis in patients with inflammatory bowel disease, a prospective population-based study (the IBSEN study). Rheumatology (Oxford) 2001;40:1256-1261.
4. Palm O, Moum B, Ongre A, Gran JT. Prevalence of ankylosing spondylitis and other spondyloarthropathies among patients with inflammatory bowel disease: a population study (the IBSEN study). Journal of Rheumatology 2002;29:511-515.
5. De Vos M, De Keyser F, Mielants H, Cuvelier C, Veys E. Review article: bone and joint diseases in inflammatory bowel disease. Alimentary Pharmacology and Therapy 1998;12:397-404.
6. Schorr-Lesnick B, Brandt LJ. Selected rheumatologic and dermatologic manifestations of inflammatory bowel disease. American Journal of Gastroenterology 1988;83:216-223.
7. Veloso FT, Carvalho J, Magro F. Immune-related systemic manifestations of inflammatory bowel disease. A prospective study of 792 patients. Journal of Clinical Gastroenterology 1996;23:29-34.
8. Oostenbrug LE, van Dullemen HM, te Meerman GJ, Jansen PL. IBD and genetics: new developments. Scandinavian Journal of Gastroenterology Supplement 2003:63-68.
9. Schultz M, Scholmerich J, Rath HC. Rationale for probiotic and antibiotic treatment strategies in inflammatory bowel diseases. Digestive Diseases 2003;21:105-128.
10. Guarner F, Malagelada JR. Role of bacteria in experimental colitis. Best Practical Research and Clinical Gastroenterology 2003;17:793-804.
11. Sellon RK, Tonkonogy S, Schultz M, Dieleman LA, Grenther W, Balish E, Rennick DM, Sartor RB. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infection and Immunity 1998;66:5224-231.
12. Sartor RB. Targeting enteric bacteria in treatment of inflammatory bowel diseases: why, how, and when. Current Opinion in Gastroenterology 2003;19:358-365.
193
13. Fuller R, Gibson GR. Modification of the microflora using probiotics and prebiotics. Scandinavian Journal of Gastroenterology 1997;Supplement:28-31.
14. Kasper H. Protection against gastrointestinal diseases--present facts and future development. International Journal of Food Microbiology 1998;41:127-131.
15. Hespell RB, Akin DE, Dehority BA. Bacteria, fungi and protozoa of the rumen. In: Mackie RL, ed. New York: Chapman and Hall, 1997.
16. Rolfe RD. Colonization Resistance. In: Mackie RL, ed. New York: Chapman and Hall, 1997.
17. Tannock G. Influences of the microbiota on the animal host. In: Mackie RL, ed. New York: Chapman and Hall, 1997.
18. Fuller R. Probiotics in human medicine. Gut 1991;32:439-442.
19. Ouwehand AC, Conway PL. Purification and characterisation of a component produced by Lactobacillus fermentum that inhibits the adhesion of K88 expressing Escherichia coli to porcine ileal mucus. Journal of Applied Bacteriology 1996;80:311-318.
20. Lehto EM, Salminen SJ. Inhibition of Salmonella typhimurium adhesion to Caco-2 cell cultures by Lactobacillus strain GG spent culture supernate: only a pH effect? FEMS Immunology and Medical Microbiology 1997;18:125-132.
21. Drago L, Gismondo MR, Lombardi A, de Haen C, Gozzini L. Inhibition of in vitro growth of enteropathogens by new Lactobacillus isolates of human intestinal origin. FEMS Microbiology Letters 1997;153:455-463.
22. Collins MD, Gibson GR. Probiotics, prebiotics and synbiotics: approaches for modulating the microbial ecology of the gut. American Journal of Clinical Nutrition 1999;69:1052S-1057S.
23. Conway PL. Probiotics and gastrointestinal microbiota. In: Hashimoto K, ed. Shiozawa, Japan: XII ISG Publishing Committee, 1996.
24. Yildirim Z, Johnson MG. Characterization and antimicrobial spectrum of bifidocin B, a bacteriocin produced by Bifidobacterium bifidum NCFB 1454. Journal of Food Protection 1998;61:47-51.
25. Callewaert R, Holo H, Devreese B, Van Beeumen J, Nes I, De Vuyst L. Characterization and production of amylovorin L471, a bacteriocin purified from Lactobacillus amylovorus DCE 471 by a novel three-step method. Microbiology 1999;145:2559-2568.
26. Wilson KH. Biota of the human gastrointestinal tract. In: Mackie RL, ed. New York: Chapman and Hall, 1997.
194
27. Pucciarelli MG, Siebers A, Finley BB. Bacterial pathogen translocation across the gastrointestinal barrier. In: Mackie RL, ed. New York: Chapman and Hall, 1997.
28. Conway PL. Development of the intestinal microbiota. In: Mackie RL, ed. New York: Chapman and Hall, 1997.
29. Reddy BS. Prevention of colon cancer by pre- and probiotics:evidence from laboratory studies. British Journal of Nutrition 1998;80:S219-S223.
30. Ward PB, Young GP. Dynamics of Clostridium difficile infection. Control using diet. Advance Experimental and Medical Biology 1997;412:63-75.
31. Bovee-Oudenhoven I, Van der Meer R. Protective effects of dietary lactulose and calcium phosphate against Salmonella infection. Scandinavian Journal of Gastroenterology Supplement 1997;222:112-114.
32. Brouns F, Kettlitz B, Arrigoni E. Resistant starch and "the butyrate revolution". Trends in Food Science & Technology 2002;13:251-261.
33. Pool-Zobel BL, Bertram B, Knoll M, Lambertz R, Neudecker C, Schillinger U, Schmezer P, Holzapfel WH. Antigenotoxic properties of lactic acid bacteria in vivo in the gastrointestinal tract of rats. Nutrion and Cancer 1993;20:271-281.
34. Tahri K, Crociani J, Ballongue J, Schneider F. Effects of three strains of bifidobacteria on cholesterol. Letters Applied Microbiology 1995;21:149-151.
35. Tahri K, Grill JP, Schneider F. Bifidobacteria strain behavior toward cholesterol: coprecipitation with bile salts and assimilation. Current Microbiology 1996;33:187-193.
36. Cebra JJ. Influences of microbiota on intestinal immune system development. American Journal of Clinical Nutrition 1999;69:1046S-1051S.
37. Talham GL, Jiang HQ, Bos NA, Cebra JJ. Segmented filamentous bacteria are potent stimuli of a physiologically normal state of the murine gut mucosal immune system. Infection and Immunity 1999;67:1992-2000.
38. Lopez-Boado YS, Wilson CL, Hooper LV, Gordon JI, Hultgren SJ, Parks WC. Bacterial exposure induces and activates matrilysin in mucosal epithelial cells. Journal of Cell Biology 2000;148:1305-1315.
39. Cong Y, Weaver CT, Lazenby A, Elson CO. Bacterial-reactive T regulatory cells inhibit pathogenic immune responses to the enteric flora. Journal of Immunology 2002;169:6112-6119.
40. Brandwein SL, McCabe RP, Cong Y, Waites KB, Ridwan BU, Dean PA, Ohkusa T, Birkenmeier EH, Sundberg JP, Elson CO. Spontaneously colitic
195
C3H/HeJBir mice demonstrate selective antibody reactivity to antigens of the enteric bacterial flora. Journal of Immunology 1997;159:44-52.
41. Das KM, Sakamaki S, Vecchi M, Diamond B. The production and characterization of monoclonal antibodies to human colonic antigen associated with ulcerative colitis: cellular localization of the antigen by using the monoclonal antibody. Journal of Immunology 1987;139:77-84.
42. Lichtman SN, Sartor RB, Keku J, Schwab JH. Hepatic inflammation in rats with experimental small intestinal bacterial overgrowth. Gastroenterology 1990;98:414-423.
43. Sartor RB, Rath HC, Lichtman SN, van Tol EA. Animal models of intestinal and joint inflammation. Baillieres Clinical Rheumatology 1996;10:55-76.
44. Sartor RB, Herfarth H, Van Tol EAF. Bacterial Cell Wall Polymer-Induced Granulomatous Inflammation. Methods 1996;9:233-247.
45. Mahler M, Bristol IJ, Leiter EH, Workman AE, Birkenmeier EH, Elson CO, Sundberg JP. Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis. American Journal of Physiology 1998;274:G544-551.
46. Garcia-Lafuente A, Antolin M, Guarner F, Crespo E, Salas A, Forcada P, Malagelada J. Derangement of mucosal barrier function by bacteria colonizing the rat colonic mucosa. European Journal of Clinical Investigation 1998;28:1019-1026.
47. MacDonald TT. Breakdown of tolerance to the intestinal bacterial flora in inflammatory bowel disease (IBD). Clinical and Experimental Immunology 1995;102:445-447.
48. Duchmann R, Schmitt E, Knolle P, Meyer zum Buschenfelde KH, Neurath M. Tolerance towards resident intestinal flora in mice is abrogated in experimental colitis and restored by treatment with interleukin-10 or antibodies to interleukin-12. European Journal of Immunology 1996;26:934-938.
49. Duchmann R, Marker-Hermann E, Meyer zum Buschenfelde KH. Bacteria-specific T-cell clones are selective in their reactivity towards different enterobacteria or H. pylori and increased in inflammatory bowel disease. Scandinavian Journal of Immunology 1996;44:71-79.
50. Kallinowski F, Wassmer A, Hofmann MA, Harmsen D, Heesemann J, Karch H, Herfarth C, Buhr HJ. Prevalence of enteropathogenic bacteria in surgically treated chronic inflammatory bowel disease. Hepatogastroenterology 1998;45:1552-1558.
196
51. Madsen KL, Doyle JS, Jewell LD, Tavernini MM, Fedorak RN. Lactobacillusspecies prevents colitis in interleukin 10 gene-deficient mice. Gastroenterology 1999;116:1107-1114.
52. Shiba T, Aiba Y, Ishikawa H, Ushiyama A, Takagi A, Mine T, Koga Y. The suppressive effect of bifidobacteria on Bacteroides vulgatus, a putative pathogenic microbe in inflammatory bowel disease. Microbiology and Immunology 2003;47:371-378.
53. Rath HC, Herfarth HH, Ikeda JS, Grenther WB, Hamm TE, Jr., Balish E, Taurog JD, Hammer RE, Wilson KH, Sartor RB. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats. Journal of Clinical Investigation 1996;98:945-953.
54. Balish E, Warner T. Enterococcus faecalis induces inflammatory bowel disease in interleukin-10 knockout mice. American Journal of Pathology 2002;160:2253-2257.
55. Kleessen B, Kroesen AJ, Buhr HJ, Blaut M. Mucosal and invading bacteria in patients with inflammatory bowel disease compared with controls. Scandinavian Journal of Gastroenterology 2002;37:1034-1041.
