Module 1.2: Coeliac, CRC, IBD + other intestinal Flashcards
Define Coeliac Disease.
GLUTEN SENSITIVE ENTEROPATHY.
A chronic small intestinal immune mediated enteropathy precipitated by exposure to gluten in genetically suspected individuals
- Oslo Definitions, Ludvigsson et al, Gut, 2013
Name the three types of gluten
A gliadin - wheat
Hordein - barley
Secalin - rye
Frequent features of coeliac (50%)
malaise diarrhoea steatorrhoea weight loss anaemia low folate low Fe
Common features of coeliac (25%)
anorexia abdo pain oral ulcers distension and bloating flatulence low B12 low albumin low vitD high PTH
Occasional features of coeliac (<25%)
Nausea Muscle pains (due to low vitD and Ca) Tetany Bone pain bruising (low vitK) oedema constipation rashes lymphoma
Endoscopic features of CeD
mosaic surface
scalloped (flattened) mucosa
Histology:
subtotal villous atrophy with crypt hyperplasia
Grading system in CeD
Marsh Grading, Gastroenterology 1992
0 - Normal mucosa
1 - increased number of intra-epithelial lymphocytes
2 - proliferation of the crypts of liberkuhn
3 - variable villous atrophy
3a - partial
3b - subtotal
3c - totla
4 -hypoplasia of the small bowel architecture
Serology in CeD
Antigliadin antibodies
- less specific than endomysial antibodies
Endomysial antibodies
- IgA class antibodies (except in selective IgA deficiency, then IgG, 1 in 80 people)
- detected by indirect immunofluorescence
- high specificity (>95%) and sensitivity
Tissue transglutaminase
- shown to be the autoantigen recognised by endomysial antibodies
- tTG cross links glutamine residues including those in gliadin and produces neo-antigens
- IgA antibodies measured by ELISA (easier than immunofluorescence)
- tTG done first and endomysial antibodies done second to confirm
Deamidated-gliadin peptide antibodies
- may be as useful as TTG
Volta et al 2010 suggests that the combined search for IgA tTGA and IgG DGP-AGA provides the best diagnostic accuracy for CD
Burgin-Wolf et al, 2002:
- All those positive for tTG will have positive EMA antibodies
- Antibodies fall as pts started on gluten free diet”
Diagnosis of CeD in patients
ESPGAN working group recommendation, 2012
IF SYMPTOMATIC:
Based on symptoms + positive serology + histology consistent with CeD
If IgA tTG2 Ab titres > 10x upper limit of normal, CeD can be diagnosed without duodenal biopsy by applying strict protocol in further diagnostic tests
IF ASYMPTOMATIC:
based on + serology and histology
HLA-DQ2/8 useful in excluding diagnosis
Prevalence of CD in GP
1%
Prevalence of undiagnosed CeD - 10x the general population
Evidence for silent disease in CD
- Dermititis herpetiformis - 30% have no GI symptoms
- Population screening
- – Catassi C, 1994 - showed a prevalance of 3.3 per 1000, silent:known cases ratio 1:5
- – West et al, 2003 - sero-prevalence of 1.2%
- – Sanders et al, 2003 - 1% prevalence, commoner in IBD, IDA and fatigue)
- – Bingley et al, 2004 - 1% sero-prevalence at 7 years old, comparable to adults, suggesting you don’t develop CD in adulthood
Groups with prevalence of CeD between 1-5%
T1DM
- Cronin et al, 1997
IBS
- Sanders et al, 2007 - OR 7 for coeliacs with IBS
Osteoporosis
- Sanders et al, 2005 - overall 3x increase in CeD prevalence in those with bone problems
Treatment of CeD
GLUTEN FREE DIET - no wheat, barley, rye
Treatment of nutrient deficiencies
Complications in CeD
Dermatitis Herpetiformis
nutrient malabsorption and impaired nutritional status
Osteoporosis + osteopenia
Small bowel malignancy
Describe dermatitis herpetiformis
Vesicular rash
Intense pruritus
On skin biopsy:
- granular IgA deposits
Associated villous atrophy and gluten sensitivity
Can potentially be much worse than intestinal disease
Types of Small bowel malignancy in CeD
Lymphoma: EATL - enteropathy associated T-cell Lymphoma
Adenocarcinoma
Pathogenesis of CeD
Shan et al, 2002:
PROLAMINES (storage proteins rich in proline (P) and glutamine (Q) ) responsible for majority of immune response.
The tight junctions of enterocytes are disrupted by a2-gliadin –> large peptides can enter circulation
a2-gliadin peptide (glutamine) is DEAMIDATED by TTG to glutamate
the deamidated peptide is ENDOCYTOSED by APC and processed to 3 distinct epitopes, which are presented by either DQ2-a1 or DQ8 presenting proteins, becoming neo-antigens for HLA-restricted T-cell clones.
(95% of pts carry DQ2, 5% DQ8)
Th-Cells are activated
- Th1 –> cytotoxic apoptosis
- Th2 –> plasma cell maturation and anti-gliadin and anti-TTG ab production
What is the risk of CeD in first degree relatives?
