Module 3.4: Microbiome & inflammation in the gut

Question 1

Epidemiology of CeD

Answer 1

1% prevalence
F:M 2:1
Peak incidence 40-50y

Question 2

Clinical Features of CD

Answer 2

• Classical CD: presence of significant GI symptoms abdominal pain, diarrhoea with or without malabsorption.
• Symptomatic CD: anaemia (iron deficiency and chronic disease), osteoporosis and unexplained fatigue.

• Conditions associated with CD
o Dermatitis Herpetiformis – gluten induced sensitivity in the skin
o Type 1 diabetes
o Autoimmune thyroid disease

Question 3

Diagnosis of CD: Serology Tests

Answer 3

• > 90% of patients with CD have IgA antibodies to a protein called tissue transglutaminase 2 (TTG2)
• IgA TTG2 can be detected using either ELISA or Indirect Immunofluoresence tests.
• The TTG2 antibodies disappear when a patient goes on a gluten free diet: can be used to check if patients are compliant with their diet

Question 4

Diagnosis of CD: Histology

Answer 4

– Gold standard

• Loss of normal villus architecture: total or partial villus atrophy
• Increased CD8+ T cell intraepithelial infiltrate
• Increased mononuclear cell infiltrate in lamina propria (CD4 T cells)
• Crypt hyperplasia

Question 5

Susceptibility of CD – Genetics

Answer 5

• CD clusters in families (85% concordance in MZ studies)
• 5-11% of 1st degree relatives will have CD
• 2.5% of 2nd degree relatives
• 90% of patients with CD carry genes encoding the HLA-DQ2 allele
• There is an association between prevalence of CD and frequency of wheat consumption and risk MHC haplotypes

Question 6

summarise the MHC system

Answer 6

• Class I
o All nucleated cells
o Single polymorphic alpha chain bound to b2-microglobulin
o 3 major subtypes: A, B, C
o Peptide-MHC complex recognised by CD8 T cells

• Class II
o APC (dendritic cells, B-cells, monocytes), epithelial cells of thymus
o Consists of polymorphic alpha and beta chains
o 3 major subtypes: DR, DQ, DP
o Peptide-MHC complex recognised by CD4 T cells

•	Nomenclature of system:
o	Name of gene (DQ)
o	Alpha or beta chain (a/b)
o	Family number of alpha or beta chain:
	Asterisk * (0000)
	First 2 digits: allelic variant (based on similarity to other alleles or serological specificity)
	Last two digits: order of discovery of allelic variant
•	DQA1*05:01	DQB1*02:01

Question 7

Describe the biology of the MHC system

Answer 7

o T cell recognition of processed peptides depends on ability of MHC encoded proteins to bind small peptides
o Self peptide-MHC complexes select T cell repertoire in the thymus (assess tolerance)
o Non self-peptide-MHC complexes are recognised by T cells during an adaptive immune response (host defence)
o MHC class I/II are extremely polymorphic: each allele encodes HLA proteins with different peptide properties
 Immune system can recognise and bind to enormous number of foreign proteins
 Immune evasion to all MHC molecules is extremely unlikely

o HLA gene expression is co-dominant: protein from both chromosomes expressed on surface of cell – most individuals heterozygous for each HLA locus
o HLA gene sequences can be inherited in DISCRETE GENE SEGMENTS – which encode for several different alleles: the HLA haplotype
o Frequency among NW Europeans of HLA-A0101 is 20% + HLA-B0801 is 16% + HLADRB1*0301 is 16%
o If each allele was inherited independently, the frequency of this haplotype would be:
 20 x 16 x 16 = 0.052%
 The observed frequency of this haplotype, however is 9% - showing that genes are inherited together in gene segments to have combined effect
 The linkage disequilibrium difference value (difference between frequency observed for a particular combination of alleles and that frequency expected for each individual allele) is 9 – 0.05 = 8.5
 PS: this is known as the autoimmune haplotype as it has been tied to a number of autoimmune diseases

Question 8

Describe the structure of the MHC Class II

Answer 8

o Variable regions in alpha and beta chains of the MHC Class II protein bind to peptide fragments
o MHC Class II region highly polymorphic as a as a result of differences in amino acids that bind to the floor or sides of peptide binding groove
o HLA Class II proteins
 HLA DRA1 (1 allele) can pair with HLA DRB1 (500 alleles) and HLA DRB3 (60 alleles)
 HLA DQA (25 alleles) pairs with HLADQB (72 alleles)
 HLA DPA (16 alleles) pairs with HLA DPB (118 alleles)
o Each individual may express 4 different HLA DQ polypeptide chains derived from genes inherited from both maternal and paternal chromosomes
o The beta2 domain binds to CD4 molecules on T cells
o The region is highly polymorphic as a result of differences in amino acids that bind to the floor or sides of the peptide binding groove
o Each individual may express 4 different HLA DP and DQ proteins and 4 distinct HLA-DR protein based on pairing between gene products of both maternal and paternal chromosomes
 HLA DRA1 can bind with HLA DRB1 + HLA DRB3
 HLA DQA pairs with HLA DQB
 HLA DPA pairs with HLA DPB

Question 9

HLA genes in CD

Answer 9

• DQ2.5 (DQa10501, DQb10201) is expressed in 90% of patients with CD – expressed 30% in population
• DQ8 (DQa10301, DQb10302) expressed in 5% of CD patients
• In the remaining 5%, at least one of the proteins encoded by either DQ2 and DQ8 expressed by APC
• Expression of HLA DQ2 allele explains 40% of RISK for CD
• Significance: HLA-DQ2 and HLA-DQ8 bind negative charged peptide fragments (see next page).

HLA DQ2 homozygous individuals have a 5X increased risk of developing CD than DQ2 heterozygous subjects (hence thought that there is a threshold i.e. the more DQ2.5 protein, the greater the likelihood of CD development as one is more likely to be able to switch on T-Cells)

• This is due to 4 receptors being produced from the 4 allelic regions from the 2 chromosomes from the paternal and maternal side
• The more DQ2 proteins an individual expresses, the more likely that one will induce gluten specific immune responses to CD4 T cells

Question 10

Non-HLA genes in CD

Answer 10

• GWAS DNA microarray studies have identified 42 non HLA regions implicated
• Contribution of individual CD associated SNP to disease risk is small (however overall effect explains 14% of genetic susceptibility to CD)
• Most of the genes are associated with T and B function and are known risk factors for other autoimmune risks (T1DM, RA)
• RNA sequencing studies have also identified non-coding RNA regions with CD
• Factors involved:
o Dendritic cell activation
o T cell stimulation
o Gluten specific Th cell function
o Recruitment of B cell and antibody production
o Cross talk between CD8 T cells and GI epithelial cells