56. Yoshida M, Watanabe T, Usui T, Matsunaga Y, Shirai Y, Yamori M, Itoh T, Habu S, Chiba T, Kita T, Wakatsuki Y. CD4 T cells monospecific to ovalbumin produced by Escherichia coli can induce colitis upon transfer to BALB/c and SCID mice. International Immunology 2001;13:1561-1570.
57. Sartor R. The role of luminal bacteria in colitis: more than an antigenic drive. European Journal of Clinical Investigation 1998;28:1027-1029.
58. Rath HC, Ikeda JS, Linde HJ, Scholmerich J, Wilson KH, Sartor RB. Varying cecal bacterial loads influences colitis and gastritis in HLA-B27 transgenic rats. Gastroenterology 1999;116:310-319.
59. Kim SC, Tonkonogy SL, Albright CA, Tsang J, Balish EJ, Braun J, Huycke MM, Sartor RB. Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria. Gastroenterology 2005;128:891-906.
60. Stimpson SA, Esser RE, Carter PB, Sartor RB, Cromartie WJ, Schwab JH. Lipopolysaccharide induces recurrence of arthritis in rat joints previously injured by peptidoglycan-polysaccharide. Journal of Experimental Medicine 1987;165:1688-1702.
61. Lichtman SN, Keku J, Schwab JH, Sartor RB. Evidence for peptidoglycan absorption in rats with experimental small bowel bacterial overgrowth. Infection and Immunity 1991;59:555-562.
197
62. Stadnicki A, DeLa Cadena RA, Sartor RB, Bender D, Kettner CA, Rath HC, Adam A, Colman RW. Selective plasma kallikrein inhibitor attenuates acute intestinal inflammation in Lewis rat. Digestive Diseases and Sciences 1996;41:912-920.
63. Herfarth H, Scholmerich J. IL-10 therapy in Crohn's disease: at the crossroads. Treatment of Crohn's disease with the anti-inflammatory cytokine interleukin 10. Gut 2002;50:146-147.
64. von Ritter C, Sekizuka E, Grisham MB, Granger DN. The chemotactic peptide N-formyl methionyl-leucyl-phenylalanine increases mucosal permeability in the distal ileum of the rat. Gastroenterology 1988;95:651-656.
65. Obermeier F, Dunger N, Deml L, Herfarth H, Scholmerich J, Falk W. CpG motifs of bacterial DNA exacerbate colitis of dextran sulfate sodium-treated mice. European Journal of Immunology 2002;32:2084-2092.
66. Collins MT, Lisby G, Moser C, Chicks D, Christensen S, Reichelderfer M, Hoiby N, Harms BA, Thomsen OO, Skibsted U, Binder V. Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls. Journal of Clinical Microbiology 2000;38:4373-81.
67. Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, Collins MJ, Jr., Gorelick PL, Ward JM. Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. Journal of Clinical Microbiology 1994;32:1238-45.
68. Cahill RJ, Foltz CJ, Fox JG, Dangler CA, Powrie F, Schauer DB. Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus. Infection and Immunity 1997;65:3126-3131.
69. Foltz CJ, Fox JG, Cahill R, Murphy JC, Yan L, Shames B, Schauer DB. Spontaneous inflammatory bowel disease in multiple mutant mouse lines: association with colonization by Helicobacter hepaticus. Helicobacter 1998;3:69-78.
70. D'Haens GR, Geboes K, Peeters M, Baert F, Penninckx F, Rutgeerts P. Early lesions of recurrent Crohn's disease caused by infusion of intestinal contents in excluded ileum. Gastroenterology 1998;114:262-267.
71. Campieri M, Gionchetti P. Probiotics in inflammatory bowel disease: new insight to pathogenesis or a possible therapeutic alternative? Gastroenterology 1999;116:1246-1249.
72. Peppercorn MA. Is there a role for antibiotics as primary therapy in Crohn's ileitis? Journal of Clinical Gastroenterology 1993;17:235-237.
198
73. Peppercorn MA. Are antibiotics useful in the management of nontoxic severe ulcerative colitis? Journal of Clinical Gastroenterology 1993;17:14-7.
74. Linskens RK, Huijsdens XW, Savelkoul PH, Vandenbroucke-Grauls CM, Meuwissen SG. The bacterial flora in inflammatory bowel disease: current insights in pathogenesis and the influence of antibiotics and probiotics. Scandinavian Journal of Gastroenterology Supplement 2001:29-40.
75. Sanderson IR. Nutritional factors and immune functions of gut epithelium. Proceedings of Nutrition Society 2001;60:443-447.
76. Strober W, Ludviksson BR, Fuss IJ. The pathogenesis of mucosal inflammation in murine models of inflammatory bowel disease and Crohn disease. Annals Internal Medicine 1998;128:848-856.
77. Strober W, Kelsall B. To be responsive or not to be responsive, that is the mucosal question. Gastroenterology 1998;114:214-217.
78. Farhadi A, Banan A, Fields J, Keshavarzian A. Intestinal barrier: an interface between health and disease. Journal of Gastroenterology and Hepatology 2003;18:479-497.
79. Pickard KM, Bremner AR, Gordon JN, MacDonald TT. Microbial-gut interactions in health and disease. Immune responses. Best Practical Research and Clinical Gastroenterology 2004;18:271-285.
80. Guarner F, Casellas F, Borruel N, Antolin M, Videla S, Vilaseca J, Malagelada JR. Role of microecology in chronic inflammatory bowel diseases. European Journal of Clinical Nutrition 2002;56 Suppl 4:S34-38.
81. Furrie E, Macfarlane S, Cummings JH, Macfarlane GT. Systemic antibodies towards mucosal bacteria in ulcerative colitis and Crohn's disease differentially activate the innate immune response. Gut 2004;53:91-98.
82. Macpherson A, Khoo UY, Forgacs I, Philpott-Howard J, Bjarnason I. Mucosal antibodies in inflammatory bowel disease are directed against intestinal bacteria. Gut 1996;38:365-375.
83. Pirzer U, Schonhaar A, Fleischer B, Hermann E, Meyer zum Buschenfelde KH. Reactivity of infiltrating T lymphocytes with microbial antigens in Crohn's disease. Lancet 1991;338:1238-1239.
84. Swidsinski A, Ladhoff A, Pernthaler A, Swidsinski S, Loening-Baucke V, Ortner M, Weber J, Hoffmann U, Schreiber S, Dietel M, Lochs H. Mucosal flora in inflammatory bowel disease. Gastroenterology 2002;122:44-54.
85. Bjorksten B, Naaber P, Sepp E, Mikelsaar M. The intestinal microflora in allergic Estonian and Swedish 2-year-old children. Clinical and Experimental Allergy 1999;29:342-346.
199
86. Holt PG, Jones CA. The development of the immune system during pregnancy and early life. Allergy 2000;55:688-697.
87. Christensen HR, Frokiaer H, Pestka JJ. Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells. Journal of Immunology 2002;168:171-178.
88. Isolauri E, Sutas Y, Kankaanpaa P, Arvilommi H, Salminen S. Probiotics: effects on immunity. American Journal of Clinical Nutrition 2001;73:444S-450S.
89. McCracken VJ, Lorenz RG. The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota. Cell Microbiology 2001;3:1-11.
90. Ishizuka K, Sugimura K, Homma T, Matsuzawa J, Mochizuki T, Kobayashi M, Suzuki K, Otsuka K, Tashiro K, Yamaguchi O, Asakura H. Influence of interleukin-10 on the interleukin-1 receptor antagonist/interleukin-1 beta ratio in the colonic mucosa of ulcerative colitis. Digestion 2001;63 Supplement 1:22-27.
91. Colombel JF, Rutgeerts P, Malchow H, Jacyna M, Nielsen OH, Rask-Madsen J, Van Deventer S, Ferguson A, Desreumaux P, Forbes A, Geboes K, Melani L, Cohard M. Interleukin 10 (Tenovil) in the prevention of postoperative recurrence of Crohn's disease. Gut 2001;49:42-46.
92. Fuss IJ, Boirivant M, Lacy B, Strober W. The interrelated roles of TGF-beta and IL-10 in the regulation of experimental colitis. Journal of Immunology 2002;168:900-908.
93. Janeway CA, Jr., Medzhitov R. Innate immune recognition. Annual Review in Immunology 2002;20:197-216.
94. Dieleman LA, Hoentjen F, Qian BF, Sprengers D, Tjwa E, Torres MF, Torrice CD, Sartor RB, Tonkonogy SL. Reduced ratio of protective versus proinflammatory cytokine responses to commensal bacteria in HLA-B27 transgenic rats. Clinical and Experimental Immunology 2004;136:30-39.
95. Hanada T, Yoshimura A. Regulation of cytokine signaling and inflammation. Cytokine Growth Factor Review 2002;13:413-421.
96. Rogler G, Andus T. Cytokines in inflammatory bowel disease. World Journal of Surgery 1998;22:382-389.
97. Barton GM, Medzhitov R. Toll-like receptors and their ligands. Current Topics in Microbiology and Immunology 2002;270:81-92.
98. Xavier RJ, Podolsky DK. How to get along-- friendly microbes in a hostile world. Science 2000;289:1483-1484.
200
99. Neish AS, Gewirtz AT, Zeng H, Young AN, Hobert ME, Karmali V, Rao AS, Madara JL. Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science 2000;289:1560-1563.
100. Hanauer SB, Present DH. The state of the art in the management of inflammatory bowel disease. Review in Gastroenterology Disorders 2003;3:81-92.
101. Lim WC, Hanauer SB. Emerging biologic therapies in inflammatory bowel disease. Review in Gastroenterology Disorders 2004;4:66-85.
102. Baert F, Vermeire S, Noman M, Van Assche G, D'Haens G, Rutgeerts P. Management of ulcerative colitis and Crohn's disease. Acta Clin Belg 2004;59:304-314.
103. Scholmerich J, Huber G. Biological therapy in IBD. Anti-tumor necrosis factor-alpha and others. Digestive Diseases 2003;21:180-191.
104. Strober W. Interactions between epithelial cells and immune cells in the intestine. Annals of New York Academy of Science 1998;859:37-45.
105. Reinecker HC, Steffen M, Witthoeft T, Pflueger I, Schreiber S, MacDermott RP, Raedler A. Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease. Clinical and Experimental Immunology 1993;94:174-181.
106. Reimund JM, Wittersheim C, Dumont S, Muller CD, Kenney JS, Baumann R, Poindron P, Duclos B. Increased production of tumour necrosis factor-alpha interleukin-1 beta, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn's disease. Gut 1996;39:684-689.
107. Braegger CP, Nicholls S, Murch SH, Stephens S, MacDonald TT. Tumour necrosis factor alpha in stool as a marker of intestinal inflammation. Lancet 1992;339:89-91.