10%
MacDonald et al, 1965
80% concordance in twin studie
Greco et al, 2002
Which chromosome is HLA coded for?
6
What are the HLA class I associations in CeD?
HLA-A1 and HLA-B8
What are the HLA class II associations in CeD?
HLA-DR3 and HLA-DQ2
Describe HLA-DQ2
heterodimeric cell surface receptor molecule on lymphocytes, in linkage disequilibrium with all the ones mentioned above.
Most Coeliacs have DQ2 but around 5% are DR4-DQ8 but these individuals have no phenotypic difference in CeD pathology
Describe the evidence for non-HLA genes in CeD
HLA-DQ2.5 found in 90% of northern European CeD pts but also in 20% of non-celiac controls.
Concordance in HLA-matched siblings is 30%
Three strategies to map non-HLA genes in CeD
GWLS - genome wide linkage studies
Candidate gene analysis
GWAS
non-HLA genes implicated in CeD in GWLS
• There were 13 studies with variable, often non-confirmed
o CELIAC 1
HLA implicated in most studies
o CELIAC 2
5q31-33 (cytokine cluster)
o CELIAC 3
2q33 (CD28-CTLA-ICOS region)
o CELIAC 4
19p13.1 (myosin 9B)
Describe the role of 2q33 in CeD
3 genes involved in immune processing: CD28, CTLA4, ICOS
CTLA4/CD8 binding regulates T-cell activation
- If there is CTLA4 with no CD8 on a T-cell, there is T-cell anergy
Hunt et al 2008
Describe the role of non-HLA loci within the MHC in CeD
strong LD found in the region of extended haplotypes mostly due to DQ2
MICA5.1 allele has shown to have an OR 8.6 in coeliacs
–> INVOLVED IN NK/CD8+ cell binding
Lopez-Vasquez et al, 2002
Describe the role of 19p13.1 in CeD
Monsuur 2005
homozygous individuals have a 2.3x higher risk of developing CeD
Von Heel et al 2007
Myosin 9B may have role in actin remodelling
Summarise the findings of candidate gene analysis in CeD
HLA class II (DQ2.5)
2q33
MICA
19p13.1
Findings of the CeD GWAS
o HLA-DQ2.5cis shown in 89.9% coeliacs patients
o CD28-CTLA4-ICOS region implicated again
o But no association with 19p13.1 (Myo9B)
HLA DQ2 Serotypes
- HLA-DQ (a10501, b102) is a heterodimeric cell surface receptor molecule on lymphocytes
- It can be encoded in DNA in a:
- Cis form – polypeptide subunits encoded on the same chromosome - -> HLA-DR3 (65-95% of N European patients) - Trans form – polypeptide subunits encoded on different chromosomes - ->HLA-DR5/DR7 (Mediterranean patients)
Function of Chromosome 4q27 in CeD
o Demonstrated further risk variants in region harbouring IL2 and IL21
o Rs13119723 = best marker
o Rs6822844 = strongest association overall in combined cohorts, coeliacs have the major allele in recent meta-analysis
o IL21
- Enhances B, T, NK cell proliferation + IFNy production
- Implicated in intestinal inflammation
- Greatly increased expression in untreated coeliac
o IL2
- Key cytokine for T cell activation and proliferation
- Modest reduction in untreated coeliac
- Synthetic region in mouse (Idd3) determines susceptibility to autoimmune diseases in the NOD mouse by influencing IL2 mRNA/protein level and CD4+ and CD25+ regulatory T cell activity
o KIAA1109
- Modest reduction in untreated coeliac
- Unknown function
o TENR/ADAD1
- Expressed in testes
Summary of initial coeliac genetic studies
- HLA-DQ
- IL2 – IL21
- 7 more risk variants and regions identified by GWAS
- Genes from 8 regions involved in immune response
- 4 of 9 regions overlap with T1DM
Further GWAS in CeD, Trynka et al 2009
• Further GWAS follow up found 2 novel coeliac regions:
o 6q23.3 (OKIG3-TNFAIP3)
o 2p16.1 (REL)
both involved in the NFkB pathway
Other environmental factors affecting development of coeliac
•Gluten
•Weaning and tolerance o DAISY study revealed age of exposure to wheat, barley or rye was important: o 1-3m = 23 hazard ratio o 4-6m = 1 (all good) o Over 7m = 4 hazard ratio
•Influences at time of exposure
o Rotavirus infection increases risk of coeliacs in children
- DAISY study revealed multiple infections with rotavirus in children increased risk ratio of coeliacs
Summarise the inherited predispositions to CRC
- Mendelian Dominant
- FAP
- Lynch snydrome - Mendelian Recessive
- MYH associated polyposis - Rare low penetrance variants
- APC T3920A - Common low penetrance variants
- identified by GWAS
What % of CRC risk is genetic?