Question 11

Gluten Chemistry

Answer 11

• Can be separated into 2 main fractions
o Alcohol soluble gliadins
o Alcohol insoluble glutenins

• Rich in proline (15%) and glutamine (30%) = prolamine

• Gliadins can be subdivided into alpha, gamma and omega subgroups
• Glutenins consist of LMW and HMW fractions

• High proline content increases resistance of gluten to enzymatic breakdown in GIT
• Means that gluten peptide fragments have increased chance to bind to HLA-proteins implicated in CD
• Glutamine and proline amino acids are often adjacent to each other – this grouping is an excellent substrate for an inflammatory enzyme: tissue transglutaminase 2 (TTG2)
• The spacing of glutamine and proline amino acids which are often adjacent to each other facilitates deamidation of glutamine to negative charged glutamic acid
• Exposure of gluten extracts containing several thousand different peptides to TTG2 contributes to pathogenesis of CD as it:
o Cross links proteins  glutamine to lysine
o Deamidates glutamine to glutamic acid

• These deamidated (-vely charged) gliadin peptides can be loaded on HLADQ2-DQ8 molecules

Question 12

Immunology of HLA DQ2 and DQ8 in CD

Answer 12

• DQ2.2, DQ2.5 and DQ8 bind readily to modified gluten peptide fragments (HLA DQ8 can also bind to native gluten polypeptides)
• The binding of gluten polypeptides to HLA-DQ2.5 is very stable – lasts up to 4d
o DQ2.5 = makes it the most susceptible as it forms the most stable compound of the HLA DQ alleles identified to be involved in pathogenesis
• Means that there is sufficient time for APC to bind gluten peptides in the intestinal mucosa: migrate to mesenteric lymph nodes and stimulate naïve T cells
• HLA DQ2.5 and DQ8 binding to cognate TCR encourages the recruitment of cross reactive T cell immune responses to gluten polypeptides
• Patients with CD tend to use the same T cell receptors to respond to modified gluten peptides – public immune response

CD4 T cells
• CD4 T cell only recognize a very small number of gluten peptide fragments
o 25 for HLA DQ2.5 and 10 for HLA-DQ8
• Almost all patients with CD have CD4 T cell immune responses to a- and w- gliadins, reactivity to g-gliadins and glutenins occurs much less frequently
• One reason is that a-gliadins are much better substrate for TTG-2 than other gluten gliadin and glutenin components
• This immunodominant gliadin epitope lies within the amino acid 57-75 of alpha gliadin which contains multiple HLA-DQ2 binding sites.

Question 13

Adaptive and Innate Immunity in CD

Answer 13

• Gluten does not usually promote an inflammatory T-cell response
• The vitamin A metabolite retinoic acid usually promotes immune tolerance however in CD it may promote intestinal inflammation
• Retinoic acid (RA) and TGF-b promotes T regulatory immune responses (suppresses intestinal inflammation)
• In the presence of high levels of IL-15 and IL-18 retinoic acid promotes IL-12 secretion by DC which induce pro-inflammatory IFN-g (Th1) CD4 immune responses.
• Trigger for induction IL-15 is unknown but postulated: gluten peptide fragment, viruses (IFN-a), and alteration in the intestinal microbiome.

Question 14

CD4 T-cells and Immune Pathology in CD

Answer 14

• Gluten specific CD4 T cell make up 0.5-2.0% of intestinal T cell in CD, however they still persist at much lower levels in patients on GFD
• Gluten specific CD4 T cell are not thought to cause tissue damage directly in CD
• Activated CD4 T cells secrete IFN-g and IL-21 which damage GI epithelial cells and also activate CD8 intra-epithelial T cells (more important and more likely to be the causative cell contributing to damage)
• Activated CD4 T cells provide B cell help to make deamidated gluten specific and TTG-2 specific IgG and IgA antibodies.

Question 15

Antigen Presenting B-Cells in CD

Answer 15

• Deamidated gluten peptides are presented by HLA DQ2 or DQ8 expressed on surface of either
o A) TG-2 reactive B cells OR
o B) Deamidated gluten reactive B cells to CD4 T cells
• Co-operation between gluten reactive T cell and B cell results in activation of both lymphocyte subsets
• Activated B cell differentiate into antibody producing plasma cells
• CD4 T cell proliferate and clonally expand

Question 16

TTG-2 IgA Antibodies in CeD

Answer 16

• Anti-TTG2 IgA are found in intestinal tissue and extra-intestinal sites before development of overt CD  though to potentially cause damage in CD
• In active CD: 5-25% of intestinal plasma cell target TTG2 and de-amidated gluten peptides (DGP)
o TTG2: DGP ratio is 10: to 1
• Antibody responses to TTG2 and DGP show following features
o Restricted use of B cell receptor variable heavy and light genes repertoire
o Relatively few somatic hyper-mutations in variable light and heavy gene
o Antibody response to TTG2 and DGP lost following introduction of GFD
o Finding suggestive of T cell independent, extra-follicular antibody response
• B cell epitopes lie within or are in close proximity to immuno-dominant T cell epitopes raising the possibility that antibodies also influence CD4 T cell recognition.

Question 17

CD8 T cells in CeD

Answer 17

• In coeliac, intraepithelial CD8 upregulate expression of NK proteins  NKG2D and NKG2C/CD94 is induced on intraepithelial CD8 T cells by IL-15 secreted from damaged epithelial cells and by inflammatory
• CD8 can then differentiate into cells with NK cell like proteins
• This population can directly bind to MIC-A to HLA-E expressed on surface of damaged or activated enterocytes results in epithelial cell apoptosis (cell death)
• IL15 is important for the development of CD8 NK like cells + upregulates HLA molecules on damaged epithelial cells to be targets for NK killing proteins  i.e. promote CD8 T-cell activation against self-proteins
• End result: CD8 cytotoxicity to SI epithelial cells and possible cytokine (IFNgamma) induced damage

Question 18

Innate Immunity in CD

Answer 18

• Activation of dendritic cells
o Expression of IL-15
o Induction of high level IL-12 cytokine secretion  promotes T/B-cells to produce IFN gamma
o Association of CD with SNP in interferon regulatory factor 1 (regulates anti-viral and bacterial immune resposnes)
• Expression of stress proteins (markers of cell damage or activation) by gastrointestinal epithelial cells
o MIC-A and the non classical HLA-E protein
o IL-15
• Secretion of inflammatory cytokines IL-15 and IFN-a

Question 19

Factors that initiate Damage in CD

Answer 19

o Induce expression of MICA and HLA-E on surface of epithelial cells
o Upregulate expression of IL-15/IL-15 by epithelial cells
o Disruption of epithelial tight junction network
o Peptide fragments differ from those inducing CD4 T cell immune responses

Question 20

Role of IL-15 in CeD

Answer 20

• Secretion of IL-15 by damaged intestinal cells  lowers TCR activation threshold for intra-epithelial CD8 T cells  increases the expression of NK cytotoxic protein by intra-epithelial CD8 T cells
• Promote survival and expansion of intra-epithelial CD8 T cells
• Secretion of IL-15 by myeloid cells and Dc in the lamina propria
• Induces expression of IL-12 by intestinal DC
• Promotes pro-inflammatory CD4 Th1 rather then tolorogenic CD4 FoxP T cells

Summary: IL15, IL21 and Type 1 IFN are the key mediators of inflammatory immune responses in CD.