108. Targan SR, Hanauer SB, van Deventer SJ, Mayer L, Present DH, Braakman T, DeWoody KL, Schaible TF, Rutgeerts PJ. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease. Crohn's Disease cA2 Study Group. New England Journal of Medicine 1997;337:1029-3105.
109. Present DH, Rutgeerts P, Targan S, Hanauer SB, Mayer L, van Hogezand RA, Podolsky DK, Sands BE, Braakman T, DeWoody KL, Schaible TF, van Deventer SJ. Infliximab for the treatment of fistulas in patients with Crohn's disease. New England Journal of Medicine 1999;340:1398-1405.
110. Rutgeerts P, D'Haens G, Targan S, Vasiliauskas E, Hanauer SB, Present DH, Mayer L, Van Hogezand RA, Braakman T, DeWoody KL, Schaible TF, Van Deventer SJ. Efficacy and safety of retreatment with anti-tumor necrosis factor
201
antibody (infliximab) to maintain remission in Crohn's disease. Gastroenterology 1999;117:761-769.
111. Sands BE, Anderson FH, Bernstein CN, Chey WY, Feagan BG, Fedorak RN, Kamm MA, Korzenik JR, Lashner BA, Onken JE, Rachmilewitz D, Rutgeerts P, Wild G, Wolf DC, Marsters PA, Travers SB, Blank MA, van Deventer SJ. Infliximab maintenance therapy for fistulizing Crohn's disease. New England Journal of Medicine 2004;350:876-885.
112. Sands BE, Tremaine WJ, Sandborn WJ, Rutgeerts PJ, Hanauer SB, Mayer L, Targan SR, Podolsky DK. Infliximab in the treatment of severe, steroid-refractory ulcerative colitis: a pilot study. Inflammatory Bowel Disease 2001;7:83-88.
113. Stokkers PC, Hommes DW. New cytokine therapeutics for inflammatory bowel disease. Cytokine 2004;28:167-173.
114. Li MC, He SH. IL-10 and its related cytokines for treatment of inflammatory bowel disease. World Journal of Gastroenterology 2004;10:620-625.
115. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E. Treatment of murine colitis by Lactococcus lactis secreting interleuki-10. Science 2000;289:1352-1355.
116. Fedorak RN, Gangl A, Elson CO, Rutgeerts P, Schreiber S, Wild G, Hanauer SB, Kilian A, Cohard M, LeBeaut A, Feagan B. Recombinant human interleukin 10 in the treatment of patients with mild to moderately active Crohn's disease. The Interleukin 10 Inflammatory Bowel Disease Cooperative Study Group. Gastroenterology 2000;119:1473-1482.
117. Schreiber S, Fedorak RN, Nielsen OH, Wild G, Williams CN, Nikolaus S, Jacyna M, Lashner BA, Gangl A, Rutgeerts P, Isaacs K, van Deventer SJ, Koningsberger JC, Cohard M, LeBeaut A, Hanauer SB. Safety and efficacy of recombinant human interleukin 10 in chronic active Crohn's disease. Crohn's Disease IL-10 Cooperative Study Group. Gastroenterology 2000;119:1461-1472.
118. Tilg H, van Montfrans C, van den Ende A, Kaser A, van Deventer SJ, Schreiber S, Gregor M, Ludwiczek O, Rutgeerts P, Gasche C, Koningsberger JC, Abreu L, Kuhn I, Cohard M, LeBeaut A, Grint P, Weiss G. Treatment of Crohn's disease with recombinant human interleukin 10 induces proinflammatory cytokine interferon gamma. Gut 2002;50:191-195.
119. Guslandi M. Antibiotics for inflammatory bowel disease: do they work? Eur J Gastroenterology and Hepatology 2005;17:145-147.
120. Greenberg GR. Antibiotics should be used as first-line therapy for Crohn's disease. Inflamm Bowel Disease 2004;10:318-20.
202
121. Beaugerie L, Petit JC. Microbial-gut interactions in health and disease. Antibiotic-associated diarrhoea. Best Practice Research and Clinical Gastroenterology 2004;18:337-352.
122. Buchman AL, Rao S. Pseudomembranous collagenous colitis. Digestive Diseases and Sciences 2004;49:1763-1777.
123. Summers RW, Elliott DE, Urban JF, Jr., Thompson R, Weinstock JV. Trichuris suis therapy in Crohn's disease. Gut 2005;54:87-90.
124. Summers RW, Elliott DE, Urban JF, Jr., Thompson RA, Weinstock JV. Trichuris suis therapy for active ulcerative colitis: a randomized controlled trial. Gastroenterology 2005;128:825-32.
125. Shanahan F. Probiotics and inflammatory bowel disease: is there a scientific rationale? Inflammatory Bowel Disease 2000;6:107-115.
126. Shanahan F, O'Sullivan GC. How do intestinal T cells sense the dietary and microbial environment? Gastroenterology 2000;118:444-446.
127. Tannock GW, Munro K, Harmsen HJ, Welling GW, Smart J, Gopal PK. Analysis of the fecal microflora of human subjects consuming a probiotic product containing Lactobacillus rhamnosus DR20. Applied Environmental Microbiology 2000;66:2578-2588.
128. Borriello SP, Hammes WP, Holzapfel W, Marteau P, Schrezenmeir J, Vaara M, Valtonen V. Safety of probiotics that contain lactobacilli or bifidobacteria. Clinical and Infectious Diseases 2003;36:775-780.
129. Goldin BR. Health benefits of probiotics. British Journal of Nutrition 1998;80:S203-207.
130. Conway PL, Henriksson A. Strategies for the isolation and characterisation of functional probiotics. In: Gibson SA, ed. London: Springer Verlag, 1994.
131. Madsen K, Cornish A, Soper P, McKaigney C, Jijon H, Yachimec C, Doyle J, Jewell L, De Simone C. Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology 2001;121:580-591.
132. Malin M, Suomalainen H, Saxelin M, Isolauri E. Promotion of IgA immune response in patients with Crohn's disease by oral bacteriotherapy with Lactobacillus GG. Annals Nutrion and Metabolism 1996;40:137-145.
133. Kruis W, Schutz E, Fric P, Fixa B, Judmaier G, Stolte M. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. Alimentary Pharmacology Therapeutics 1997;11:853-858.
203
134. Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon AT. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 1999;354:635-639.
135. Gionchetti P, Rizzello F, Venturi A, Brigidi P, Matteuzzi D, Bazzocchi G, Poggioli G, Miglioli M, Campieri M. Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 2000;119:305-309.
136. Rachmilewitz D, Katakura K, Karmeli F, Hayashi T, Reinus C, Rudensky B, Akira S, Takeda K, Lee J, Takabayashi K, Raz E. Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis. Gastroenterology 2004;126:520-528.
137. Gionchetti P, Rizzello F, Helwig U, Venturi A, Lammers KM, Brigidi P, Vitali B, Poggioli G, Miglioli M, Campieri M. Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial. Gastroenterology 2003;124:1202-1209.
138. Kruis W, Fric P, Pokrotnieks J, Lukas M, Fixa B, Kascak M, Kamm MA, Weismueller J, Beglinger C, Stolte M, Wolff C, Schulze J. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 2004;53:1617-1623.
139. Osman N, Adawi D, Ahrne S, Jeppsson B, Molin G. Modulation of the effect of dextran sulfate sodium-induced acute colitis by the administration of different probiotic strains of Lactobacillus and Bifidobacterium. Dig Dis Sci 2004;49:320-7.
140. Dieleman LA, Goerres MS, Arends A, Sprengers D, Torrice C, Hoentjen F, Grenther WB, Sartor RB. Lactobacillus GG prevents recurrence of colitis in HLA-B27 transgenic rats after antibiotic treatment. Gut 2003;52:370-376.
141. Guslandi M, Mezzi G, Sorghi M, Testoni PA. Saccharomyces boulardii in maintenance treatment of Crohn's disease. Digestive Diseases and Sciences 2000;45:1462-4.
142. Venturi A, Gionchetti P, Rizzello F, Johansson R, Zucconi E, Brigidi P, Matteuzzi D, Campieri M. Impact on the composition of the faecal flora by a new probiotic preparation: preliminary data on maintenance treatment of patients with ulcerative colitis. Aliment Pharmacology and Therapeutics 1999;13:1103-1108.
143. Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition 1995;125:1401-1412.
144. Erickson KL, Hubbard NE. Probiotic immunomodulation in health and disease. Journal of Nutrition 2000;130:403S-409S.
204
145. Roberfroid MB. Prebiotics and synbiotics: concepts and nutritional properties. British Journal of Nutrition 1998;80:S197-202.
146. Crittendon R. Prebiotics. In: Tannock GW, ed. Wymondham, UK: Horizon Scientific Press, 1999.
147. Kleessen B, Stoof G, Proll J, Schmiedl D, Noack J, Blaut M. Feeding resistant starch affects fecal and cecal microflora and short-chain fatty acids in rats. Journal of Animal Science 1997;75:2453-62.
148. Wang X, Conway PL, Brown IL, Evans AJ. In vitro utilization of amylopectin and high-amylose maize (Amylomaize) starch granules by human colonic bacteria. Applied Environmental Microbiology. 1999;65:4848-4854.
149. Wang X, Brown IL, Evans AJ, Conway PL. The protective effects of high amylose maize (amylomaize) starch granules on the survival of Bifidobacterium spp. in the mouse intestinal tract. Journal of Applied Microbiology. 1999;87:631-639.
150. Wang X, Brown IL, Khaled D, Mahoney MC, Evans AJ, Conway PL. Manipulation of colonic bacteria and volatile fatty acid production by dietary high amylose maize (amylomaize) starch granules. Journal of Applied Microbiology. 2002;93:390-397.
151. Oyarzabal OA, Conner DE. Application of direct-fed microbial bacteria and fructooligosaccharides for salmonella control in broilers during feed withdrawal. Poultry Science 1996;75:186-190.
152. Oli MW, Petschow BW, Buddington RK. Evaluation of fructooligosaccharide supplementation of oral electrolyte solutions for treatment of diarrhea: recovery of the intestinal bacteria. Digestive Diseases and Sciences 1998;43:138-147.
153. Conway PL. Prebiotics and human health: The state-of-art and future perspectives. Scandinavian Journal of Nutrition/Naringsforskning 2001;45:13-21.
154. Zopf D, Roth S. Oligosaccharide anti-infective agents. Lancet 1996;347:1017-1021.
155. Zopf D, Simon P, Barthelson R, Cundell D, Idanpaan-Heikkila I, Tuomanen E. Development of anti-adhesion carbohydrate drugs for clinical use. Advance Experimental and Medical Biology 1996;408:35-38.
156. Gibson GR, Wang X. Regulatory effects of bifidobacteria on the growth of other colonic bacteria. Journal of Applied Bacteriology 1994;77:412-420.
