Role of Intestinal Microbiota in Ulcerative Colitis

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Statistics PCA were generated by SAS JMP data analytical software (version 6.0.2; SAS Institute Inc., North Carolina, USA). Univariate ANOVA was used to analyze differences in ages and differences in DGGE bands of the three groups (version 5.03; GraphPad Software Inc., California, USA). Statistical analysis of the quantitative PCR data was performed with OriginPro software (version 8.1; OriginLab Corporation, Northampton, USA). Data was analyzed by univariate ANOVA. Where ANOVA indicated a significant difference multiple comparisons of means (Fisher’s least significant different test) were used to determine significant differences among disease groups, assuming equal variances. The mean of the triplicate feces sample for each subject was used for the statistical calculations. PCR measurements were log‐transformed before statistical analysis, if the data did not follow a normal distribution. Normal distribution was assessed using D'Agostino & Pearson omnibus normality test (GraphPad Prism version 5.03). Tests were considered statistically significant if P‐values lower than 0.05 were obtained. Results DGGE analysis with universal primers and sequencing of DGGE bands Microbial diversity of fecal samples was assessed by DGGE of 16S rRNA genes. The average number of bands (mean±SD) in the fecal profiles from the three groups (healthy 10.0±2.0, UC patients in remission 9.0±2.7 and UC patients in relapse 9.8±2.7) did not differ significantly (P > 0.10). Additionally, Dice cluster analysis revealed that the triplicates taken from each individual fecal sample were clustered together, demonstrating that the three extractions from each sample were similar (data not shown). Additionally, Dice cluster analysis was performed using one of the triplicate samples from each individual (See additional file 1). The dendrogram from this Dice cluster analysis showed two large clusters revealing that the DGGE profiles from the UC patients are more similar to each other than to the healthy controls. Cluster I contains ten samples all from UC patients, Cluster II contains five samples from healthy controls and two from UC patients, and a single (healthy) sample does not belong to either of the clusters. Part of this effect could be explained by the occurrence of three prominent bands present in either the profile of the healthy 8
controls or the UC patients. Band no. 1 was more prevalent in the profiles of the UC patients in relapse (5 out of 6 individuals) compared to the profiles of the healthy controls (2 out of 6 individuals) and the UC patients in remission (1 out of 6 individuals). Sequencing of the band revealed that it represented a species within the Gram‐negative genus Bacteroides (See additional file 2). Band no. 2 was more prevalent in the healthy control profiles (4 out of 6 individuals) compared to the other two UC profiles (remission; 2 out of 6 individuals and relapse; 2 out of 6 individuals). Sequencing of the band revealed that it represented a species belonging to the gram‐ negative genus Alistipes (See additional file 2). Band no. 3 was more prevalent in the UC profiles (remission; 4 out of 6 individuals and relapse; 4 out of 6 individuals) compared to the profiles of the healthy controls (0 out of 6 individuals). The band was found to represent members of the Gram‐negative genus Prevotella (See additional file 2). Principal component analysis of the relative quantities of the fecal microbiota QPCR was applied for measurement of 16S rRNA gene and 16S‐23S intergenic spacer region content of selected bacterial taxa. The data were analysed using PCA. As seen on Figure 1, the PCA plots of UC patients in remission indicated only low levels of systematic variation, with two patients showing similar profile as the healthy controls, and four patients showing similar profile as the UC patients in relapse. However, a separation between the healthy controls and the UC patients in relapse could be observed from PC1 and PC2 (29.37% and 21.82% of explained variance, respectively). This was attributed to Prevotella spp. in the first direction (PC1) in combination with Bacteroides spp., Bacteroides uniformis, Bacteroides fragilis and Bacteroides thetaiotaomicron versus Bacteroidetes, Bacteroides distasonis, Akk. muciniphila and Lactobacillus spp. in the second direction (PC2). PCA of the Gram‐negative bacteria and Gram‐positive bacteria showed a complete separation between the healthy control group and UC patients in relapse with respect to the composition of the Gram‐negative bacteria (Figure 2). The variation was mainly attributed in the second direction (PC2) with a higher positive score for Bacteroidetes, Akk. muciniphila, Desulfovibrio spp., and Bac. distasonis in the healthy controls, versus Bacteroides spp., Prevotella spp., Bac. thetaiotaomicron and B. uniformis in the UC patients in relapse. PCA of the UC patients in remission showed no clear grouping of the subjects. 9
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Role of Intestinal Microbiota in Ul
