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9/6/2010 1 Group 2: GUT MICROBIOTA AND DISEASE Castelldefels August 29, 2010 GUT MICROBIOTA AND DISEASE Francisco Guarner, Chair University Hospital Vall d'Hebron Spain James Versalovic, Co-Chair Texas Children'sHospital USA John Bienenstock McMaster Brain-Body Institute Canada Dusko Ehrlich INRA France Inna Eiberger Merck Selbstmedikation GmbH Germany Kirsty Hunter Nottingham Trent University UK Annett Klinder University of Reading UK Krishna Madduri The Dow Chemical Company USA Chays Manichanh University Hospital Vall d’Hebron Spain Peggy Martini Kraft Foods USA Ravi Menon General Mills Inc USA Alicia Murcia University Hospital Vall d´Hebron Spain Arthur Ouwehand Danisco Finland Junjie Qin Beijing Genomics Institute China Yehuda Ringel University of North Carolina USA Ian Rowland University of Reading UK Saskia van Hemert Winclove Netherlands Johan van Hylckama Vlieg Danone Research France
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Group 2: GUT MICROBIOTA AND DISEASE - ISAPP · Group 2: GUT MICROBIOTA AND DISEASE Castelldefels August 29, ... Ian Rowland University of Reading UK ... 2 GUT MICROBIOTA AND DISEASE

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Page 1: Group 2: GUT MICROBIOTA AND DISEASE - ISAPP · Group 2: GUT MICROBIOTA AND DISEASE Castelldefels August 29, ... Ian Rowland University of Reading UK ... 2 GUT MICROBIOTA AND DISEASE

9/6/2010

1

Group 2:

GUT MICROBIOTA AND

DISEASE

Castelldefels

August 29, 2010

GUT MICROBIOTA

AND DISEASE

Francisco Guarner, Chair University Hospital Vall d'Hebron Spain

James Versalovic, Co-Chair Texas Children's Hospital USA

John Bienenstock McMaster Brain-Body Institute Canada

Dusko Ehrlich INRA France

Inna Eiberger Merck Selbstmedikation GmbH Germany

Kirsty Hunter Nottingham Trent University UK

Annett Klinder University of Reading UK

Krishna Madduri The Dow Chemical Company USA

Chays Manichanh University Hospital Vall d’Hebron Spain

Peggy Martini Kraft Foods USA

Ravi Menon General Mills Inc USA

Alicia Murcia University Hospital Vall d´Hebron Spain

Arthur Ouwehand Danisco Finland

Junjie Qin Beijing Genomics Institute China

Yehuda Ringel University of North Carolina USA

Ian Rowland University of Reading UK

Saskia van Hemert Winclove Netherlands

Johan van Hylckama Vlieg Danone Research France

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GUT MICROBIOTA AND DISEASECastelldefels 2010

Aims:

• To define traits/characteristics/structure of a

“normal” gut microbiota

• To review clinical conditions associated with

dysbiosis. Is dysbiosis a pathogenetic factor of

disease?

Dusko Ehrlich:

Definition of a normal structure of the human gut microbiota

Jim Versalovic:

The intestinal microbiota in children

Junjie Qin:

Microbiota in Chinese Type II Diabetes patients and control individuals

Udi Ringel:

Microbiota in IBS (human studies)

John Bienenstock:

Microbiota and IBS (animal studies)

Ian Rowland:

Microbiota in colon cancer

Francisco Guarner:

Microbiota in IBD

Kirsty Hunter:

Gut microbiota in physical stress

Chaysavanh Manichanh:

Transplantation of gut microbiota in experimental models

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The « healthy » intestinal microbiome

Can we define health? A question for doctors!

Health not black and white. There is a range. Arthur C. Ouwehand

Health microbiome may also not be black and white - can’t we expect that there is a range?

Ehrlich, ISAPP 2010

The Human Gut Metagenome

# of genes

Non-redundant gene set 3,299,822

Common (>50% of individuals) 294,110

Rare (<20% of individuals) 2,375,655

Average gene set per individual 590,384

Qin et al, Nature 2010

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The landscape of « healthy » intestinal

microbiome

What is the form of the landscape?