157. Kanauchi O, Hitomi Y, Agata K, Nakamura T, Fushiki T. Germinated barley foodstuffs improves constipation induced by loperamide in rats. Bioscience, Biotechnology and Biochemistry 1998;62:1788-1790.
205
158. Jacobasch G, Schmiedl D, Kruschewski M, Schmehl K. Dietary resistant starch and chronic inflammatory bowel diseases. International Journal of Colorectal Disease 1999;14:201-211.
159. Videla S, Vilaseca J, Antolin M, Garcia-Lafuente A, Guarner F, Crespo E, Casalots J, Salas A, Malagelada JR. Dietary inulin improves distal colitis induced by dextran sodium sulfate in the rat. American Journal of Gastroenterology 2001;96:1486-1493.
160. Welters CF, Heineman E, Thunnissen FB, van den Bogaard AE, Soeters PB, Baeten CG. Effect of dietary inulin supplementation on inflammation of pouch mucosa in patients with an ileal pouch-anal anastomosis. Distal Colon and Rectum 2002;45:621-627.
161. Teramoto F, Rokutan K, Kawakami Y, Fujimura Y, Uchida J, Oku K, Oka M, Yoneyama M. Effect of 4G-beta-D-galactosylsucrose (lactosucrose) on fecal microflora in patients with chronic inflammatory bowel disease. Journal of Gastroenterology 1996;31:33-39.
162. Mao Y, Nobaek S, Kasravi B, Adawi D, Stenram U, Molin G, Jeppsson B. The effects of Lactobacillus strains and oat fiber on methotrexate-induced enterocolitis in rats. Gastroenterology 1996;111:334-344.
163. Pizarro TT, Arseneau KO, Bamias G, Cominelli F. Mouse models for the study of Crohn's disease. Trends in Molecular Medicine 2003;9:218-222.
164. Elson CO, Cong Y, Lorenz R, Weaver CT. New developments in experimental models of inflammatory bowel disease. Current Opinion in Gastroenterology 2004;20:360-367.
165. Garcia-Lafuente A, Antolin M, Guarner F, Crespo E, Salas A, Forcada P, Laguarda M, Gavalda J, Baena JA, Vilaseca J, Malagelada JR. Incrimination of anaerobic bacteria in the induction of experimental colitis. American Journal of Physiology 1997;272:G10-G15.
166. Neurath MF, Fuss I, Kelsall BL, Stuber E, Strober W. Antibodies to interleukin 12 abrogate established experimental colitis in mice. Journal of Experimental Medicine 1995;182:1281-1290.
167. Elson CO, Beagley KW, Sharmanov AT, Fujihashi K, Kiyono H, Tennyson GS, Cong Y, Black CA, Ridwan BW, McGhee JR. Hapten-induced model of murine inflammatory bowel disease: mucosa immune responses and protection by tolerance. Journal of Immunology 1996;157:2174-2185.
168. Neurath MF, Fuss I, Schurmann G, Pettersson S, Arnold K, Muller-Lobeck H, Strober W, Herfarth C, Buschenfelde KH. Cytokine gene transcription by NF-kappa B family members in patients with inflammatory bowel disease. Annals of New York Academy of Science 1998;859:149-159.
206
169. Inagaki-Ohara K, Chinen T, Matsuzaki G, Sasaki A, Sakamoto Y, Hiromatsu K, Nakamura-Uchiyama F, Nawa Y, Yoshimura A. Mucosal T cells bearing TCRgammadelta play a protective role in intestinal inflammation. Journal of Immunology 2004;173:1390-1398.
170. Holma R, Juvonen P, Asmawi MZ, Vapaatalo H, Korpela R. Galacto-oligosaccharides stimulate the growth of bifidobacteria but fail to attenuate inflammation in experimental colitis in rats. Scandinavian Journal of Gastroenterology 2002;37:1042-1047.
171. Kennedy RJ, Hoper M, Deodhar K, Kirk SJ, Gardiner KR. Probiotic therapy fails to improve gut permeability in a hapten model of colitis. Scandinavian Journal of Gastroenterology 2000;35:1266-1271.
172. Bennink RJ, van Montfrans C, de Jonge WJ, de Bruin K, van Deventer SJ, te Velde AA. Imaging of intestinal lymphocyte homing by means of pinhole SPECT in a TNBS colitis mouse model. Nuclear Medicine and Biology 2004;31:93-101.
173. Newman R, Cuan N, Hampartzoumian T, Connor SJ, Lloyd AR, Grimm MC. Vasoactive intestinal peptide impairs leucocyte migration but fails to modify experimental murine colitis. Clinical and Experimental Immunology 2005;139:411-420.
174. Duchmann R, Neurath MF, Meyer zum Buschenfelde KH. Responses to self and non-self intestinal microflora in health and inflammatory bowel disease. Research in Immunology 1997;148:589-594.
175. Geboes K, Riddell R, Ost A, Jensfelt B, Persson T, Lofberg R. A reproducible grading scale for histological assessment of inflammation in ulcerative colitis. Gut 2000;47:404-409.
176. Tanaka M, Riddell RH, Saito H, Soma Y, Hidaka H, Kudo H. Morphologic criteria applicable to biopsy specimens for effective distinction of inflammatory bowel disease from other forms of colitis and of Crohn's disease from ulcerative colitis. Scandinavian Journal of Gastroeneterology 1999;34:55-67.
177. Dohi T, Fujihashi K, Rennert PD, Iwatani K, Kiyono H, McGhee JR. Hapten-induced colitis is associated with colonic patch hypertrophy and T helper cell 2-type responses. Journal of Experimental Medicine 1999;189:1169-1180.
178. Caradonna L, Amati L, Magrone T, Pellegrino NM, Jirillo E, Caccavo D. Enteric bacteria, lipopolysaccharides and related cytokines in inflammatory bowel disease: biological and clinical significance. Journal of Endotoxin Research 2000;6:205-214.
179. Lochner M, Forster I. Anti-interleukin-18 therapy in murine models of inflammatory bowel disease. Pathobiology 2002;70:164-169.
207
180. Braat H, Peppelenbosch MP, Hommes DW. Interleukin-10-based therapy for inflammatory bowel disease. Expert Opinion in Biol Ther 2003;3:725-731.
181. Borg S, Bjorkman J, Eriksson S, Syk A, Andersson DI, Schesser K, Rhen M, Pettersson S, French NS. Novel Salmonella typhimurium properties in host--parasite interactions. Immunology Letters 1999;68:247-249.
182. Galvez J, Rodriguez-Cabezas ME, Zarzuelo A. Effects of dietary fiber on inflammatory bowel disease. Molecular Nutrition and Food Research. 2005. Electronic publication prior to print publication.
183. Scheppach W. Effects of short chain fatty acids on gut morphology and function. Gut 1994;35:S35-38.
184. Tappenden KA MM. Systemic short-chain fatty acids rapidly alter gastrointestinal structure, function, and expression of early response genes. Digestive Diseases and Sciences. 1998;43:1526-1536.
185. Barcelo A, Claustre J, Moro F, Chayvialle JA, Cuber JC, Plaisancie P. Mucin secretion is modulated by luminal factors in the isolated vascularly perfused rat colon. Gut 2000;46:218-224.
186. Zambell KL, Fitch MD, Fleming SE. Acetate and butyrate are the major substrates for de novo lipogenesis in rat colonic epithelial cells. Journal of Nutrition 2003;133:3509-3515.
187. Nakanishi S, Kataoka K, Kuwahara T, Ohnishi Y. Effects of high amylose maize starch and Clostridium butyricum on metabolism in colonic microbiota and formation of azoxymethane-induced aberrant crypt foci in the rat colon. Microbiology and Immunology 2003;47:951-958.
188. Gibson GR, Wang X. Enrichment of bifidobacteria from human gut contents by oligofructose using continuous culture. FEMS Microbiology Letters 1994;118:121-127.
189. Wang JF, Zhu YH, Li DF, Wang Z, Jensen BB. In vitro fermentation of various fiber and starch sources by pig fecal inocula. Journal of Animal Science 2004;82:2615-2622.
190. Brown IL, Wang X, Topping DL, Playne MJ, Conway PL. High amylose maize starch as a versatile prebiotic for use with probiotic bacteria. Food Australia 1998;50:603-610.
191. Ferguson LR, Tasman-Jones C, Englyst H, Harris PJ. Comparative effects of three resistant starch preparations on transit time and short-chain fatty acid production in rats. Nutrion and Cancer 2000;36:230-237.
192. Alles MS, Katan MB, Salemans JM, Van Laere KM, Gerichhausen MJ, Rozendaal MJ, Nagengast FM. Bacterial fermentation of
208
fructooligosaccharides and resistant starch in patients with an ileal pouch-anal anastomosis. American Journal of Clinical Nutrition 1997;66:1286-1292.
193. Ameho CK, Adjei AA, Harrison EK, Takeshita K, Morioka T, Arakaki Y, Ito E, Suzuki I, Kulkarni AD, Kawajiri A, Yamamoto S. Prophylactic effect of dietary glutamine supplementation on interleukin 8 and tumour necrosis factor alpha production in trinitrobenzene sulphonic acid induced colitis. Gut 1997;41:487-493.
194. Hartemink R, Rombouts FM. Comparison of media for the detection of bifidobacteria, lactobacilli and total anaerobes from faecal samples. Journal of Microbiological Methods 1999;36:181-192.
195. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Press, 1989.
196. Moreau NM, Champ MM, Goupry SM, Le Bizec BJ, Krempf M, Nguyen PG, Dumon HJ, Martin LJ. Resistant starch modulates in vivo colonic butyrate uptake and its oxidation in rats with dextran sulfate sodium-induced colitis. Journal of Nutrition 2004;134:493-500.
197. Rodriguez-Cabezas ME, Galvez J, Camuesco D, Lorente MD, Concha A, Martinez-Augustin O, Redondo L, Zarzuelo A. Intestinal anti-inflammatory activity of dietary fiber (Plantago ovata seeds) in HLA-B27 transgenic rats. Clinical Nutrition 2003;22:463-471.
198. Rodriguez-Cabezas ME, Galvez J, Lorente MD, Concha A, Camuesco D, Azzouz S, Osuna A, Redondo L, Zarzuelo A. Dietary fiber down-regulates colonic tumor necrosis factor alpha and nitric oxide production in trinitrobenzenesulfonic acid-induced colitic rats. Journal of Nutrition 2002;132:3263-3271.
199. Bernhard AE, Field KG. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Applied Environmental Microbiology 2000;66:1587-1594.
200. Dick LK, Field KG. Rapid estimation of numbers of fecal Bacteroidetes by use of a quantitative PCR assay for 16S rRNA genes. Applied Environmental Microbiology. 2004;70:5695-5697.