15% - Nordic twin studies, Mucci et al 2016
Summarise the features of FAP
Autosomal dominant
>100 polyps/adenomas
Will progress to CRC by 40-45y
10% has no FHx –> High mutation rate
Extracolonic features of FAP
GARDNER’S –> Osteomas and epidermoid cysts
TURCOT’S –> medulloblastomas
Summarise the features of APC mutations
APC is a tumour suppressor gene on Ch5
1st hit = genetic
2nd hit = inactivates the normal allele
Controls proliferation of colonic epithelial cells via the Wnt pathway which affects Myc expression
Occurs early in tumorigenesis
APC is frequently mutated in sporadic CRC
Types of mutation in APC
TRUNCATING MUTATIONS –> polyposis (avg age 40)
POINT MUTATIONS (e.g. T3920A) –> fewer polyps
3’ and 5’ mutations –> attenuated polyposis (avg age 50-60)
Management of FAP
referral to genetic/polyposis registry of the family
full colectomy if polyposis arises
There may be a role for aspirin in FAP
Summarise the features of Lynch Syndrome
Autosomal dominant
early age of onset
Lifetime risk of CRC is 35% and endometrial cancer of 34% - Bonadona et al 2011
NICE: all those diagnoses with CRC tested for this
2-3% of CRCs - Hampel et al 2005
Right sided - 2/3 occur in proximal colon
Caused by defective DNA mismatch repair, which leads to diploid tumours with MICROSATELLITE INSTABILITY
proportion of BRCA1 and BRCA2 mutations in those with Lynch syndrome - Yurgelin et al 2015
Genes involved in Lynch Syndrome
MLH1 + PMS2 - lost together
MSH2 + MSH6 - lost together
EPCAM
Lynch Classification
Lynch I - Familial colon cancer
Lynch II - other cancers of the GI, extra colonic sites: endometrial, ovarian, small bowel, ureter, renal pelvis
Diagnosis of Lynch syndrome
Genetic testing - MSI profiling - BAT25 used to identify MSI
Immunohistochemistry for mismatch repair gene expression
Management of Lynch syndrome
GENETIC TESTING of family members!!!
Colonoscopy every 2 years
Surveillance for associated cancers
Risk reducing surgery - esp oophorectomy and hysterectomy for women who have had families
Chemoprevention studies:
- CAPP2, Burn et al 2009/2011
- – Aspirin usage can reduce risk by up to 50% in 6y
- – No difference at 2y
Name 4 hmartomatous polyposis syndromes and their causative genes
Peutz-Jeghers - LKB1 pathway
Juvenile Polyposis - SMAD4 pathway 20% and BMPR1A in 20%
Cowden’s - PTEN
Hereditary mixed polyposis - GREM1
Summarise the features of MYH associated polyposis
only autosomal recessive inherited CRC
mutation: DNA base excision repair gene which repairs oxidative damage
- Due to transversion
Multiple adenomas
What is transversion and transition
Transversion: purine to pyrimidine or vice versa
Transition: purine to purine or prymidine to prymidine
Summarise the genetic pathways in CRC tumorigenesis
CHROMOSOMAL INSTABILITY (85%)
- APC gene mutation
- Aneuploid tumours
- Growth control genes e.g APC, GREM1
MICROTELLATE INSTABILITY
- methylation MLH1 gene promoter
- diploid tumours
- DNA repair:
- mismatch repair
- base excision repair
- polymerase proofing
Roles of POLE and POLD1 in CRC
POLD1 - encodes DNA polymerase delta catalytic subunit enzyme
POLE - encodes DNA polymerase epsilon enzyme
Both involved in DNA replication and repair
Mutations in them occur in the PROOFREADING domains leading to a DEFECT IN CORRECTION OF MISPAIRED BASES
These tumours are MICROSATTELITE STABLE
MSI is the phenotypic evidence that DNA mismatch repair is not functioning
Summarise PPAP
Polymerase proofreading associated polyposis
multiple adenomas early onset CRC duodenal adenomas and carcinomas POLD1 endometrial cancers accellerated tumourigenesis
Two types of CRC according to Cancer Genome Atlas 2012
NON-HYPERMUTABLE - 84%
- chromosomal instability
- <1 mutation/10^6 bases
HYPERMUTABLE - 16%
- MSI/diploid
- – 75% hypermethylation of MLH1 mismatch repair gene
- – 25% mutation o POLE (MS stable)
- > 100 mutations/10^6 bases
- fare better with chemo
Explain the role of immunotherapy and personalised medicine in CRC
hypermutable cancers have many somatic mutations that lead to multiple new non-self antigens
these tumours are characterised by lymphocyte infiltration
these can be targeted by immunotherapy
PROGRAMME DEATH 1 PATHWAY is an immune checkpoint that prevents autoimmunity. This is upregulated in MSI tumours to prevent cytotoxic cells from killing tumour cells
PEMBROLIZUMAB - PD1 checkpoint inhibitor -> 80% response rate and increases survival to 40%
Definition of phenotype
the physical characteristics of something living resulting from the interaction between its environment and genetic make-up
Phenotypes of Crohn’s
Sometimes spares rectum Mouth-to-anus transmural (full thickness) patchy (skip lesions) F>M 3:2 granulomas in 60% fistulae and strictures common
Phenotypes of UC
always involves rectum confined to colon superficial continuous M=F no granulomas fistulae and strictures rare
Advantages of phenotyping