- a continuum – any combination of microbial

species is possible

- discrete states – some microbial community

structures are favoured

Ehrlich, ISAPP 2010

The Distal Gut Microbiomes of Healthy Children

Differ from Healthy Adults

• Healthy

Children

• Healthy

Adults

V1V3 regions only (1 replicate/sample)

11 samples from healthy adults

(18-40 yo) sequenced

by HGSC at Baylor.

29 samples from healthy children

(7-12 yo) sequenced by HGSC

at Baylor

454 16S metagenomic sequencing

14-16K reads per sample

Versalovic et al, 2010

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Gene catalog update

884,577 novel genes assembled from Chinese samples

124 EU samples

73 CN samples

Contribution to gene catalog Contribution to sequencing read

Qin et al, 2010

Intestinal Microbiota and IBS

• Only few studies

=> Results are not consistentand sometimes conflicting

• Mostly small studies

• Considerable limitations (lack of information on subtypes, use of antibiotics, hospitalized patients)

Intestinal Microbiota and IBSIntestinal Microbiota and IBS

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• Significantly lower levels oflactobacillus ssp. in D-IBSvs. C-IBS

Malinen et al, Am J Gastroenterol 2005

qPCR analysis

• Significantly higher levelsof veillonella spp. in C-IBSvs. controls

• RT-PCR of 20 bacterialgroups covering 300different species

Intestinal Microbiota and IBSIntestinal Microbiota and IBS

• The observations seen on theIBS subtype analyses did nothold on the mixed group (pooled samples) analysis.

Malinen et al, Am J Gastroenterol 2005

• Results were reported by genomic DNA in 1g of wet weight fecal samples

qPCR analysis

=> Results may depend on theIBS subtype.

Intestinal Microbiota and IBSIntestinal Microbiota and IBS

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We need

Clinical implications

Conclusions and Personal Perspective

• High quality studies to better characterize the intestinal microbiota in well definedpatients with IBS.

• More studies to help determine the reasons for the observed alteration in themicrobiota (cause or effect?).

• More studies on the host-microbiota interaction to help determine the mechanismsby which the intestinal microbiota affect GI function and functional GI symptoms.

• The current data together with recent studies indicating beneficial effects of manipulation of the intestinal microbiota by prebiotics, probiotics, and antibiotics,provide the rational for targeting intestinal bacteria in the treatment of IBS.

• The enthusiasm for the use of this approaches in IBS has outpaced the scientificevidence.

FGID - Bacteriological and Luminal Factors

Ringel, ISAPP 2010

Kamiya et al: Gut 2006 55 191

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Effect of lactobacillus reuteri on HR response to CRD (80 mm Hg)

92

94

96

98

100

102

rest 10s 20s 30s 40s 50s 60s post 1 post 2

time

% r

esti

ng

hea

rt r

ate broth

lactobacillus

Kamiya et al: Gut 2006 55 191

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GF vs CV animalsGF vs CV animals

�� In CV rats tumour formation more rapid In CV rats tumour formation more rapid and more [malignant] and more [malignant] tumourstumours [Reddy 1974][Reddy 1974]

�� CV rats develop more & larger CV rats develop more & larger tumourstumours[[VanucciVanucci 2008]2008]

�� TcellTcell receptorreceptor--/p53/p53-- mice. CV mice higher mice. CV mice higher tumour incidence (70%) than GF (0%) tumour incidence (70%) than GF (0%) [[KadoKado2001]2001]

�� HFA rats had 2x more DNA adducts than HFA rats had 2x more DNA adducts than CV rats CV rats [[RumneyRumney 1993]1993]

Bacteria & colorectal cancer-2Bacteria & colorectal cancer-2

Molecular methods:Molecular methods:

��CRC risk (CRC risk (vsvs healthy controls) associated withhealthy controls) associated with

�� ��DiversityDiversity of C of C leptumleptum, , C.coccoidesC.coccoides subgroups subgroups (no effect on (no effect on BactBact fragilisfragilis group) group) (Scanlon et al Environ (Scanlon et al Environ MicrobiolMicrobiol 10:1382; 2008; DGGE)10:1382; 2008; DGGE)