Question 21

Other Environmental Factors Implicated in CD

Answer 21

• Breast feeding and infant feeding practice
o Results of initial epidemiological studies not confirmed  thought that timing of gluten introduction is no longer a trigger

• Microbiome
o Alteration in composition even in patients on GFD
o Increase in bifdobacterium bifidum content

• Gastro-intestinal infections
o Reovirus: in murine models activate DC and induce IRF1 immune responses (does not cause disease in humans)
 Subset of patients with CD have increased exposure to reovirus and activating SNP in IRF-1
o Rotavirus
o Camplyobacter

Question 22

Current Expectations for iBD Therapy

Answer 22

• Induce clinical remission
• Maintain clinical remission
• Improve patient quality of life
• Heal mucosa
• Decrease hospitalisation/ surgery and overall cost
• Minimise disease and therapy related complications

Question 23

Types of UC and Spectrum of Disease

Answer 23

• Variability in extent and severity of disease
• Some may have only proctitis whilst others can have extensive pancolitis  over time patients can also develop more severe disease for example, a patient can first present only with proctitis but eventually develop pancolitis within a few years
• Severity can also vary

o Mild  moderate  severe (severe = deep ulceration, edema, narrow lumen and spontaneous bleeding)

• Dependent on disease severity and extent of disease, these patients are treated differently

Question 24

Types of Crohn’s Disease and Spectrum of Disease

Answer 24

• Same principle as UC  variability in extent and severity of disease will mean different treatments
• Types: most severe from a patient perspective is perianal disease
• Severity: mild (aphtous ulcers)  moderate  severe (edema, cobblestoning of colonic mucosa, ulceration)

Question 25

Summary of Medications in IBD

Answer 25

• Steroids
• 5-ASAs
• Immune suppressants
• Biological Therapies
• Others – diet, FMT, antibiotics, probiotics, novel agents (currently in development)

Question 26

Mechanism of action of steroids

Answer 26

Diffuse and bind in nucleus to glucocorticoid responsive elements (GRE)

GRE interacts with specific DNA sequences  inhibits NFkB

Increase in anti-inflammatory gene products

Block pro-inflammatory genes

• Binding to and blocking promoter sites of pro-inflammatory genes e.g. IL1α and IL1β
• Inhibition of the synthesis and secretion of inflammatory cytokines
• Suppressing production of inflammatory eicosanoids in phagocytic cells
• Suppressing the synthesis of cyclooxygenase-2 (COX-2), the inducible isoform of cyclooxygenase primarily responsible for production of prostaglandins at sites of tissue injury and inflammation

Question 27

Mode of Delivery of steroids in IBD

Answer 27

o	IV (hydrocortisone 100mg QDS), oral (prednisolone 40mg OD, budesonide 9mg per day), rectal (enemas/suppositories)
o	Short-term use, as a bridge in acutely unwell patients

Question 28

Side effects of steroids

Answer 28

MAIN PROBLEM

Cushingoid features: euphoria/psychotic symptoms (steroid induced psychosis), HTN, increased abdominal fat and obesity, skin thinning, poor wound healing, muscle wasting, osteoporosis/osteonecrosis, increased susceptibility to infection, risk of gastric ulceration, interference with glycaemic control

Question 29

Mechanism of action of 5-ASA Therapy in IBD

Answer 29

Several pathways implicated

Inhibition of proinflammatory cytokines  IL-1 and TNF-a

Inhibition of the lipo-oxygenase pathway i.e. prostaglandin and leukotrienes

Scavenging of free radicals

Inhibition of NF-kB/ TLR via PPAR-gamma induction (perioxisome proliferator activated receptor-gamma)

Some immunosuppresive activity – inhibiting T cell proliferation, activation and differentiation

Impairs neutrophil chemotaxis and activation

Question 30

Mode of delivery of 5-ASA in IBD

Answer 30

o Oral, modified release, rectal (suppositories, liquid and foam enemas)
o Generally not easily absorbable  different modes of delivery allow for topical administration
o More evidence of effects in UC over Crohns  why? mucosal effects (drug-wise) hence works better in a mucosal disease such as UC

Question 31

Side effects of 5-ASA

Answer 31

generally well-tolerated and are very good at both inducing and maintaining remission in many patients. However, many side effects have been noted:

Intolerance, diarrhoea, renal impairment, headache, malaise, pancreatitis, pneumonitis

Renal Impairment: Patel H et al, Can J Gastroenterol, 2009  found dose-dependent and duration-dependent decline in renal function for patients using 5-ASAs in 171 pts

Question 32

Exapmples to Immune Modulators in IBD tx

Answer 32

2nd Line Therapy

• Azathioprine/ 6MP – mechanism, mode of delivery, speed of action, side effects
• Thioguanine
• Methrotrexate (MTX)– mechanism, mode , speed, side effects
• Ciclosporin

Question 33

Azathioprine/6-Mercaptopurine in IBD Tx

Answer 33

• Mechanism of Action
o 6-TG interfere with adenine and guanine ribonucleotide production
o Results in reduced numbers of B and T-lymphocytes, Immunoglobulins and interleukins
o Another pathway potentially results in apoptosis of T-cells

• Side effects
o GI disturbances (nausea, vomiting), hepatotoxicity? Nodular regenerative hyperplasia, infection, allergic-type reaction fever, rash, arthralgias, myalgias, fatigue, pancreatitis, BM suppression, malignancy (lymphoma)

•	Checks
o	TPMT (heterozygotes will have reduced activity of this enzyme leading to increased toxicity)
o	Hep B and C, HIV
o	Chicken pox
o	Vaccinations
o	TB
All infections mentioned above need to be checked to increased risk of reactivation of diseases.
o	Frequent bloods on starting 
o	Maintenance bloods

Need LFTs and BM monitored due to side effects

Question 34

Methotrexate in IBD Tx

Answer 34

•	Clinical uses
o	Takes 3 months
o	Need history re. liver abnormalities
o	Monitor LFTs, FBC
o	Advise NO pregnancy (teratogenic)
o	Folic acid supplements (reduces side effects) 
o	Weekly dose

• Mechanism of Action – unclear
o Thought to interfere with DNA synthesis and cell reproduction
o Increased adenosine levels (anti-inflammatory)
o Increased apoptosis of peripheral T-cells observed

• Side effects – similar to Azathioprine
o Rash, nausea, mucositis, diarrhoea, BM suppression, hypersensitivity pneumonitis, raise LFTs, hepatic fibrosis/cirrhosis, known abortifacient, no documented increased risk of lymphoma or cancer

N.B: Methotrexate and Azathioprine take a long time to work hence they are only considered for maintenance therapy, hence need 5-ASAs/steroids needed to induce remission.