provide an insight into pathogenesis with respect to genetic predisposition
defining responses to treatment and so enabling clincians to offer the right treatment
help define prognostics, improving the Tx plan
Disadvantages of phenotyping
concept defined by humans so may not represent all biologically important categories
may change over time so only true for a particular instance
Vienna and Montreal Classification for Crohn’s
VIENNA - Age A1 below 40 A2 above 40 - Location L1 ileal L2 colonic L3 ileocolonic L4 upper - Behaviour B1 non-stricturing B2 stricturing B3 penetrating
MONTREAL - Age A1 below 16 A2 16-40 A3 40+ - Location L1 ileal L2 colonic L3 ileocolonic L4 isolated upper disease (modifier that can be added to L1-L3) - Behaviour B1 non-stricturing B2 stricturing B3 penetrating p Perianal disease modifier (can be added to B1-B3)
Montreal Classification of UC
EXTENT and SEVERITY
E1 - ulcerative procritis
E2 - left sided (distal) UC
E3 - extensive UC - pancolitis
S0 - clinical remission - asymptomatic
S1 - mild UC - passage of 4 or less stools/day with out without blood, no systemic illness and normal ESR
S2 - moderate UC - +4 passage/day, minimal systemic toxicity
S3 - severe UC - at least 6 bloody stools/day, pulse +90 bpm, temp >37.5, Hb <10.5, ESR >30
Antibodies implicated in UC and Crohns:
UC = pANCA crohns = ASCA
examples to the genes implicated in IBD
NOD2, HLA, ATG
What are the methods available to investigate the aetiology of IBD
- genetic studies
- studies of bacterial flora - difficult as only 40% can be colonised
- surrogate markers
- mechanistic studies
Summarise the extraintestinal manifestations of IBD (KEY KNOWLEDGE)
Arthritis - axial (ankylosing spondylitis) or peripheral
Skin - erythema nodosum, pyoderma gangrenosum
– erythema nodosum: up to 10% of patients, raised nodules on shins, associated with HLA-B62
Eyes - anterior uveitis, episcleritis, iritis
— associated with HLA-B27, HLA-B58, HLA-DR103
Liver - PSC, autoimmune hepatitis
Summarise the two types of peripheral arthopathies in UC and Crohn
TYPE 1 - UC - pauciarticular
- less than 5 joints
- self-limiting episodes
- associated with HLA-B27 and HLA-DR103
TYPE 2 - Crohns - polyarticular
- more than 5 joints
- persistent symptoms independent of IBD
- associated with HLA-B44
Define Linkage disequilibrium and its significance in IBD.
the likelihood of two genes being inherited together
The genes that determine the individual EIMs might be in LD
TNFa its between HLA-DR and HLA-B, close to MICA
- TNFa is proinflammatory cytokine. Treatment with infliximab is effective in Crohns, RA and AS
- – explains overlapping clinical syndromes in practice
Describe the role of IBD1/NOD2 in IBD
Ogura and Hugot 2001:
NOD2 is the specific gene whose frameshift mutation incurs suceptibility to Crohns
- LRRs interact with bacteria LPS
- – once the LPS is bound to LRRS on NOD2, this oligomerises and binds to CARD ROCK.
- – Rick then oligomerises to transmit the signal to IKK complex, activating NFkB pathway –> cytokine secretion
Revised studies suggest that these have a truncated NOD2 which downregulates NFkB activity and the immun sytstem???
poorly understood
Give examples to the enviornmental factors influencing IBD
smoking
appendicectomy
vit D
Microbiota
Epidemiology of UC and CD
West > East
North > South
UC > CD
-Rising incidence in the east –> westernisation Ng Sc, 2013
How does population movement affect the prevalence of IBD?
Li X et al 2011
first generation immigrants from low incidence countries show low incidence compared to native population
Benchimol et al 2015
younger age at time of arrival to western country increased risk to that of native country
children born to immigrants in western country had the same incidence as native children
Effect of smoking on IBD and why?
Mahid SS et al 2007
Makes you suceptible to CD (OR 1.76) and less suceptible to UC (OR 0.58)
Smoking increases NO in endothelial cells –> increansed guy permeability –> access to immune cells granted to microbiota –> inflammation
Also alters microbiota
Effect of appendicectomies on IBD and why?
protective in UC
Andersson et al 2001
increased risk of CD with appendicectomies that drops over time
Kaplan et al 2008
May be due to alteration to microbiota and hense to immune response following appendicectomy –> Roblin 2012
Effect of vitamin D on IBD and why?
higher levels of Vit D –> reduced risk of CD
Effect of microbiota on IBD and why?
altered composition of gut flora in IBD
- reduction in diversity - Frank et al 2007
- reduction in beneifical species - Sartor 2008
- Increase in pathogenic species - Feller 2007
Describe how the microbiota directs immune response in IBD
affected by the number of IL-17/Th17 cells in the mucosa
T0 cells, if primed by IL-23, cause a Th17 response. If primed by IL-2, cause a Th1 response. Both are pro-inflammatory.