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Role of Intestinal Microbiota in Ul
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Preface Preface This thesis present
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Summary Summary The microbiota of t
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Dansk sammendrag Dansk sammendrag M
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Introduction and objectives Introdu
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List of Manuscripts Not included in
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List of contents List of Centents P
Page 18 and 19: List of Centents Methodology append
Page 21 and 22: 1. The intestinal environment Theor
Page 23 and 24: Theoretical part 5 1. The intestina
Page 25 and 26: 2. The colonic environment Theoreti
Page 27 and 28: Theoretical part 9 2. The colonic e
Page 29 and 30: Table 1: The presence of glycoside
Page 31 and 32: Theoretical part Figure 3: The colo
Page 33 and 34: 3. Inflammatory Bowel disease Theor
Page 35 and 36: Theoretical part 17 3. Inflammatory
Page 37 and 38: Theoretical part 19 4. Modulation o
Page 43 and 44: Table 4: Clinical trials on the pre
Page 45 and 46: Theoretical part 5. Production of p
Page 51: Methodology part
Page 54 and 55: Methodology part 6. Methodology, co
Page 60 and 61: Introduction Methodology part 42 Pa
Page 62 and 63: Abstract Background Detailed knowle
Page 64 and 65: depending the level of disease acti
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Page 70 and 71: The PCA of the Gram‐positive bact
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Page 74 and 75: Acknowledgements The authors thank
Page 76 and 77: Table 2 ‐ 16S rRNA gene and 16S
Page 78 and 79: 1. Firmicutes phylum 2. Bacteroidet
Page 80 and 81: Supplementary Figure S1. Dice clust
Page 82 and 83: Reference List 1. Ahmed S, Macfarla
Page 84 and 85: 32. Matsuki T, Watanabe K, Fujimoto
Page 87 and 88: Methodology part Paper 2 Fecal lact
Page 89 and 90: Fecal lactobacilli and bifidobacter
Page 91 and 92: Introduction The mucus layer lining
Page 93 and 94: efore enrolment and there was no si
Page 95 and 96: (Bio‐Rad Labs, Hercules, Californ
Page 97 and 98: Microbial community analysis using
Page 99 and 100: difference from the luminal microbi
Page 101 and 102: that C. coccoides group and C. lept
Page 103 and 104: Table 1 ‐ 16S rRNA gene of phylum
Page 105 and 106: Table 2 ‐ Preference of bacterial
Page 107 and 108: Figure 1. A) Schematic overview of
Page 109 and 110: A. B. Figure 3. Principal component
Page 111 and 112: 15. Fooks LJ, Gibson GR. (2002) In
Page 113 and 114: 47. Ouwehand AC, Suomalainen T, Tol
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8338 VIGSNÆS ET AL. APPL. ENVIRON.
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Methodology part Introduction The a
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Journal of Agricultural and Food Ch
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Methodology part Paper 5 Paper 5 Ma
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Appl Microbiol Biotechnol (2011) 90
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Methodology part Paper 6 Tailored e
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Process Biochemistry 46 (2011) 1039
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Table 1 List of enzymes. Enzyme Sou
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J. Holck et al. / Process Biochemis
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Intensity 50 0 C1 175.05 J. Holck e
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J. Holck et al. / Process Biochemis
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[29] Bauer S, Vasu P, Persson S, Mo
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Methodology part 150
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Methodology part 152 Appendix 1 hor
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Appendix 3 Methodology part Appendi
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Methodology part 156 Appendix 3
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7. Discussion and perspectives Disc
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Discussion and conclusion 161 7. Di
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References References Abreu,M.T., V
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References Derrien,M., Vaughan,E.E.
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References Haarman,M. and Knol,J. (
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References Lantz,P.G., Matsson,M.,
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References Matsuki,T., Watanabe,K.,
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References Pullan,R.D., Thomas,G.A.
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References Tannock,G.W. (2010). Ana
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National Food Institute Technical U
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Role of Intestinal Microbiota in Ulcerative Colitis

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