201. Scott TM, Rose JB, Jenkins TM, Farrah SR, Lukasik J. Microbial source tracking: current methodology and future directions. Applied Environmental Microbiology 2002;68:5796-5803.
202. Luhrs H, Gerke T, Schauber J, Dusel G, Melcher R, Scheppach W, Menzel T. Cytokine-activated degradation of inhibitory kappaB protein alpha is inhibited by the short-chain fatty acid butyrate. International Journal of Colorectal Disease 2001;16:195-201.
209
203. Huuskonen J, Suuronen T, Nuutinen T, Kyrylenko S, Salminen A. Regulation of microglial inflammatory response by sodium butyrate and short-chain fatty acids. British Journal of Pharmacology 2004;141:874-880.
204. Short chain fatty acid regulation of signaling genes expressed by the intestinal epithelium. Nutrition 2004;134:2450S-2454S.
205. Segain JP, Raingeard de la Bletiere D, Bourreille A, Leray V, Gervois N, Rosales C, Ferrier L, Bonnet C, Blottiere HM, Galmiche JP. Butyrate inhibits inflammatory responses through NFkappaB inhibition: implications for Crohn's disease. Gut 2000;47:397-403.
206. Noakes M, Clifton PM, Nestel PJ, Le Leu R, McIntosh G. Effect of high-amylose starch and oat bran on metabolic variables and bowel function in subjects with hypertriglyceridemia. American Journal of Clinical Nutrition 1996;64:944-951.
207. Yin L, Laevsky G, Giardina C. Butyrate suppression of colonocyte NF-kappa B activation and cellular proteasome activity. Journal of Biological Chemistry 2001;276:44641-44646.
208. Wachtershauser A, Stein J. Rationale for the luminal provision of butyrate in intestinal diseases. European Journal of Nutrition 2000;39:164-171.
209. Bergeron RJ, Wiegand J, Weimar WR, Nguyen JN, Sninsky CA. Prevention of acetic acid-induced colitis by desferrithiocin analogs in a rat model. Digestive Diseases and Sciences. 2003;48:399-407.
210. Blum S, Alvarez S, Haller D, Perez P, Schiffrin EJ. Intestinal microflora and the interaction with immunocompetent cells. Antonie Van Leeuwenhoek 1999;76:199-205.
211. Blum S, Schiffrin EJ. Intestinal microflora and homeostasis of the mucosal immune response: implications for probiotic bacteria? Current Issues in Intestinal Microbiology 2003;4:53-60.
212. Monteleone I, Vavassori P, Biancone L, Monteleone G, Pallone F. Immunoregulation in the gut: success and failures in human disease. Gut 2002;50 Supplement 3:III60-64.
213. Colpaert S, Vastraelen K, Liu Z, Maerten P, Shen C, Penninckx F, Geboes K, Rutgeerts P, Ceuppens JL. In vitro analysis of interferon gamma (IFN-gamma) and interleukin-12 (IL-12) production and their effects in ileal Crohn's disease. European Cytokine Network 2002;13:431-437.
214. Nahar IK, Shojania K, Marra CA, Alamgir AH, Anis AH. Infliximab treatment of rheumatoid arthritis and Crohn's disease. Annals of Pharmacotherapy 2003;37:1256-1265.
210
215. Tomoyose M, Mitsuyama K, Ishida H, Toyonaga A, Tanikawa K. Role of interleukin-10 in a murine model of dextran sulfate sodium-induced colitis. Scandinavian Journal of Gastroenterology 1998;33:435-440.
216. Bernet MF, Brassart D, Neeser JR, Servin AL. Lactobacillus acidophilus LA1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut 1994;35:483-489.
217. Matsumoto M, Ohishi H, Benno Y. Impact of LKM512 yogurt on improvement of intestinal environment of the elderly. FEMS Immunology and Medical Microbiology 2001;31:181-186.
218. Pochard P, Gosset P, Grangette C, Andre C, Tonnel AB, Pestel J, Mercenier A. Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients. Journal of Allergy and Clinical Immunology 2002;110.
219. Madsen KL, Doyle JS, Jewell LD, Tavernini MM, Fedorak RN. Lactobacillusspecies prevents colitis in interleukin 10 gene-deficient mice. Gastroenterology 1999;116:1107-1114.
220. Konrad A, Mahler M, Flogerzi B, Kalousek MB, Lange J, Varga L, Seibold F. Amelioration of murine colitis by feeding a solution of lysed Escherichia coli.Scandinavian Journal of Gastroenterology 2003;38:172-179.
221. Di Giacinto C, Marinaro M, Sanchez M, Strober W, Boirivant M. Probiotics ameliorate recurrent Th1-mediated murine colitis by inducing IL-10 and IL-10-dependent TGF-beta-bearing regulatory cells. Journal of Immunology 2005;174:3237-3246.
222. Shibolet O, Karmeli F, Eliakim R, Swennen E, Brigidi P, Gionchetti P, Campieri M, Morgenstern S, Rachmilewitz D. Variable response to probiotics in two models of experimental colitis in rats. Inflammatory Bowel Diseases 2002;8:399-406.
223. Tozawa K, Hanai H, Sugimoto K, Baba S, Sugimura H, Aoshi T, Uchijima M, Nagata T, Koide Y. Evidence for the critical role of interleukin-12 but not interferon-gamma in the pathogenesis of experimental colitis in mice. Journal of Gastroenterology and Hepatology 2003;18:578-587.
224. Regueiro M, Kip KE, Cheung O, Hegazi RA, Plevy S. Cigarette smoking and age at diagnosis of inflammatory bowel disease. Inflammatory Bowel Diseases 2005;11:42-47.
225. Mylonaki M, Langmead L, Pantes A, Johnson F, Rampton DS. Enteric infection in relapse of inflammatory bowel disease: importance of microbiological examination of stool. European Journal of Gastroenterology and Hepatology 2004;16:775-778.
226. Franz CM, Holzapfel WH, Stiles ME. Enterococci at the crossroads of food safety? International Journal of Food Microbiology 1999;47:1-24.
211
227. Vanek NN, Simon SI, Jacques-Palaz K, Mariscalco MM, Dunny GM, Rakita RM. Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism. FEMS Immunology and Medical Microbiology 1999;26:49-60.
228. Meyer AM, Ramzan NN, Loftus EV, Jr., Heigh RI, Leighton JA. The diagnostic yield of stool pathogen studies during relapses of inflammatory bowel disease. Journal of Clinical Gastroenterology 2004;38:772-775.
229. Weber P, Koch M, Heizmann WR, Scheurlen M, Jenss H, Hartmann F. Microbic superinfection in relapse of inflammatory bowel disease. Journal of Clinical Gastroenterology 1992;14:302-308.
230. Darfeuille-Michaud AB. High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease. Gastroenterology 2004;127:412-421.
231. Prantera C, Scribano ML, Falasco G, Andreoli A, Luzi C. Ineffectiveness of probiotics in preventing recurrence after curative resection for Crohn's disease: a randomised controlled trial with Lactobacillus GG. Gut 2002;51:405-9.
232. Schultz M, Timmer A, Herfarth HH, Sartor RB, Vanderhoof JA, Rath HC. Lactobacillus GG in inducing and maintaining remission of Crohn's disease. BMC Gastroenterology 2004;4:5.
233. Ishikawa H, Akedo I, Umesaki Y, Tanaka R, Imaoka A, Otani T. Randomized controlled trial of the effect of bifidobacteria-fermented milk on ulcerative colitis. Journal of American College of Nutrition 2003;22:56-63.
234. Li P. Potential effects of the administration of probiotics and resistant starch on the faecal microbial profiles and symptoms of irritable bowel syndrome. School of Microbiology and Immunology. Sydney, Australia: University of New South Wales, 2001.
235. Kang S. The adhesive characteristics of L. fermentumVRI 003 to Peyer's patches and its effects on the immune response in mice. School of Biotechnology and Biomolecular Sciences. Sydney, Australia: University of New South Wales, 2005.
236. Playford RJ, Macdonald CE, Johnson WS. Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders. American Journal of Nutrition 2000;72:5-14.
237. Ebrahim GJ. Clinical application of peptide growth factors first identified in breastmilk. Journal of Tropical pediatrics 2004;50:3-4.
238. Labeta MO, Vidal K, Nores JER, Arias M, Vita N, Morgan BP, Guillemot JC, Loyaux D, Ferrara P, Schmid D, Affolter M, Borysiewicz LK, Donnet-Hughes
212
A, Schiffrin EJ. Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14. Journal of Experimental Medicine 2000;191:1807-1812.
239. Wold AE, Adlerberth I. Breast feeding and the intestinal microflora of the infant---implications for protection against infectious diseases. Advances in Experimental Medicine and Biology 2000;478:77-93.
240. Petschow BW, Talbott RD, Batema RP. Ability of lactoferrin to promote the growth of Bifidobacterium spp in vitro is independent of receptor binding capacity and iron saturation level. Journal of Medical Microbiology 1999;48:541-549.
241. Saarinen KM, Vaarala O, Klemetti P, Savilahti E. Transforming growth factor-beta1 in mother’s colostrums and immune responses to cow’s milk proteins in infants with cows’ milk allergy. Journal of Allergy and Clinical Immunology 1999;104:1093-1098.
242. Takahashi N, Eisenhuth G, Lee I, Schachtele C. Nonspecific antibacterial factors in milk from cows immunized with human oral bacterial pathogens. Journal of Dairy Science 1992;75:1810-1820.
243. Travassos WJ, Cheifetz AS. Infliximab: use in inflammatory bowel diseases. Current Treatments Options in Gastroenterology 2005;8:187-196.
244. Gareth AO, Thomas JR, Ingram JR. Mechanisms of disease:nicotine—a review of its actions in the context of gastrointestinal disease. Nature Clinical Prattice Gastroenterology and Hepatology 2005;2:536-544.
245. Pallant J. SPSS Survival Manual. In: Allen & Uwin, ed. New York: Chapman and Hall, 2001.
246. Blumberg RS, Saubermann LJ, Strober W. Animal models of mucosal inflammation and their relation to human inflammatory bowel disease. Current Opinion in Immunology 1999;11:648-656.
247. Videla S, Vilaseca J, Guarner F, Salas A, Tereserra F, Crespo E, Antolin M, Malagelada JR. Role of intestinal microflora in chronic inflammation and ulceration of the rat colon. Gut 1994;35:1090-1097.
248. Burkitt DP, Walker ARP, Painter NS. Effect of dietary fiber on stools and transit times, and its role in the causation of disease. Lancet 1972;2:1408-1412.
249. Jenkins DJA, Vuksan V, Kendall CWC, Wursch P, Jeffcoat R, Waring S, Mehling CC, Vidgen E, Agustin LSA, Wong E. Physiological effects of resistant starch on fecal bulk, short chain fatty acids, blood lipids and glycemic index. Journal of the American College of Nutrition 1998;17:609-616.