Th17 cells are potent effectors of inflammation
Differentiation from naive T-cells require TGFb, IL-6, IL-21, and IL-23
they produce pro-inflammatory IL-17 and IL-22 –> role in patholgenesis of Crohns
Th17 cells do not develop in the absence of microbiota
Autophagy in IBD
ATG16L1 codes for mechanisms of autophagy
essential process for maintenance of cellular homeostasis
engulf waste and deliver to lysosome for degradation
Phases: initiation–> nucleation –> elongation –> maturbation
ATG16L1 is important in elongation and cargo selection
If one has defective autopahgy –> defective homeostasis in the intestines
- – increased bacterial permeability
- – decreased bacterial defense
- – decreased bacterial clearance
- – decreased peripheral T-cell numbers
- – decreased Ab production
- – Expansion of microbiota
Describe the familial aggregation of IBD
5-22% have an IBD-affected relative
1st degree relatives have 10-15x increased risk (more CD than UC)
TWIN CONCORDANCE - BMJ 1996
Monozygotic twins:
- CD 35%, UC 11%
Dizygotic twins:
- CD 7%, UC 3%
Examples to genes of interest in CD
CARD15 (NOD2)
ATG26L1
IL23R
TLLs
Examples to genes of interest in UC
HLA II
IL23R
Role of CARD15/NOD2 in CD
CARD15 encodes NOD2 on ch16
There are three main variants of this gene
x1 NOD2 variation increases CD risk x2-4
x2 NOD2 variation increased CD risk x20-40
These variants aren’t present in Asian CD pts –> there are other genes
Describe the function of CARD15/NOD2
codes for a protein present in monocytes, macrophages, Dendritic cells, epithelial and paneth cells
Involved in recognising bacterial MDP through its LRRs –> NFkB secretion to protect host from invasion
The variants cause a loss of function of this pathway –> decreased clearance of bacteria
Role of ATG16L1 in CD
found on ch2
protein forms part of autophagy complex
variant has reduced capacity to capture bacteria
Role of IL23R in IBD
variant present in 14% of healthy europeans
associated with DECREASED risk of IBD –> PROTECTIVE
IL23 is a pro-inflammatory cytokine
Role of Toll Like Receptors in CD
TLR4 recognises LPS in G-ve bacterial cell walls
Polymorphysm increases CD risk
TKR5 recognises flagellin on motile gut bacteria
Polymorphysim protective in CD
Role of HLA Region IBD3 in IBD
Chr6
IBD associated with HLA II
Chrohns with:
- DRB*0701
- DRB1*0103
- DRB1*04
UC with:
- DRB1*0103
- DRB1*1502
It is likely that HLA genes play a disease-modifying role rather than affecting suceptibility
summarise the anatomy of the small intestine
villous hight about 3x crypt depth
muscularis mucosa
Another layer of muscles found in human: transverse and longitudinal muscles involved in pushing contents along
mucosal surface amplified by folds of Kerkring (x3), villi (x10) and microvilli (x20) so total surface area >200m2
Mechanisms of absorption in the small intestine
Disgestive enzymes: gastric, intestinal and pancreatic + biliary secretions
transport of products through the intestinal mucosa
Villous tip
- ACTIVE TRANSPORT
- abundant brush border hydroxylases, high nutrient transporter and involved in water and ion absorption
Crypt
- high secretion of net water and ions
- highly permeable
- passive permeability
Where are stem cells found in the small intestine
3-4 cells up from the crypt
What types of cells can small intestinal stem cells make?
Enteroendocrine cells - involved in making gastrin, GIP, GLP1/2, PYY, secretin
Goblet cells - produce mucus
Absorptive enterocytes
Paneth cells
M-cells - involved in immune system (antigen sampling) - Peyer’s patch in ileum
What are other cell types that originate elsewhere in the body in the small intestine?