Question 35

Ciclosporine in IBD

Answer 35

• Clinical uses
o Used in UC  IV infusion in acute severe not responding to IV steroids (i.e. salvage treatment before patient would have a colectomy as pt not responding to usual treatment)
o aTNF is an alternative  similar efficacy in head-to-head trials
o Continuous infusion (or oral as a bridge)
o Can take 8 days to work

• Mechanism of Action
o Blocks transcription of cytokine genes in activated T cells
o Binds to calcineurin resulting in reduced IL2
o IL2 stimulates T cell production

• Side effects
o Nephrotoxicity, hepatotoxicity, HTN, neurological excitability, immune suppression, hirsutism, gum hypertrophy

N.B: Lots of side effects meaning pts need to be carefully monitored and these drugs are only used in the short term to induce remission and not maintain remission.

Question 36

Biological Therapies in IBD

Answer 36

• Anti-TNFα – infliximab, adalimumab,
• Anti- α4β7 – Vedolizumab
• Anti-IL12/IL23 – Ustekinumab
• More and more on the way – and some that have been and gone!

N.B.: Efficacy of biological therapies: 25-30% remission, 50-60% response (pts feel better but not necessarily full remission)

Question 37

The effects of TNFa

Answer 37

MACROPHAGES - increased proinflammatory cytokines and chemokines –> increased inflammation

ENDOTHELIUM - increased adhesion molecules –> increased cell infiltration

FIBROBLASTS

• increased acute phase response –> increased CRP in serum
• increased metallopreoteinase synthesis + decreased collagen production –> tissue remodeling

EPITHELUM - increased ion transport and permeability –> compromised barrier function

Question 38

Types of anti-TNFs for IBD Tx

Answer 38

•	Types:
IV infliximab (remicade), Remsima.
	in hospital – less frequent
	Induction 0,2,6 weeks
	Maintenance 8 weekly
S/C adalimumab (humira)
	160/80/ 40mg EOW 
	At home – more frequent

Used in Crohns > UC

Question 39

proposed mechanism of action of anti-TNFs

Answer 39

o Direct neutralisation of soluble and membrane-bound (predominant factor in IBD) TNF-alpha
o Reverse signalling
o Antibody-dependent and complement dependent cytotoxicity
o Upregulation of regulatory macrophages and downregulation +/- direct and indirect apoptosis of effector cascades

Question 40

Side effects of anti-TNFs

Answer 40

o Opportunistic infections, infusion or site reactions, infusion reactions, neutropenia, infections, demyelinating disease, heart failure (HF), cutaneous reactions (including psoriasis), malignancy, induction of autoimmunity
o Similar checks needed as with AZT/Methotrexate

•	Caution
o	Infection (particularly TB)
o	Cancer
o	Old age
o	Other comorbidities (COPD, HF, SLE, MS)
o	Side effects (psoriasis, SLE, MS)

•	Monitoring
o	Clinical response (primary non-response and secondary loss of response)
o	Drug reactions
o	Opportunistic infections
o	Drug levels
o	Antibody levels
o	De-escalation therapy

Question 41

Combination Therapy in IBD

Answer 41

AZA/6MO and anti-TNFa act synergistically

o Combination is superior in inducing and maintaining response and remission
o Reduces rates of antibody formation

Question 42

Describe Ustekinemab uses in IBD

Answer 42

anti-IL12/23

• Mechanism of Action
o Inhibits the common p40 subunit of Il12 and Il23
o Inhibits these cytokine’s receptors on T cells, NK cells and AP cells
o Currently licensed for the treatment of psoriasis
o Newly licensed for Crohn’s disease early 2017

• Dosage
o IV loading dose of 6mg/Kg
o Maintenance 8-12 weekly S/C
o Well tolerated and similar or slightly less major adverse reactions to anti TNFs

• Efficacy: similar to anti-TNF
• Good for pts with specific contra-indications (TNF induced psoriasis, FH/PH of MS, PH of TB. SLE, comorbidities)
• Likely effective against fistulising disease and EIMs (extra-intestinal manifestations)
• Still unclear when to use this as first-line above anti-TNF

Question 43

Describe Vedolizumab uses in IBD

Answer 43

anti-integrin

• Mechanism of action
o A4b7 integrin inhibitor  a4b7 interacts with MAdCAM-1 in the endothelium directing lymphocytes to the gut thereby contributing to chronic inflammation observed in CD and UC
o Hence: anti-integrin inhibits inflammatory cells from getting into the gut tissues

• Speed of action
o Slower than other biological therapies and reason is thought to be due to the fact that it does not affect lymphocytes already present in the gut hence takes longer to start working
o Studies show better response at week 10 than week 6.

• Clinical Uses
o Active moderate to severe non-fistulising CD as an alternative to anti-TNF therapy
o Moderate to severe UC when anti-TNF has failed
• Dosage
o Induction – 300mg IV at weeks 0, 2, 6
o IV Maintenance 8 weekly

• Side effects
o Drug is gut-specific hence limited side effects, better for certain populations e.g. elderly, patients with PMH of cancer, other co-morbidities
o Not so good for fistulising disease and EIMs
o Slow? – maybe suitable for steroid dependent patients (not steroid resistant)?

Question 44

Diet therapy in IBD

Answer 44

•	Dietary therapy
o	Liquid therapy diet
o	Increased use in children
o	As effective as steroids
o	Use in small bowel
o	Crohns disease
o	Weeks

Question 45

Antibiotics and FMT in IBD

Answer 45

•	Anti-biotics
o	No hard evidence
o	Good for sepsis
•	FMT
o	Lots research into the role of the microbiome

Question 46

Bacteriology of c dificile

Answer 46

• Gram positive anaerobic, spore forming bacillus bacterium
o Spores: allows bacteria to survive in inanimate objects for long periods of time and resist heat, acid, antibiotics

• First described in 1935
o Isolated from stool samples of new-born babies

• Mid 1980s recognised cause of antibiotic-associated diarrhoea and colitis
• Trasmission: faeco-oral route

Question 47

Mechanism of Disease in C dif

Answer 47

• Causes disease through production of two protein exotoxins
• Toxins disrupt the actin cytoskeleton (inactive GTPases) resulting in cell death of colonic epithelial cells
• Toxin A

   - Binds to apical side of cells 
   - Internalised and disrupts cell structure firstly by inactivating the action of GTPases
   - This causes changes cytoskeletal changes that disrupt tight junctions/ loosen epithelial barrier 
   - This results in colonocyte cell death/ production of inflammatory mediators that attract neutrophils.