213
250. Phillips J, Muir JG, Birkett A. Effect of resistant starch on fecal bulk and fermentation-dependent events in humans. American Journal of Clinical Nutrition 1995;62:121-130.
251. Cummings JH, Beatty ER, Kingman SM, Bingham SA, Englyst HN. Digestion and physiological properties of resistant starch in the human large bowel. British Journal of Nutrition 1996;75:733-747.
252. Birkett A, Muir JG, Phillips J, Jones GP, O’Dea K. Resistant starch lowers fecal concentrations of ammonia and phenols in humans. American Journal of Clinical Nutrition 1996;63:766-772.
253. Peach S, Lock MR, Katz D, Todd IP, Tabaqchali S. Mucosal-associated bacterial flora of the intestine in patients with CD and in a control group. Gut 1978;19:1034-1042.
254. Hartley MG, Hudson MJ, Swarbick ET, Hill MJ, Gent AE, Hellier MD, Grace RH. The rectal mucosa-associated microflora in patients with ulcerative colitis. Journal of Medical Microbiology 1992;36:96-102.
255. Fabia R, Ar’Rajab A, Johansson ML, Andersson R, Willen R, Jeppsson B, Molin G, Bengmark S. Impairment of bacterial flora in human ulcerative colitis and experimental colitis in the rat. Digestion 1993;54:248-255.
256. Schultsz C, Van den Berg FM, Ten Kate FW, Tytgat GN, Dankert J. The intestinal mucus layer from patients with inflammatory bowel disease harbors high numbers of bacteria compared with controls. Gastroenterology 1999;117:1089-1097.
257. Zinkevich V, Beech, IB. Screening of sulfate-reducing bacteria in colonoscopy samples from healthy and colitic human gut mucosa. FEMS Microbiology Ecology 2000;34:147-155.
258. Fite A, Macfarlane GT, Cummings JH, Hopkins MJ, Kong SC, Furrie E, Macfarlane S. Identification and quantitation of mucosal and faecal desulfovibrios using real time polymerase chain reaction. Gut 2004;53:523-529.
259. Schultsz C, Moussa M, van Ketel R, Tytgat GNJ, Dankert D. Frequency of pathogenic and enteroadherent Escherichia coli in patients with inflammatory bowel disease and controls. Journal of Clinical Pathology 1997;50:573-579.
260. Ott SJ, Musfeldt M, Wenderoth DF, Hampe J, Brant O, Folsch UR, Timmis KN, Schreiber S. Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease. Gut 2004;53:685-693.
261. Swidsinski A, Weber J, Loening-Baucke V, Hale LP, Lochs H. Spatial organization and composition of the mucosal flora in patients with
214
inflammatory bowel disease. Journal of Clinical Microbiology 2005;43:3380-3389.
262. Matsuda H, Fujiyama Y, Andoh A, Ushijima T, Kajinami T, Bamba T. Characterization of antibody response against rectal mucosa-assocaited bacterial flora in patients with ulcerative colitis. Journal of Gasroenterology and Hepatology 2000;15:61-68.
263. Delahooke DM, Barclay GR, Poxton IR. Reappraisal of the biological activity of Bacteroides lipopolysaccharide. Journal of Medical Microbiology. 1995;42:102-112.
264. Giaffer MH, Holdsworth CD, Duerden BI. Virulence properties of Escherichia coli strains isolated from patients with inflammatory bowel disease. Gut 1992;33:646-650.
265. Ljungh A, Eriksson M, Eriksson O, Henter JI, Wadstrom T. Shiga-like toxin production and connective tissue protein binding of Escherichia coli from a patient with ulcerative colitis. Scandinavian Journal of Infectious Diseases 1988;29:443-446.
266. Roediger WEW, Moore J, Babidge W. Colonic sulfide in the pathogenesis and treatment of ulcerative colitis. Digestive Diseases and Sciences 1997;42:1571-1579.
267. Wilson KH. Biota of the human gastrointestinal tract. In Gastrointestinal Microbiology. Ed. Mackie RI, White BA, Isaacson RE. New York:Chapman and Hall,1997.
268. Savage DC. Microbial ecology of the gastrointestinal tract. Annual Reviews of Microbiology 1977;31:107-133.
269. Klassen HLBM, Koopman JP, van den Brink ME, van Wezel HPN, Beynen AC. Intestinal, segmented, filamentous bacteria. FEMS Microbiology Review 1992;88:165-180.
270. Savage DC, Dubos R, Schaedler RW. The gastrointestinal epithelium and its authocthonous bacterial flora. Journal of Experimental Medicine 1968;127:67-76.
271. Conway PL. Microbiota: development, characterization and ecology. In Gastrointestinal Microbiology. Ed. Mackie RI, White BA, Isaacson RE. New York:Chapman and Hall,1997.
272. Moore WEC, Holdeman LV. Special problems associated with the isolation and identification of intestinal bacteria in fecal flora studies. American Journal of Clinical Nutrition 1974;27:1450-1455.
215
273. Dubos R, Schaedler RW, Costello R, Hoet P. Indigenous, normal and autochtonous flora of the gastrointestinal tract. Journal of Experimental Medicine 1965;22-76.
275. Englyst HN, Kingman SM, Cummings JH. Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition 1992;46(Supplement):S33-S50.
276. Brown I, Conway PL, Topping D. The health potential of resistant starches in foods: An Australian perspective. Scandinavian Journal of Nutrition 2000;44:53-58.
277. Topping DL, Clifton PM. Short-chain fatty acids and human colonic function: roles of resistant starch and non-starch polysaccharides. Physiology Review 2001;81:1031-1064.
278. Cummings JH, Beatty ER, Kingman SM, Bingham SA, Englyst HN. Digestion and physiological properties of resistant starch in the human bowel. British Journal of Nutrition 1996;75:733-747.
279. Phillips J, Muir JG, Birkett A, Zhong XL, Jones PJ, O’Dea K, Young GP. Effect of resistant starch on faecal bulk and fermentation-dependent events in humans. American Journal of Clinical Nutrition 1995;62:121-130.
280. Birkett A, Muir J, Phillips J, Jones G, O’Dea K. Resistant starch lowers fecal concentrations of ammonia and phenols in humans. American Journal of Clinical Nutrition 1996; 63:766-772.
281. Muir JG, Yeow EGW, Keogh J, Pizzey C, Bird AR, Sharpe K, O’Dea K, macrae FA. Combining wheat bran with resistant starch has more beneficial effects on fecal indexes than does wheat ran alone. American Journal of Clinical Nutrition 2004;79:1020-1028.
282. Kleesen B, Stoof G, Prell J, Schmiedl D, Noack J, Blaut M. Feeding resistant starch affects fecal and cecal microflora and short chain fatty acids in rats. Journal of Animal Science 1997;75:2453-2462.
283. Moreau NM, Martin LJ, Toquet CS, laboisse CL, Nguyen PG, Siliart BS, Dumon HJ, Champ MMJ. Restoration of the integrity of rat caeco-colonic mucosa by resistant starch, but not by fructo-oligosaccharides, in dextran sulfate sodium-induced experimental colitis. British Journal of Nutrition 2003;90:75-85.
284. Morita T, Tanabe H, Takahashi K, Sugiyama K. Ingestion of resistant starch protects endotoxin influx from the intestinal tract and reduces D-galactosamine-induced liver injury in rats. Journal of Gastroenterology and Hepatology 2004;19:303-313.
216
285. Tatsumi Y, Lichtenberger LM. Molecular association of trinitrobenzenesulfonic acid and surface phospholipids in the development of colitis in rats. Gastroenterology 1996;110:780-789.
286. Klemm P. Fimbraie: adhesion, genetics, biogenesis, and vaccines. In: Per Klemm, ed. USA:CRC Press, 1994.
287. Rachmilewitz D. Coated mesalazine (5-aminosalicylic acid) versus sulphasalazine in the treatment of active ulcerative colitis: a randomized trial. BMJ 1989; 298:82-86.
217
Appendix I
Case Reports
Subject UC-2
Case History
Subject UC-2 is a 65 year old, Australian male who was clinically diagnosed in year
2001. He has no known family history of IBD. He was a smoker for 10 years but
stopped on year 1975. He regularly consumes alcohol with an average of 3 bottles per
day. He has a moderate, proctitis type of UC. He has an average bowel movement of
3-4 per day. His standard medication for UC is 500 mg sulphasalazine.
Treatment
Group A - received placebo treatment first then L. fermentum VRI 003
Observations
Subject UC-2 tolerated both placebo and L. fermentum VRI 003 treatments very well.
No side effects were noted with their use. On the third month of taking the placebo
treatment, participant felt a heavy sensation in the stomach which he suspected to be
due to forgetting to take Pyrolin after his meals. This condition lasted for a couple of
days but intensity subsided until it disappeared. In total, he missed taking the
investigational capsules for 3 days during the placebo period.
Figure A.1A shows that during placebo treatment frequency of bowel movement
increased and shifting to L. fermentum VRI 003 treatment (Figure A.1B) resulted to a
regular bowel movement during this treatment period. No abdominal pain was
experienced in both treatments (Figures A.1C-D) and felt generally well on both
treatments (Figures A.1E-F).
218
A
y = 0.0039x + 1.6921R2 = 0.0782
01
23
456
78
910
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = 0.0009x + 2.1489R2 = 0.0049
012345
6789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
A
y = 3E-06x + 0.0049R2 = 4E-06
012
34567
89
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
B
y = 1E-04x + 0.147R2 = 2E-05
01
2345
678
910
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
A
y = 10R2 = #N/A
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
B
y = 10R2 = #N/A
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.1. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-2. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
C D
E F
219
Subject UC-3
Case History
Subject UC-3 is a 34 year old English male with no history of IBD. He has been a
smoker for 12 years before quitting in 2000. He consumes an average of 1 bottle of
alcohol per day. He was diagnosed with UC on 2002 and had active, colitis for 8
months in the same year he was diagnosed with UC. He has severe case of colitis that
resulted to 10-20 bowel movements per day. His UC is localized on the left side (L-
side colitis). His standard medication includes 1g Salofalk taken 3x a day and 200 mg
Imuran.
Treatment
Group A - received placebo treatment first then L. fermentum VRI 003
Observations
Subject felt that his colitis was returning on two occasions, 2nd month and 5th month,
during the placebo treatment. He observed that there was an increase in bowel
movements, appearance of mucus and blood in stool and unsettled stomach for which
he suspected can either be due to introduction of new food in the diet, viral infection
or from the two consecutive big activities he attended. Inspite of these observations,
there was a dramatic decrease in the trend of bowel movement when in the placebo
course of treatment (Figure A.2A). During the active phase of treatment no side
effects were reported his bowel habits and general well being. His wife gave birth
when he was on active treatment. Improvements observed during the L. fermentum
VRI 003 were continued decrease in the frequency of bowel movements (Figures
A.2B), maintained effect of placebo treatment on abdominal pain and continued
absence of abdominal pain during the active period (Figure A.2C-A.2D) and
increased feeling of general well being at the end of L. fermentum VRI 003 course
(Figure A.2F).