intraepithelial lymphocytes lamina propria lymphocytes DCs monocytes myofibroblasts vascular and lymphatic cells neuronal cells - two plexi that allow movement of contents: myoenteric and submucosal plexi
Describe the process of cell turnover in the small intestine
cell death occurs at the top of villi
Stem cells divide and move up from crypt to the tip of villi to replace those lost
Turnover time: 48-72h
No of cells lost = no of cels formed
Cells are linked up in a linear fashion
Describe the locations of absorption of different nutrients in the small intestine
glucose: mainly in duodenum and jejunun with little in ileum
protein: mostly in jejunum and ileum, 20% not absorbed
fat: most in jejunum, some in ileum, 5% not absorbed
Describe the digestive process in the stomach
food enters digestive system
VAGUS leads to production of ACh –> HISTAMINE production –> GASTRIN production (in antrum)
Parietal cells make H+ –> low pH
pepsinogen I and I made to be activated later
Fundic cells make gastric lipase –> Some lipolysis in stomach (20-30%)
Triglycerides + phospholipids –> diglycerides + FAs
Most lipolysis in JEJUNUM
Describe the process of JEJUNAL lipolysis
COLIPASE: TG –> FA + MG
PHOSPHOLIPASE A2: PL –> FA + lysolecithin
CHOLESTEROL ESTERASE: cholesterol ester –> FA + cholesterol
These are then put into mycelles which carry lipids to BRUSH BORDER by FACILITATED DIFFUSION and bind to specific transporters allowing to move into enterocytes
Inside enterocytes, lipids are RE-ESTERIFIED –> TG + PL + CE reformed into a lipid vesicle
(enterocytes only form chylomicrons and VLDL)
LIPID VESICLES come together with APOLIPOPROTEINS (formed in ER)
Travel to golgi –> form secretory vesicle containing chylomicrons and VLDL
fuse with cell membrane and release chylomicrons and VLDL
go into LACTEAL and into LYMPHATIC SYSTEM –> released to blood
Describe the structure of mycelles
hydrophilic -OH outside, lipophilic parts inside lipid bilayers with lipids carried in the centre
Describe the different classes of amino acids
All proteins have an -NH2 an a -COOH side chain
ACIDIC: glutamine, glutamate, aspartate
BASIC: Lysine, hystadine
NEUTRAL:
- aliphatic: leucine, valine
- aromatic: tyrosine, tryptophan
- Imino: glycine, proline
- Sulfur: cyteine, methionine
How many AAs are there?
How many essential AAs are there?
21
8
Describe the process of proteolysis
ENTEROPEPTIDASE activates trypsinogen –> trypsin
Trypsin activates all other pro-proteases to their active forms:
Chymotrypsinogen –> chymotrypsin
Proelastase –> elastase
Procarboxypeptidase A/B –> carboxypeptidase A/B
NOTE: carboxypeptidases are EXOPEPTIDASES - they work on the end of the chain
Proteins are split up into peptides and aa’s
These are further broken down to oligopeptides
AA diffuse via specific transporters into villus capillaries at the basolateral membrane
Once these diffuse into the blood, the liver breaks them down further
AA transporters can be proton-coupled OR sodium coupled
The transport across the membrane is:
- active transport
- facilitated diffusion
- passive transport
- vectorial flow
Describe the different pumps and their uses in the GI system
these pumps are ATPases
Na/K ATPase –> uses 30% of ATP production in the body. K into cell, Na out
Ca2+ ATPase –> 1000fold difference between inside and outside
H+/K+ ATPase –> works in the stomach to produce low pH
Pumps for counter-transport:
- Na+/H+
- Cl-/HCO3-
- – these use existing gradients to exchange molecules
Co-transport:
- Na+/glucose cotransporter (SGLT1)
- Na+/AA cotransport
- H+/AA cotransport
Passive transport:
- Ca channels
- K channels
Describe the role of the Na/K/2Cl cotransporter in the small intestine
important in JEJUNUM
Cl- exits enterocytes at the apical membrane via passive transport through a channel.
K+ exits through a channel on the apical membrane whilst Na is pumped out using Na+/K+ ATPase
In CF, there are problems regulating this Cl channel
Summarise the absorption of nutrients in the intestines
sugars:
broken down to glucose, galactose and fructose by enzymes at the brush border. these come into the cell via channels or co-transported with Na
Proteins:
broken down by proteases on the brush border and enter the cell via Na+ or H+ coupled absorption. Peptidases break these down in enterocytes to AA which are taken out of cell to blood via channels
Fats:
broken down on the brush border into FA + MG and transported into enterocyte via transporters (FATptr). Then attached to a FABP to allow them to be present in cytoplasm. The apolipoptoteins are transported to golgi via MTTP. Both APOs and FAs join in Golgi to make TGs
Describe the means of abnormal digestion in the intestines
reduced gastric tissue/secretion
loss of pancreatic tissue
impaired bile secretion
reduction in intestinal brush-border enzymes
Describe the means of abnormal absorption in the intestines
loss of functional enterocytes
pre and post mucosal effects
single gene disorders
Describe the tests for malabsorption
Tubeless tests
- Xylose absorption
- pancreolauryl test
- schilling test
Fecal Pancreatic Elastasr 2
- stable during passage through GI.