• Toxin B

   - Binding to basolateral membrane
   - Facilitates binding of biphasic toxins onto gut mucosal cells, facilitating the entry of toxin A

• Both toxins are cytotoxic, and promote release of immunomodulatory mediators from epithelium/ phagocytes/ mast cells.
• Result:

   - Diarrhoea
   - Colitis
   - Loss of intestinal barrier function 

• Third toxin? thought to be involved (‘binary toxin’) although role is currently poorly defined

Question 48

Immunology of C. Difficile

Answer 48

Rupnik et al, Nat Rev Microbiol 2009

• IgG and IgA response post-infection, with antibodies directed at toxins.
• High IgG antitoxin levels are protective.
• The problem lies in that not everyone will be able to form IgG antibodies against C. difficile toxins for example: the old and immunosuppressed (see right)
• Those C. diff stains that do not produce toxin are not of importance as don’t cause damage and do not cause symptoms (see right)

Question 49

Complications of C. Difficile Infection

Answer 49

• Toxic megacolon.
Inflammation paralyses the neuronal network around the colon, reducing parasympathetic drive –> dilation

• Perforation. –> if megacolon perforates
• Renal failure.

•	Systemic inflammation/ sepsis.  
pseudomembranous colitis (neutrophils found in these little membranes) à systemic inflammatory process and can cause death through MO

Question 50

Diagnosis of C dif

Answer 50

• Enzyme immunoassay (EIA) for glutamate dehydrogenase (GDH) antigen.
• Toxin A and B testing EIA (more expensive than GDH, considerable false positive and false negative rate)
• PCR of toxin gene (sensitive test but it can have false positives + difficulty in interpretation)
• Cytotoxin/cytotoxigenic culture as ‘gold standard’ à difficult to culture in lab (anaerobic organism)
• Combination of tests currently used, as all tests have pros and cons PCR of gene does not necessarily imply active infection, e.g. post-treatment PCR may still be positive due to DNA still present

Question 51

Treatment of C Dif

Answer 51

• Little change between 1970s up until the past few years.

• Medical:
o Metronidazole (IV/ PO).
o Vancomycin (PO/ PR)  IV given for MRSA only
o Intravenous immunoglobulin (IVIg)  works in those who cannot form IgG against C. Diff

• Surgical:
o Colectomy – used if recurrent (although now not the only available option)

Question 52

Problems with Conventional Treatment of C. dif

Answer 52

• Non-response to antibiotics – Kelly and LaMont NEJM 2008
o Increased resistance to metronidazole – ¼ -1/3 cases do not respond
o Small increase in resistance to vancomycin (not as rapid as metronidazole)

• Recurrent Disease (increasing, 1/3 of cases now recur)

• New Hyper-virulent strains
o E.g. NAP1/027 – produces large numbers of aggressive toxin, non-response to antibiotics

Question 53

Potential New Approaches to c dif tx

Answer 53

• Current developments focusing on different aspects, including: - Padua and Pothoulakis, Expert Rev Gastroenterol Hepatol, 2016
o Immune support: vaccination?
o Barrier support: using non-toxigenic C. Diff or FMT to outcompete virulent toxigenic C. Diff?
o Antibacterial: creating new antibiotics?

Question 54

Fidaxomicin in c dif tx

Answer 54

• Poorly-absorbed, bactericidal macrolide, spectrum limited to C. difficile and other Gram+ anaerobes.
• Inhibits bacterial RNA polymerase – perhaps anti-inflammatory effects?

• Promising trial results but still concerns:
o Borderline significance for multiple recurrences – reduces secondary infection better (borderline) compared to vancomycin for first recurrence, but could not be statistically proven for more than one recurrence [Cornely et al, CID, 2012]
o No effect on recurrence against 027?
o Expensive.
o No evidence in fulminant colitis.

Question 55

Other Antibacterials used in c dif

Answer 55

• Antibiotics – rifaximin, tigecycline, others at trial stage.
• Antibiotic inactivation in the gut: Ribaxamase = beta-lactamase to break down IV Abx in the gut.
• Toxin binders: no apparent benefit from cholestyramine/ other anion binding resins.

Question 56

Passive Immunisation in c dif tx

Answer 56

Actoxumab and Bezlotoxumab

• Neutralisation of toxin A and B appears to be necessary to prevent infection in rodents, but neutralisation of toxin B alone appears sufficient in piglets.
o Actoxumab: anti-tox A antibody.
o Bezlotoxumab: anti-tox B antibody – reduced rate of recurrence of disease only seen with this (reference below)

• Now RCT evidence of potential of this route – Wilcox et al, NEJM, 2017
o But main side effect of diarrhoea.
o Expensive.
o Affects cure in the first place?

Question 57

Active Immunisation in c dif tx

Answer 57

Wilcox et al, NEJM, 2017
• Current Phase 3 study into active vaccination in people at risk of CDI
• Concern about reduced rates in seroconversion in elderly in earlier Phase studies…
o Two initial phase 3 trials, one continued and one stopped

Question 58

Role of Microbiota in c dif

Answer 58

Colonisation Resistance

• CDI is associated with loss of diversity of the gut microbiota – Rupnik et al, Nat Rev Micribiol, 2009 + Chang et al, J Infect Dis, 2008
• Restoration of the gut microbiota restores colonisation resistance - a potential treatment?

Question 59

Probiotics in c dif

Answer 59

• Latest systematic reviews/ meta-analyses: May prevent antibiotic-associated diarrhoea/ CDI (reduction by 64%?), probably not treat CDI  Goldenberg et al, Cochrane Database Syst Rev, 2013
• Still same rates of C. difficile colonisation – probiotics treat symptoms rather than affect colonisation?
• Saccharomyces boulardii (yeast) potentially of benefit BUT:
o Not available in UK.
o Conflicting data.
o May cause fungaemia even in immunocompetent patients.

Question 60

Non-Toxigenic C. Difficile in tx

Answer 60

• M3 strain lacks genes for toxin production and frequently found in asymptomatically colonised inpatients.
• Prevents CDI in hamsters when challenged with toxigenic C. difficile.

Double-blind RCT of 168 patients – Gerding et al, JAMA, 2015
o Colonisation with NTCD-M3 in 69% of patients (highest with highest doses).
o CDI recurrence rate of 30% receiving placebo vs 11% receiving NTCD-M3.
o Reduced recurrence correlated with colonisation.
o However – colonisation transient, and all patients had lost this by 22 weeks.
o Concern: Can non-toxigenic strains gain toxigenic genes in vivo?