220
A
y = -0.0087x + 4.5964R2 = 0.1301
01
23
456
78
910
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = -0.0009x + 3.2799R2 = 0.002
012
34567
89
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = -0.0037x + 0.4826R2 = 0.1292
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = 0.0001x + 0.0109R2 = 0.0007
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = 0.0002x + 7.8546R2 = 0.0006
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = 0.0013x + 7.5816R2 = 0.0062
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.2. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-3. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
221
Subject UC-4
Case History
Subject UC-4 is a 58 year old, Australian, male, who does not have any family history
of IBD. He was a smoker for 20 years but stopped in 1976. Average alcohol
consumption in a day is 6 bottles. He was diagnosed with UC in 1986 and at present
has the mild form of colitis. He has an average of 6 bowel movements and a
maximum of 18 on a bad day. He takes 5 mg of prednisone once a day and
methatrexate once a week.
Treatment
Group B – received L. fermentum VRI 003 first then placebo
Observations
He reported that whilst on L. fermentum VRI 003 treatment his degree of his urgency
dramatically changed from 6 to 2 bowel movements a day. However, on the 5th month
after consuming lots of chocolates resulted to an increase in bowel motions and
appearance of mucus in the stool. His gastroenterologist prescribed 25 mg prednisone
after this event and after which his condition stabilized. Entry to the second period of
the treatment, placebo, increased urgency factor and been going to the toilet from 5 to
10 times a day. By the 11 month he started having arthritic pain and his
gastroenterologist increased dosage of prednisone to 50 mg and methatrexate to 15
mg. Thus, L. fermentum VRI 003 (Figure A.3A) dramatically decreased frequency of
bowel movements while placebo treatment increased this event (Figure A.3B). The
patient did not experience any abdominal pain during active treatment (Figure A.3C)
but had bouts of abdominal pain simultaneous with the arthritic pain when placebo
treatment was received (Figure A.3D).
222
A
y = -0.0128x + 5.4176R2 = 0.2309
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = 0.0006x + 4.7996R2 = 0.0006
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = -0.0007x + 0.129R2 = 0.0023
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = 0.0029x - 0.1448R2 = 0.0669
012
34567
89
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = 0.0004x + 9.9341R2 = 0.0054
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = 0.0004x + 9.9396R2 = 0.0117
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.3. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-4. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
223
Subject UC-5
Case History
Subject 5 is a 72 year old Australian male who has been a smoker for 20 years but
stopped in 2002. He does not have a family history of IBD but had been suffering
from colitis for 35 years. His colitis was first diagnosed in 1998. Activity of UC is
mild at this stage and experiences an average of 4 bowel movements a day.
Treatment
Group A – received placebo treatment first then L. fermentum VRI 003
Observations
Participant felt constipated during the first month of taking the placebo treatment. On
the second month, there was a delay in the delivery of investigational materials for 9
days. The participant noticed an increase in bowel movements and appearance of
blood in his stool while he was off the investigational capsules. After receiving the
investigational capsules he was able to control his bowel motions. Subject UC-5
regularly consumes alcohol and he noticed that limiting alcohol intake to 2-3 stubbies
a day resulted to having bowel movements of 2-3 times a day. He observed that he
may go to the toilet 3-4 times a day but has better control of his bowel actions when
he was on L. fermentum VRI 003 treatment (Figure A.4B). At the end of the L.
fermentum VRI 003 period, he reported an 80% control over his bowel motions and
had overall big improvement with L. fermentum PCC™. Moreover, his abdominal
pain intensified during placebo treatment (Figure A.4C) but this reduced during active
treatment (Figure A.4D) and remained stable until the end of L. fermentum VRI 003
period. His general well being score dropped by the end of placebo treatment (Figure
A.4E) and improved when L. fermentum VRI 003 started (Figure A.4F).
224
A
y = 0.0066x + 2.5836R2 = 0.1042
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = 0.0069x + 2.803R2 = 0.1592
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = 0.0103x + 0.1199R2 = 0.163
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = 0.0018x + 0.8304R2 = 0.0069
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = -0.0033x + 9.2356R2 = 0.0563
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10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng
F
y = -0.0014x + 9.2403R2 = 0.0134
0123456789
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0 50 100 150 200
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Gen
eral
Wel
l Bei
ng
Figure A.4. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-5. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
225
Subject UC-6
Case History
Subject UC-6 is a 55 year old, male, Australian with no history of IBD. He had been
diagnosed with UC in 1993. He suffers from a mild form UC at the time of enrolment
and experiences an average bowel movement of 1 per day. He has been a smoker for
16 years but ceased in 1980. He has an average intake of 6 bottles of alcohol per day.
Treatment
Group B – received L. fermentum VRI 003 first then placebo treatment
Observations
Participant had bad arthritic experience during the first 3 months of the active
treatment. His gastroenterologist advised to increase his dosage of prednisone to 15
mg but did not provide any relief and brought it back down to 10 mg. Participant then
sought alternative therapy to relieve him of the arthritis pain by seeing an
acupuncturist. Afterwhich, the participant did not complain of any pain associated
from arthritis until the end of the first treatment period. He also went into antibiotic
therapy for a hand injury during the active treatment period. He then experienced
recurrent loose bowel motions for two consecutive months after starting on the
placebo treatment. He felt to be having a relapse from colitis during this period. His
arthritis came back during this time then his specialist increased prednisone dosage to
20 mg which he took for 10 days and the dose was reduced to 15 mg. He had
increased bowel movements during the latter part of the placebo treatment which is
partly due to colonoscopy preparation. Overall, he had an average of 1-2 bowel
movements while on active treatment (Figure A.5A) but it dramatically increased
during the placebo treatment period (Figure A.5B). Although the trend of severity of
abdominal pain remain unchanged for both treatments (Figure A.5C-A.5D), Subject
UC-6 had more pronounced abdominal pain during the 4th month of placebo treatment
(Figure A.5D) which was not seen in active treatment. Both treatments did not make
him feel better (Figure A.5E – A.5F).
226
A
y = 0.0009x + 1.4479R2 = 0.0074
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Num
ber o
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el A
ctio
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B
y = 0.008x + 1.4986R2 = 0.0629
0
5
10
15
20
25
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = 5E-05x + 0.0316R2 = 0.0002
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10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = 0.0004x + 0.0697R2 = 0.002
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10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = -0.002x + 4.9705R2 = 0.0608
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng
F
y = -0.0017x + 5.0321R2 = 0.0307
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng
Figure A.5. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-6. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
227
Subject UC-7
Case History
Subject UC-7 is a 55 year old Australian, non-smoker, female who has family history
of IBD. She has been suffering from UC for 25 years but was only diagnosed of the
condition in 1997. She has a mild form of proctitis at the time of enrolment. Her
medications are Avapno HCT 300/12 5 tablets taken once daily, Mesasal taken twice
a day and Tryptanol taken twice nocte.
Treatment
Group A – received placebo treatment first then L. fermentum VRI 003
Observations
The first six months of the clinical program was stressful and depressing for Subject
UC-7 as these were the times that her husband was diagnosed with cancer, undergone
chemotherapy, cared for him, admitted to the hospital and eventually passed away and
has to cope from her loss. She had a worse relapse, as indicated by loose bowel
movement and blood in the stools, when her husband was admitted to the hospital and
missed taking her medications. By the time, she was in the L. fermentum VRI 003 she
was feeling better although she occasionally felt depressed and missed taking her
medication. An unfortunate event of falling off the ladder which hurt her ankle
happened during this period and she took panadol for the pain. She also had to take
antibiotics, Amoxil, prior to her dental work during the active treatment period.
Because of these personal events, the results shown in the graphs (Figure A.6A to
A.6F) may not necessarily reflect the real effects of the placebo and L. fermentum
VRI 003.
228
A
y = 0.002x + 2.2549R2 = 0.0134
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0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = 0.0006x + 2.4585R2 = 0.0017
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = -0.0006x + 0.1459R2 = 0.0094
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = -0.0005x + 0.0884R2 = 0.0151
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = -0.0202x + 9.0199R2 = 0.423
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = 0.0009x + 7.0925R2 = 0.0024
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10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.6. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-7. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
229
Subject UC-8
Case History
Subject 8 is a 56 year old, female from an Italian background who had UC since 1991.
She has no family history of IBD. She was a smoker for 25 years but quitted in 2002.
She can consume 2 bottles of alcohol per day. She has a mild form of UC and has
bowel actions at an average of 4 times a day. She takes 150 mg Imuran per day,
Celephor granules thrice a day, 100 mg Oestrogen/Progesteron, 50 mg Inderal,
Vitamin D once daily and 260 mg Calcium twice daily.
Treatment
Group A – received placebo treatment then L. fermentum VRI 003
Observations
Some changes on her medications were made during placebo treatment. She got off
Inderal at the end of the first month, changed from Salofalk granules to 250 mg
Dipentum on the second and fourth months but reverted back again to Salofalk when
bowel motions increased, and took 1000 mg Ostelin 4 times instead on once a day.
After consumption of rich Italian food, blood was observed in her stools then started
using predsol suppositories. Persistence of blood in her stool was observed for a
month then she went for colonoscopy. She started having Salofaulk enemas on the
first month of L. fermentum VRI 003 treatment. Afterwhich, the enema treatment was
ceased. In the next three months no signs of relapse were observed during the L.
fermentum VRI 003 treatment period. A month prior to the end of the last treatment
course, bleeding was detected in the stools but was controlled upon the use of
Salofalk enemas. Similar trend was observed in the visiual analogue scale of
frequency of bowel movements, abdominal pain and general well being scores in both
treatments (Figure A.7A - A.7F).
230
A
y = 0.0024x + 1.8301R2 = 0.0219
012345
6789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = 0.0025x + 1.8774R2 = 0.0316
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = 0R2 = #N/A
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10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = 0.0007x - 0.0112R2 = 0.0145
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = 10R2 = #N/A
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = -0.0012x + 10.02R2 = 0.0141
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.7. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-8. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
231
Subject UC-10
Case History
Subject UC-10 is a 46 year old, non-smoking Australian male with no history of IBD.
His UC was diagnosed in 2001 and moves his bowels at average of 2 a day.