- small sample of stool collected
- high sensitivity and specificity to diagnose pancreatic insufficiency
- values below 200ug elastase/g stool indicate exocrine insufficiency
Explain how B12 insufficiency can be assessed
MACROCYTOSIS or NEURO SYMPTOMS –> measure B12
If B12 LOW:
- Intrinsic factor +ve –> pernicious anaemia
- Pt pregnant –> discuss with haematologist
- IF -ve –> consider other causes
- – gastric/ileal/pancreatic disease
- – drug induced (PPI, H2 receptor antagonists, chronic alcoholism)
- – dietary (strict vegan)
- – biological competition (bacterial overgrowth, fish tapeworm infestation)
Causes of malabsorption
Coeliac (most common)
Small bowel bacteria overgrowth - caused by fistulae e.g. crohns, strictures, impaired peristalsis
Pancreatic insufficiency
LESS COMMON:
chronic infections, lymphoma, radiation enteritis, intestinal lymphangiectasia, drugs, allergic, immunodeficiency
Describe abetalipoproteinaemia
recessive
presents in early childhood
malabsorption of fat and fat-soluble vitamins
Abnormal MTTP –> failure to form chylomicrons and subsequent fat droplets in enterocytes
due to mutations in Apo-B gene (C -> T)
Explain the prinicples of the sugar breath test in lactose intolerance
H2 produced by bacterial action on sugars
Fasting breath H2 <20 ppm
Sugar taken by mouth
Breath samples every 30m for 2-3h
increase of 20 ppm significant
Lactose is ingested orally. When it reaches the caecum, if it has not been absorbed, then lactobacilli will use this to make H2
Low lactase activity in the small intestinal brush-border membrane results in failure to digest lactose
Describe the mechanisms of lactase persistance
Neonates have high lactase
Lactase non-persistance is the usual adult human phenotype worldwide
Lactase persistance occurs in most Northern Europeans
Majority of those affected with lactose intolerance originate from Asian/African countries
The tolerance of lactose has allowed for the domestication of dairy animals which have provided milk–> transition from hunter-gatherer to agriculture
Genetic basis of lactase persistance
Protein active in childhood but not in adults
polymorphysms in laftase gene
Heterozygote studies showed cid acting elements
Transcription factors regulating expression of lactase
DEVELOPMENTAL SWITCHES
Enattah 2002 - polymorphysm associated with persistance/non-persistnce in Finnish population
Describe the findings of Glucose-Hydrogen breath test for small intestinal bacterial overgrowth
Breath H2 high
small intestinal bacterial count is high
Some glucose is metabolised to H2
Causes of small intestinal bacterial overgrowth
gastric surgery jejunal diverticula intestinal blind loops after surgery intestinal strictures fistulae impaired peristalsis
Describe the principles of the lactulose hydrogen breath test
lactulose not digested or absorbed by small intestine
Broken down by bacteria to give H2
Rise in breath H2 measures
Examples to functional GI disorders
psychogenic vomiting
functional dyspepsia
chronic abdo pain
IBS
Describe the classification
ROMA IV
A. Oesophageal
B. Gastroduodenal
C. Bowel
— IBS
D. Centrally Mediated Disorders of GI pain
E. Gallbladder and sphincter of oddi
F. Anorectal
G. Neonate/Toddler
H: Child/Adolescent
Summarise the epidemiology of IBS
WW prevalence 11.2%
Incidence 1.35-1.5%
F>M
Younger people more likely to be affeced
Summarise the types of IBS
Abdominal pain AND bloating
- gas production - MAJOR factor
- bacterial fermentation
- rapid small bowel transit
- Visceral hypersensitivity
Can be classed as
- CONSTIPATION PREDOMINANT
- DIARRHOEA PREDOMINANT
- MIXED
What are the functional bowel disorders as classified by Rome IV?
C1 - IBS
C2 - functional constipation
C3 - functional diarrhoea
C4 - functional abdominal bloating/distension
C5 - unspecified functional bowel disorders
C6 - opiod induced constipation
What are the diagnostic criteria for IBS?
recrurrent abdominal pain on average 1/week in the last 3 months
AND at least 2 of:
- related to defecation
- change in frequency of stool
- change in appearance of stool
Diagnosis of a functional bowel disorder –> presumes absence of a structural or biochemical explanation
Summarise the Bristol Stool Chart
Type 1 - like nuts Type 2 - sausage shape, lumpy Type 3 - sausage with cracks Type 4 - sausage, smooth and soft Type 5 - soft blobs, clear edges Type 6 - fluffy pieces with ragged edges, mushy Type 7 - watery, no solid pieces
Symptoms associated with IBS-related psychiatric disorders
panic disorder palpitations early insomnia ruminative thinking diaphoresis constant worries agoraphobia depression change in appetite late insomnia loss of interest loss of libido fatigue
Investigations and Tx in diarrhoea predominant IBS
Nat Rev Gastroenterol Hepatol 2014
Exclude carcinoma!!! - faecal calprotectin, colonoscopy etc
Exclude coeliac & other malabsorption - TTG Abs etc
Consider spurious diarrhoea - colonic transit x-ray
Consider bile acid diarrhoea - SeHCAT
Anti-diarrhoeals - loperimide, codeine
Psychological support
consider dietary review and modifications
Describe the dietary factors in IBS
Constipation predominant: water, fibre
Diarrhoea predominant: lactose, fat/bile malabsorption
Abdo pain and bloating: meal size, gas consumption, gas production
Summarise the Tx of constipation predominant IBS
exclude obstruction, hypothyroid, drugs
increase fibre and fluid
BULKING AGENTS: ispaghula, cellulose
Osmotic laxatives: Mg2+ salts, lactulose, movicol
evacuation assessment, biofeedback/training
psychological support
Summarise the Tx of abdominal pain/bloating in IBS
exclude obstruction, ulcers, gallstones, ischaemia, chronic pancreatitis
dietetic advice - meal size, gas consumption
Treat constipation/urgency/diarrhoea
Anti-spasmodics - MEBEVERINE
Anticholinergics/SSRIs
CBT to deal with pain
What are FODMAPs?