Question 61

Guidelines of FMT in C dif

Answer 61

• Stool saved from donors and infused as enema afterwards to treat pseudomembranous colitis
• Few studies
o Van Nood et al, NEJM, 2013 - 81% of patients with recurrent CDI got better with transplant

• Current Guidelines – NICE and Public Health England, 2014
o Evidence on efficacy and safety of FMT for recurrent CDI is adequate to support use of procedure  only used if patients with recurrent CDI have failed to respond to antibiotics or other therapies
o Encouragement of further research specifically into optimal dosage, mode of transmission and choice of donor
 Mullish et al, QJM, 2014: devised a questionnaire to screen FMT donors  still questions to be answered: Immunosuppressed ? severe disease? Other infections?

Question 62

Preparation and delivery of FMT in C dif

Answer 62

o Current gold-standard: colonoscopy/NG tube/ND tube/Upper GI endoscopy (i.e. can be given upper or lower GI)
o Other strategies have been refined:
 Capsules developed from stool powder and recently found to be just as effective (Kao et al, JAMA, 2017)  but disadvantages still exist: having to take many tablets, possible infection transmission)
 Capsules have been given with spores from bacteria (Khanna et al, JID, 2016)
 Sterile faecal filtrates (no intact organisms, no spores only bacteriophages, viruses and metabolites) was found to be sufficient to treat recurrent CDI in 5 patients (Ott et al, Gastroenterology, 2017)
 Raises other possibilities, given that alive bacteria are not essential for FMT to work… bacteriophages? Metabolites? Bacterial components e.g. proteins?

Question 63

Mechanisms of Action of FMT in C Dif

Answer 63

Khorotus and Sadowsky, Nat Rev Gastroenterol Hepatol, 2016
o Competition with indigenous gut microbiota:
 Competitive niche exclusion – restored microbiota outcompetes C. difficile for nutrients within the colon.
 Micro-organism production of antimicrobial peptides – e.g. Bacillus thuringiensis produces thuricin CD, a bacteriocin active against C. difficile.
o Immune-mediated:
 Microbiota induce cytokine/ immunological reactions important for gut barrier function?
o Microbiota-host metabolism interaction:
 Bile acid pathways – found that commensal bacteria from healthy donors promote secondary bile acid formation (e.g. ↑ deoxycholic acid and ↑BSH activity)
 which was also found in successful FMT patients (for reccurent CDI) Britton & Young. Trends Microbio. 2012

Question 64

Omic Approaches

Answer 64

• Metataxonomics
o Uses 16S rRNA gene that is widely conserved in various bacteria to make community-wide taxonomic classifications  this helps to identify species within the gut, BUT not strain
• Metagenomics
o Uses next-generation sequencing to sequence DNA/gene content found in a sample and therefore identify strains of species
• Metabolomics/Metabonomics
o Uses biological chemistry and seperation technologies (e.g. mass spectroscopy) to identify what is present in a specific tissue or bacterium (metabolomics) or what is present in a complex system (metabonomics)
• Transcriptomics/Metatranscriptomics and Proteomics/Metaproteomics are not used often because they rely on really good genomic data  which needs to be annotated however much of the genome is still yet “unknown” hence you cannot generate a transcript or protein

Question 65

Metataxonomics

Answer 65

• Take a faecal sample  extract the DNA/RNA from all microbes present  PCU SSU rRNA (16S rRNA gene) or any housekeeping gene  run on NGS (Illumina [2nd generation], MinION and SmidgION [3rd generation])  analyse data
• Data being analysed includes:
o Population/community dynamics
o Diversity indices
o Microbial biomarkers
o DNA – diversity (measure of how good your system is  in gut = high = good, in vagina = low = good)
o RNA – metabolic activity
• Requires very robust, strong bioinformatics support – software and databses
• £20-£50

Question 66

Metagenomics

Answer 66

• Two approaches:
o Take a faecal sample  extract DNA  NGS  bioinformatics analysis of sequence  identify functional genes
o Take a faecal sample  extract DNA and ligate and transform into surrogate host (usually E. coli) using plasmid, fosmid or BAC vector  allow it to grow and screen it (since function of E. coli is well known, if it starts to produce a new phenotype then one can identify what an unknown gene’s function is
 This is not commonly done due to time consuming nature and large cost

Question 67

Metabonomics/Metabolomics

Answer 67

• Take a faecal sample  extract metabolites e.g. urine, plasma, salva, faecal water, even solid tissue  this can then be studied using global (H NMR – proton nuclear magnetic resonance imaging) or targeted profiling (LC-MS – liquid chromatography mass spec)
o Targeted profiling = more sensitive but breaks down and will change over time
o Global profiling = produces large amounts of multivariate data but is reliable
• Analysis of these profiles will reduce data into “dots” in a 2-D space and using multivariate analysis one can determine similarity of faecal samples in terms of metabolites
o Allows generation of metabolic profiles, biomarkers, metabotypes

Question 68

Human Distal Gut Screen

Answer 68

• Complete library of metabolites screened in various bacterial clones and interest in literature has been predominantlyin bile salt hydrolases, osmotolerance, cholesterol degradation, phosphatases
o Bile salt hydrolases are particularly key because bile salts are found to be important endocrine molecules, only found in your gut and are manipulated to form secondary and tertiary bile salts without this enzyme produced by microbiome

Question 69

Examples of “Omic” Approaches in Practice

Answer 69

Bile Salt Hydrolase – Gateway Reaction in the Gut
Jones et al, Proc Natl Acad Sci USA, 2008
• BSH identified to remove a taurine or glycine from bile acids such as deoxycholic acid to form an unconjugated bile acid
• This unconjugated bile acid can then undergo a number of different reactions induced by microbiome to produce a range of molecules which have been shown to be implicated in colorectal cancer and energy balance/homeostasis (fat storage)
• BSH is the rate-limiting step that enables all these reactions to take place  without BSH activity the bile acid cannot undergo de-conjugation and form reactions

• Phylogenetic trees allow for identification of bacterial species and strains that can make BSH  by looking at origin of the gene that then disseminated through plasmid transmission
o These can then be cultured in the lab incubated with a number of different bile acids/human bile so that we can see the spectrum of profile activity that each bacteria has – right: parabacteroides only have activity in deconjugation of taurine whilst others can do both

Swann J R et al. PNAS, 2011
• Removing bacteriome and BSH impacts the host metabolic phenotype and metabotype

Question 70

Current Issues with Metagenomics

Answer 70

• In many sequence based metagenomic studies the data is dominated by unknowns and mundane functions e.g. DNA replication. In MetaHIT (consortium of many labs in Europe to complete metagenomics in healthy controls and IBD patients  produced a number of Nature papers) BUT there was no mention of:
o Butyrate synthesis – bacteria make butyrate as a by-product of fermentation, butyrate has been shown to be an important energy source of human colonocytes (fulfils definition below). Butyrate is also anti-inflammatory, anti-proliferative and it plays a key role in epigenome by switching on certain genes that make T-cells.
o Bile catabolism – BSH is produced by bacteria to protect itself from anti-bacterial aspect of bile. A the same time, this produces bile acids that interact with the host via number of receptors e.g. FXR, VDR and other nuclear receptors
o Glucuronidases – liver is able to detoxify compounds and does this by adding a glucoronate to compounds. Bacteria will cleave this off compounds to use as an energy source and utilise it. This can cause the toxin to be reactivated in the wrong place, thereby causing disease.
o These are all functions that are now known to be important to the host
• Or functions which are not easily classified, but maybe important to the host
o indole-3-propionic acid synthesis - depression
o choline catabolism – cardiovascular disease
o NF-κB modulators – innate immunity

We need to re-define what can be considered a core function: needs to relevant to the microbial host, but at the same time show an interaction with the eukaryotic host.