�� ��ErecErec; ; ��FprauFprau (4x) ((4x) (nn--Bu producers)Bu producers) ��EntEntfaecalisfaecalis((BalamuruganBalamurugan et al J Gastro et al J Gastro HepatolHepatol 23, 1298, 2008; RT PCR)23, 1298, 2008; RT PCR)

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Summary and conclusionsSummary and conclusions

�� Gut Gut microfloramicroflora is implicated in is implicated in aetiology of colorectal canceraetiology of colorectal cancer

�� Gut microbial products potentially Gut microbial products potentially involve NOC, sulphide, sec BA, involve NOC, sulphide, sec BA, ammonia, phenols, SCFAammonia, phenols, SCFA

�� Modification of gut Modification of gut microfloramicroflora by by propro-- and preand pre--bioticsbiotics alters canceralters cancer--associated events in humansassociated events in humans

Rowland, ISAPP 2010

GUT MICROBIOTA AND DISEASECastelldefels 2010

PATHOGENESIS OF IBD

Asquith & Powrie, J Exp Med 2010

Non-pathogenic Intestinal Microbia

Dysregulated

Immune Response

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Reduced Microbial Gene Diversity in IBD

Qin et al, Nature 2010

IBD CONCLUSIONSExpression of IBD may depend on 3 conditions:

• Genetic susceptibility

• Reduced diversity and instability of the intestinal ecosystem

• Dysbiosis

Imbalance between aggressive vs. protective bacteria associated with clinical relapse

Guarner, ISAPP 2010

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Exercise and immunodepression – the response to psychophysical stress

• Prolonged strenuous exercise results in:

– Increased circulating cortisol

– Postexercise neutrophilia, reduced responsiveness to LPS

– Marked decrease in circulating NK cells and cytotoxic capacity

– Reduced suppressor T cell number, complement, mucosal IgA

– Decreased expression of Toll-like receptors 1, 2, and 4 on monocytes

A heavy schedule of training and competition can lead to transient immune impairment in athletes

Nieman, IJSM, 1994

Hunter, ISAPP 2010

What do we know? Research in athletes to date is limited and inconclusive

• Clancy et al, B. J. Sports Med. 2006

– Fatigued athletes showed sig. reduced secretion of IFN – γ from T cells v healthy athletes. Fatigued athletes who consumed L. acidophilus for a month had levels comparable with healthy athletes

• Kekkonen et al, Int. J. Sport Nut. Ex. Metab. 2007

– Marathon runners consumed either L. rhamnosus GG or placebo for 3 months training then undertook a marathon. No difference in respiratory illness or GI symptoms during three month training. Shorter duration of GI symptoms for 2 weeks post marathon with placebo (no difference in GI episodes)

• Cox et al, B. J. Sports Med., 2008

– 20 elite athletes, crossover trial, L. fermentum VRI-003 v placebo

– No days respiratory illness – 30 during probiotic tmt v 72 during placebo, illness severity sig lower during probiotic tmt (p=0.06)

• Prebiotics – nothing to date

Hunter, ISAPP 2010

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EXPERIMENTAL DESIGN

• Fecal samples, 16S - V4 - pyrosequencing (>2000 sequences/sample) and real time PCR

• Sequences were analyzed with the QIIME pipeline, which providestaxonomic and phylogenetic profiles and comparisons of the 59 samples

Manichanh et al, Genome Research 2010

CLUSTERING BY TREATMENTPrincipal coordinates analysis (PCoA)

Manichanh et al, Genome Research 2010

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GUT MICROBIOTA

AND DISEASE

CONCLUSIONS:

• Too early to define characteristics/structure of a

“healthy” gut microbiota.

• Age related differences confirmed by

metagenomics.

• Microbial signatures associated with disease are

being identified by IHMC projects.

• Prospective studies needed to provide

information about cause-effect relationships.

• Interventions to change gut microbiota may

become relevant to prevent risk of chronic disorders

(Type 2 Diabetes, IBD, IBS, Atopy, Cancer).