Fermentable oligosaccharides (fructose, galactose) Disaccharides (lactose) Monosaccharides (fluctose) and Polyols (sorbitol)
How can FODMAPs contribute to the symptoms of IBS
- being osmotically active –> increased water delivery –> luminal distension
- by being rapidly fermented –> increased gas production –> luminal distension
luminal distension can cause motility changes, bloating, pain and wind
Found by Staudacher 2012 that fermentable carb restriction improved bloating, urgency and overall symptoms
What are the Rome criteria on the diagnosis and Tx of functional constipation?
straining 25% of the time
lumpy hard stool 25% of the time
sensation of incomplete emptying 25% of the time
Anorectal obstruction sensation in 25% of the time
manual manouvers to facilitate 25% of the time
fever than 3 defecations/week
any 2 of the above + insufficient criteria for IBS
Tx: exclude obstruction diet: fibre + fluids bulking agents: ispaghula, cellulose omotic laxatives psychological support
Diagnostic Rome criteria for functional bloating
BOTH of
- recurrent bloating OR distension >1d/week
- insufficient criteria for a diagnosis of IBS, functional constipation/diarrhoea or postprandial distress syndrome
Diagnostic Rome criteria for functional diarrhoea
loose mushy or watery stools WITHOUT PAIN in > 25% of defecations
Describe the normal bile acid homeostasis
bile acids synthesised in liver from cholesterol
conjugates with glycine or taurine
secreted via biliary tree into small intestine
there they solubilise lipids in micelles
REABSORPTION
- passively in jejunum if unconjugated
- actively in ileum if conjugated
Then reuptake by hepatocytes from blood and resecreted
How is bile acid diarrhoea classified?
Fromm & Malavolti 1986
TYPE 1: SECONDARY
- ileal resection, ileal disease (Crohns), bypass
TYPE 2: PRIMARY
- idiopathic BA malabsorption
- primary BA diarrhoea
TYPE 3: MISC
- radiation enteropathy, post-cholecystectomy, ulcer surgery, chronic pancreatitis, coeliac disease, SIBO
Describe the mechanism of bile acid diarrhoea
Waters 2014
Excess bile acids in colon
o Unabsorbed by the small intestine?
o Increased production?
Bacterial transformation of bile acids
o Deconjugation + dehydroxylation
Stimulation of colonic secretion if bile acid accumulates in the colon
o Anion secretion
o Watery stool
Diagnosis of bile acid malabsorption
Walters et al 2010
Fecal bile acids
- 24h stool collection
- unpopular as it’s not easy for pt or staff
SeHCAT
- synthetic 75Se radiolabelled bile acid analogue
- detected by g-camera
- limited radiation exposure
- measure bile acid retention
- – 7 day retention normal >15% hense <10% is diagnostic
7a-hydroxy-4-cholesten-3-one (C4) = CYP7A1 product
o Measure of bile acid synthesis
o Measured by HPLC
o Inversely correlates with SeHCAT
Possible mehcanisms of bile acid diarrhoea
Secondary (Type 1)
- Surgical removal of functioning ileal tissue/inflammatory changes leading to impaired ileal gene expression/
Primary (Type 2)
- Normal structure and function but abnormal transporter function perhaps due to mutant proteins or reduced expression
Summarise the absorption of bile acids in the ileum
SLC10A2 gene (apical sodium-dependent transporter – ASBT + ileal bile acid transporter – IBAT)
FABP6 gene (ileal lipid binding protein – ILBP + ileal bile acid binding protein – IBABP)
OST-alpha/OST-beta heterodimer
Idiopathic bile acid malabsorption may be caused by
- Defective BA transport (rare cases of ABST mutations)
- However there is evidence of normal BA uptake in biopsies hence it may be to do with:
- – Rapid small intestinal transit time in vivo
- – Deconjugation
- – Changes in BA pool size
- Decreased FGF19, which regulates BA synthesis
Normally BA binds FXR which upregulates FGF19 expression/secretion
- In the liver FGF19 binds FGFR4 which activates, this increases SHP which represses BA synthesis
- In decreased FGF19 there would be increased bile acid synthesis leading to more entering the colon → secretory diarrhoea
Summarise the treatment of bile acid diarrhoea
BILE ACID SEQUESTERANTS - Hofmann 1969
Questeran & Cholestid = powders
Choestagel = tablet
poor long-term compliance
bloating may worsen
can bind other drugs
optimal dosing uncertain