Question 71

Example: Metabolic Roux-en-Y Gastric Bypass Surgery and Gut Microbiota/Metabolites

Answer 71

Li et al, Gut, 2011
• Used a rat model and performed a RYGB  took metabolic profiles of gut microbiome (from urine/faeces) of rats before and after RYGB compared to SHAM models (same procedure but without actually performing the resection). They found that RYGB changes the entire metabolite profile of the host.
• All of the RYGB models had greater proportion of proteobacteria in the gut  significant (not usually found in the colon).
• Theory: allowing a lot of dietary components that would normally be absorbed by the SI to now be found in colon  creates an “SI-like” environment allowing these proteobacteria such as E. coli., to colonate the colon.
• Overgrowth can be as much as 30-35% greater
• Cross-correlation plots derived from these studies show a greater proportion of specific metabolites such as phenylacetylglycine (PAG)  function has not yet been determined but currently being investigated. Other amines such as trimethylamine = also increased  normally associated with increased risk of CV disease BUT epidemiological data shows that these patients have improved CVD risk factors. Hence never as clear cut.
• Nevertheless, it was identified that these rats had a greater susceptibility to colorectal cancer  theory: greater cytotoxicity seen in LI of RYGB rats  more inflammation. This is epidemiologically observed in humans.
• There are also altered faecal bile acid levels post RYGB  relative concentrations of unconjugated bile acid increase initially (then reduce 6-8 weeks post-surgery), and levels of conjugated/taurine-conjugated bile acids reduce massively. This is thought to be the reason for increased insulin sensitivity in these patients who were previously diabetic pre-surgical intervention.
o Watanabe et al, Bile Acid Binding Resin improves metabolic control through the induction of energy expenditure  found that BABR could be a useful strategy to manage impaired glucose tolerance in patients with metabolic syndrome as it not only lowers cholesterol levels but also reduces obesity and improves insulin resistance.

Question 72

The Dialogue between Host and Microbiome

Answer 72

• Movement of proteins and metabolites are important in maintaining a healthy context in the host  communication between microbiome and host is via proteomes and metabonomes
• While we may not be able to isolate proteases from functional metagenomics we are still able to measure their activity in vivo. This is known as degradome/degradomics – the study of enzymes which degrade proteins.
o Why? Bacterial proteases are a potential virulence factor in CRC and IBD. They have also been shown to compromise tight junction integrity.
o Steck et al, Gastroenterology, 2011: took protease from a known bacteria, gelatinase, from Enterococcus faecalis and was intubated with a monolayer of colonocytes. If cells are able to produce tight junctions, a high level of resistance should be observed. However, this is no longer seen when gelatinase is added. This model showed that proteases can contribute to host disease pathogenesis and cause a “leaky gut” by compromising tight junctions.
o Marchesi et al? Original levels of protein in the guts of IBD and healthy controls are not statistically significantly different. However when proteins substrates are added to the microbiome of IBD and healthy guts such as casein, collagen and keratin, a higher % relative activity of proteases is observed. Use of targeted protease inhibitors for each of bacteria, mammalian, fungal and plant proteases revealed that the greatest proportion of proteases acting in the gut of IBD individuals originate from bacteria and fungal species present in their guts.
 Interestingly, however, there is a wide range of protease activities observed in the healthy population on trans-epithelial resistance (i.e. there are some people who have increased proteolytic activity that would lead to reduced barrier function but are still healthy). This provides an area of research to find targets for treatment for IBD patients. It has also been observed that in monozygotic twins where one has the IBD and the other does not, the IBD twin always has a higher degree of proteolytic activity.
 Philippe Langella has developed a genetically modified organism to deliver elafin (protease inhibitor)  i.e. could be therapeutic in IBD?

This observation of degradome in IBD raises an interesting question – what is the significance of high faecal protease levels? If we accept high FP causes loss of barrier function does this mean the liver gets exposed to more endotoxins and becomes more inflamed? Does high FP lead to inflammation of the intestine too? Could it be the cause for NASH – (gut barrier integrity is compromised thereby leading to increased LPS being able to enter the bloodstream and endotoxin reaching the liver, high levels of endotoxin has been associated with NASH)?
However, it has been recently identified by Campbell et al, PNAS, 2010 that intestinal alkaline phosphatase produced by bacteria in our gut also play a role in detoxifying LPS and influencing inflammation. Could this play a role in disease?

Question 73

Epidemoiolgoy of CRC

Answer 73

Epidemiology
• UK: Overall increased incidence since 70s
• Largest increase in 80s-90s, then fall in early 2000s
• Small increase 2006-10 with with opening of BCSP
• World view: rapid increases in countries that have made recent change from low income to high income economy
• This rapid upward trajectory cannot simply be explained by established models based on human genetics

Question 74

Risk factors of CRC

Answer 74

• 50% of those affected will die from their disease
• Prognosis depends on stage of disease
• Incidence increases with age
• Lifetime risk: 1 in 20
• One 1st degree relative 8%
• Two 1st degree relative 20-25%
• One 1st degree relative <45yrs 20-25%
• Diet high in red meat, low in fibre
• Overweight
• Smoking
• Age
• Genetics

Question 75

Adenoma – Carcinoma Sequence

Answer 75

• Co-exist in same patient
• Seen in similar distribution
• Adenomas precede cancers by 10-15yrs
• Animal models
• Tumours seen arising in adenomatous tissue
• Removing adenomas decreases incidence of cancer
• Old thinking – adenoma/carcinoma sequence. Series of genetic mutations (Tumour suppressor genes & oncogenes) leading to unregulated cell proliferation, invasion and metastasis.