Treatment
Group B – received L. fermentum VRI 003 first then placebo treatment
Observations
The subject observed that even if the frequency of his bowel movements did not
decrease, he noticed that his stools were forming and were of solid consistency during
the L. fermentum VRI 003 treatment. The first six months of the study program saw
the participant moving houses, changing jobs and did a lot of traveling. On his third
month of active treatment, he was not able to take the investigational capsules for 5
days and noticed that his bowel actions increased from 2.5 to 3.5 per day. No unusual
events in gastrointestinal symptoms were observed during the placebo treatment. L
ferementum VRI 003 and placebo treatments gave opposing effects to the participant.
L fermentum VRI 003 increased the frequency of bowel motions (Figure A.8A) with a
concurrent minimal relief from abdominal pain (Figure A.8C) while placebo
treatment reduced the number of bowel actions (Figure A.8B) but the severity of the
abdominal pain became more pronounced (Figure A.8D).
232
A
y = 0.0021x + 2.4584R2 = 0.0132
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10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = -0.0067x + 3.392R2 = 0.1035
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = -0.0028x + 5.724R2 = 0.0095
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = 0.005x + 4.6879R2 = 0.021
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = -0.0018x + 6.0966R2 = 0.0057
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = -0.0102x + 6.5402R2 = 0.1658
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
1
Figure A.8. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-10. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
233
Subject UC-11
Case History
Subject UC-11 is a 40 year old, non-smoker, Australian female with no history of
IBD. She has a moderate activity in her colitis at the time of enrolment which makes
her move her bowels 4-5 times a day. She is on 3g Salofalk three times a day,
metathrexate once a week, prednisone prescribed at 20 mg per day and to be reduced
every 5 days to 2.5 mg, folic acid to be taken 6X a week, calcitrate taken once daily
and iron tonic.
Treatment
Group A – received placebo treatment first then L. fermentum VRI 003
Observations
Participant was diagnosed with Giardia infection on the fifth month of the study
program which could have contributed to the increase of bowel motions during
placebo treatment. She was given 200 mg Flagyl, thrice daily for two weeks which
she took together with the investigational materials. She then took another course of
antibiotics (500 mg Amoxil) prior to a dental procedure at the beginning of the L.
fermentum VRI 003 treatment. Her symptoms from the Giardia infection returned, on
the fourth month of the active treatment period, after eating canned fish. She was
again prescribed to take 200 mg Flagyl, thrice a day for 8 days. There was an increase
in the frequency of bowel motions in both treatments (Figure A.9A-A.9B) which
could be attributed to the Giardia infection. However, severity of abdominal pain
lessened during the probiotic treatment (Figure A.9D) but the pain intensified several
times during placebo treatment (Figure A.9C). Moreover, L. fermentum VRI 003
promoted wellness (Figure A.9F) to Subject UC-11 during the study program but not
the placebo treatment (Figure A.9E).
234
A
y = 0.0018x + 1.8206R2 = 0.0065
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0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = 0.003x + 1.3469R2 = 0.0238
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = 0.0011x + 0.374R2 = 0.003
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = -0.0002x + 0.0459R2 = 0.002
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = -0.0042x + 8.2385R2 = 0.1169
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = 0.0005x + 7.7769R2 = 0.0031
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.9. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-11. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003
235
Subject UC-12
Case History
Subject UC-12 is a 29 year old, male, non-smoker Australian with a family history of
IBD. He was diagnosed with UC in 1998 and at the time of enrolment has moderate
colitis. He has an average bowel movement of 3 per day but increases if there are
flare-ups of symptoms. His standard medication includes 100 mg Imuran taken once a
day and 50 mg Luvox taken once a day.
Treatment
Group B – received L. fermentum VRI 003 treatment first then placebo
Observations
First 6 months
The participant was stressed, anxious and physically exhausted during the most part
of the first treatment period of the study because of buying and moving houses, living
alone, starting new work as well as taking frequent road trips from Queensland-
Sydney-Melbourne to Tasmania. On the fourth month of the study program, he started
on a low residue (LR) diet. Participant also noticed bleeding during bowel movements
which worsened and lasted for 19 days on the fifth month. He was prescribed to take
4g mesalazine enemas which helped stopped the bleeding. However, there are times
that he forgot to take mesalazine and blood was again found in his stool sample. He
also started drinking another probiotic product, Yakult, whilst in the study program.
Second 6 months
He continued taking Yakult until the second treatment period of the study. He did not
change the physical he exerts on himself making him stressed and exhausted by the
day. He developed diarrhea and his bleeding continued but kept taking mesalazine
enemas although there were times he missed taking them. On the 9th month he was
prescribed 20 mg prednisolone and was admitted to the emergency department of the
236
hospital. Gastroenetrologists who attended his case advised him to take 50mg
prednisolone, twice daily, continue taking 2g mesalazine enemas and Yakult and to
stop taking Merbentyl. He increased intake of Oxazepan from 15 mg to 30 mg as
prednisolone is keeping him awake. He lost 5 kgs because of the constant diarrhea
and bleeding. He then went to Dr Vickers of RPA Hospital in Sydney and the
specialist suggested an ileostomy in a few months after he build up his strength and
weight back as he has lost 6-7 kg from his 63 kg frame. He was then put on 150 mg
Imuran, 500 mg Dipstum thrice daily, 2 g mesalazine, 30 mg Serapox and 40 mg
prednisone which is to be reduced every 5 days to 20 mg. After this consultation he
drove down to Tasmania. He also went to see a naturopath and prescribed high doses
of Blackmores calcium, PC.73, magnesium, metagenix Koprex 6x daily for week and
slippery elm bark powder. After all these, he noticed formation of his stools and
disappearance of the bleeding. However, he still felt exhausted and tired until he
completed the study program.
Figures A.10A-A.10B and A.10C-A.10D show that both L. fermentum VRI 003 and
placebo treatments had beneficial effects in reducing the incidence of bowel actions
and the severity of abdominal pain of Subject UC-12. These results could have been
affected by the combination of treatments he has been on and may not actually be the
action of the investigational treatment. However, the participant generally felt well
during L. fermentum VRI 003 and not the placebo treatment (Figures A.10E-A.10F).
237
A
y = -0.0028x + 3.6674R2 = 0.0667
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0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = -0.0043x + 3.6601R2 = 0.0824
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = -0.0029x + 1.4981R2 = 0.0802
0123456789
10
0 50 100 150 200
Day
Abd
omim
al P
ain
Scor
e
D
y = -0.013x + 2.0388R2 = 0.3119
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = 0.0029x + 6.713R2 = 0.0826
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = -0.0044x + 7.2598R2 = 0.157
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.10. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-12. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
238
Subject UC-13
Case History
Subject UC-13 is a 70 year old, Australian male with no family history of IBD. He
has been a smoker for 20 years before quitting in 1975. He was diagnosed with UC in
1988. He has a moderate type of colitis and his bowel movements ranges from 5-6 per
day and approximately 20 bowel actions before he took methoblastin. His medication
for UC includes salofalk 500mg 2x a day, methoblastin 10mg 1x a wk, noten 50 mg
1x daily, astrix half a 100mg tablet daily, acimax 1-20 mg daily and bonvit physillium
husk 2X a day.
Treatment
Group B – received L. fermentum VRI 003 first then placebo treatment
Observations
No unusual gastrointestinal disturbances or changes in medications were reported by
Subject UC-13 for both treatments. His overall comment was that his stool
consistency improved and became compact, and never felt better in months whilst in
the study program.
Figures A.11A-A.11B reveals that the frequency of bowel movements decreased
during placebo treatment while L. fermentum VRI 003increased it. Nevertheless, L.
fermentum VRI 003 reduced severity of abdominal pain (Figure A.11C) and improved
general well being (Figure A.11E) of Subject UC-13.
239
A
y = 0.0068x + 4.4211R2 = 0.0496
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10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = -0.008x + 5.5642R2 = 0.0777
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = -0.0059x + 2.1605R2 = 0.0925
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = 0.005x + 0.9479R2 = 0.0911
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = 0.0077x + 7.7697R2 = 0.1705
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = -0.0005x + 9.0132R2 = 0.0027
0123456789
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.11. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-13. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
240
Subject UC-14
Case History
Subject UC-14 is a 22 year old, Australian male who suffers from a mild form of
colitis. He has no history of IBD and he was diagnosed with the disease in 2003. He
has been smoking for 2 years but gave it up when he was diagnosed with colitis. His
average bowel movement per day is 3.
Treatment
Group B – received L. fermentum VRI 003 first then placebo treatment
Observations
Subject UC-14 had antibiotic therapy and took salazopyrin twice during the 5th month
of the study program. Other than these, no other adverse events or changes have been
noted by the participant.
L. fermentum VRI 003 treatment lessened the frequency of bowel actions (Figure
A.12A) and reduced severity of the abdominal pain (Figure A.12C) while improving
general well being (Figure A.12E) of the participant. These effects appear to be
carried over during the placebo treatment (Figure A.12B, A.12D and A.12F).
241
A
y = -0.0018x + 1.3616R2 = 0.0516
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10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
B
y = -0.0029x + 1.4948R2 = 0.1211
0123456789
10
0 50 100 150 200
Day
Num
ber o
f Bow
el A
ctio
ns
C
y = -0.0062x + 0.9793R2 = 0.0996
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
D
y = -0.0004x + 0.059R2 = 0.0103
0123456789
10
0 50 100 150 200
Day
Abd
omin
al P
ain
Scor
e
E
y = 0.0055x + 7.0073R2 = 0.0925
0
2
4
6
8
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
F
y = -9E-05x + 8.0082R2 = 0.002
0
2
4
6
8
10
0 50 100 150 200
Day
Gen
eral
Wel
l Bei
ng S
core
Figure A.12. Linear regression line demonstrating the effect of intervention in the frequency of bowel actions (A-B), abdominal pain (C-D) and general well being of Subject UC-14. Blue-coloured data points represent placebo treatment and orange-coloured data points represent L. fermentum VRI 003.
242
Appendix II
Effect of placebo treatment on serum IL-10
Comparison of the effects of placebo treatment on serum IL-10 of UC patients in remission after 3 months of dosage with routine medication, a) amminosalicylates and immunosuppressants and b) corticosteroids and immunosuppressants.
Boxes represent placebo treatment administered prior to L. fermentum VRI 003 dosage (Placebo 1). Circles represent placebo administered after L. fermentum VRI 003 dosage (Placebo 2). Baseline 1 = commencement of the study; Baseline 2 = time at cross-over period.
a) Aminosalicylates + Immunosuppressants
0102030405060708090
Baseline 1 Placebo 1 Baseline 2 Placebo 2
IL-1
0 C
once
ntra
tion
(pg.
ml-1
)
b) Cortecosteroids + Immunosuppressants
-400
-200
0
200
400
600
800
1000
Baseline 1 Placebo 1 Baseline 2 Placebo 2
IL-1
0 C
once
ntra
tion
(pg.
ml-1
)