Question 76

Modelling gene-environment interactions in relation to personal and public healthcare

Answer 76

• Given the temporal changes in incidence, Gene-environment interaction must be important
• But v difficult to pin down mechanisms through environmental exposures cause disease, because there are so many variables
• Therefore at Imperial we are taking a systems medicine approach - employ computational science to make detailed phenotyped snapshots of human disease.
• Microbiome has evolved with us and (as I hope to show) is at the centre of the systems medicine view
• It is important that we understand it - likely to be key to future personalised medicine strategies.

Question 77

What is the Microbiome?

Answer 77

• Collection of all the microorganisms in the human body.
• Most attention on bacteria, Communities fungi and viruses too (we haven’t really begun to look at them)
• 1000x more genes than present in the human genome.
• Majority in the gut.

Question 78

16S rRNA Gene Amplicon Sequencing

Answer 78

• Microbiome studies made possible by next generation sequencing (previoulsy reliant on traditional culture, which missed the vast majority of what is there)
• 16S rRNA gene – highly conserved regions and defined variable regions (V1-9) within prokaryotes these are then matched against databases
• Problems:
  o Expensive, quite slow, Semi quantitative and opportunites for experimental bias
  o Sequence similarities – Bacillus antracis and Bacillus thurigiensis differ by 9bp over whole 16S rRNA gene – led to this letter re anthrax on New York City Subway
  o Early studies focussed on creating catalogues looking for shared core microbiomes (e.g. that distinguish disease).
  o Not that simple. Focus should be on shared bacterial functions which are conserved between individual microbiomes

Question 79

DIET, DYSBIOSIS AND CANCER

Answer 79

James Kinross, Jia Li and Prof Nicholson’s with collaborators in Pittsburgh
- To study the role of the microbiota on the effect of diet on CRC risk
o Genetically similar groups, with vastly different eating habits
 African Americans vs Africans
- Rates of CRC higher in African Americans than in rural Africans. Genetically similar but environment very different
- Assoc with high animal protein and fat, lower fibre
- 2 week food exchange highly supervised
- Hypothesised this was due to large amounts of fibre and that the reason the epidemiological studies on fibre do not work is that the fibre dose was not high enough

• Butyrate (a product of saccharolytic fermentation by bacteria in the colon) is known to be anti-inflammatory and anti-neoplastic. Increased when AAs switched to high fibre diet
• In rural Africans, Ki 67 (marker of mucosal proliferation) and other biomarkers of cancer risk in 2 weeks of changing diet you modify risk – opposite affect in African Americans
• This is a reversible change

Diet significantly influences dysbiosis
• Diets composed entirely of animal or plant products altered microbial community structure and overwhelmed inter-individual differences in microbial gene expression within 4 days
• Animal-based diet significantly decreased the products of saccharolytic fermentation, (butyrate), but increased the products of proteolytic fermentation, which have inflammatory properties

Question 80

‘Alpha Bug’ Model

Answer 80

• ApcMin/+ mouse model of intestinal tumorigenesis, Fusobacterium nucleatum increases tumor multiplicity and selectively recruits tumor-infiltrating myeloid cells, which can promote tumor progression.1
• F. nucleatum adheres to, invades, and induces oncogenic and inflammatory responses to stimulate growth of CRC cells through its unique FadA adhesin.2
• FadA binds to E-cadherin, activates β-catenin signaling, and differentially regulates inflammatory and oncogenic responses.
• F. nucleatum inversely associated with density of CD3+ T cells in CRC. High levels of T cells in tumours have been associated with better patient outcomes.3

Non-enterotoxigenic Bacteroides fragilis (NTBF) strains are usually symbionts, and those expressing polysaccharide A (PSA) have been shown to inhibit the immune responses mediated by interleukin-17 (IL-17) and T helper 17 (TH17) cells. By contrast, enterotoxigenic B. fragilis (ETBF) strains activate signal transducer and activator of transcription 3 (STAT3) signalling in the colon, leading to IL-17- and TH17 cell-dependent inflammation, which is required for colonic hyperplasia and tumour formation in the multiple intestinal neoplasia (Min) mouse model. Although ETBF secretes a pro-oncogenic toxin (B. fragilis toxin (BFT)), the participation of the colonic microbiota is necessary for colon carcinogenesis. According to the ‘alpha-bug’ hypothesis, ETBF remodels the colonic microbiota and co-opts this microbial community to induce colon cancer; however, in order for this dysbiosis to trigger tumorigenesis, it is likely that ETBF must be present in combination with particular disease modifiers and in hosts with certain genetic traits. It is currently unclear exactly how ETBF influences and interacts with the colonic microbiota to promote carcinogenesis. Moreover, it is uncertain whether the microbiota is modulated by TH17 type inflammation or, conversely, contributes to the induction of this immune response.

Fusobacterium nucleatum in CRC tissue associated with poorer prognosis

Question 81

Driver – passenger’ model

Answer 81

• Emerging theories (driver-passenger) – dysbiotic state in gut created through lifestyle, allowing pathobionts to exist in the gut. Influence oncogenic pathways which are poorly understood – leading to the development of cancer.

Question 82

Bacteria linked to CRC

Answer 82

S. gallolyticus.
- COX-2 mediated inflammatory response

E. Faecalis

1) Bystander effect – Induction of mucosal macrophages to produce clastogens that cause DNA damage through a
2) Free radical and superoxide production

E. coli

1. Toxin production – Colibactin breaks double-strand DNA through polyketide synthase (pks)
2. Toxins production – cyclomodulin
3. Genotoxicity induced by outer membrane vesicles (OMVs)

Bacteroides Fragilis

1. Stat3 activation of mucosal IL-17 response, (TH 17 adaptive immune response)
2. B.fragilis toxin (or fragilysin). Metalloproteinase stimulates cleavage of the tumour suppressor protein E-cadherin.

Fusobacterium nucleatum
1. FadA adhesin bindsnextracellular domain of E-cadherin, triggering cellular invasion of organism and activation of β catenin/Wnt signalling.

Question 83

Methodological Problems in microbiota analysis

Answer 83

• Considerable disagreement about the best sampling methods(1,2):
• mucosal biopsies
• stool
• rectal swabs

• Few studies have controlled for the use of bowel preparation, which appears to have a transient effect of the microbiota(3)

Unweighted UniFrac principal component analysis; the microbiota of healthy controls, individuals with polyps and individuals with cancer (A) as well as from individuals with distal, including rectal, and proximal cancers (C) was significantly different; no difference was found in microbiota composition of tumour and paired non-tumour tissues (B); the faecal microbiota of both cancer and control individuals was different from the mucosal microbiota (D). CRC, colorectal cancer.

Marked temporary drop in bacterial load following bowel preparation based on stool samples. This recovers within two weeks.

Diet, lifestyle (exercise affects your microbiome), drugs, age have all been shown to affect microbiome coposition and function. Microbiome sits at the interface of host gene-environment interactions – cannot be ignored