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Autoimmunity in Inflammatory Bowel Diseases. Dimitrios P. Bogdanos. Professor of Immunopathology The Sheila Sherlock Medalist. Disclosure statement. 2008-2013

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slide1

Autoimmunity in Inflammatory Bowel Diseases

Dimitrios P. Bogdanos

Professor of Immunopathology

The Sheila Sherlock Medalist

slide2

Disclosure statement

2008-2013

I have received in the past Lecture Honoraria, Consultation Fees, Expert Panel Fees, Accommodation/Travel Expenses Coverage

INOVA, EUROIMMUN, Generic Assays, FALK, BIORAD,

(King’s College Hospital Charitable Trust)

Part of travel/accommodation expenses are covered by the Organizers

I do not have shares or any other relevant financial or other relationship with a commercial organization that could influence the content of my presentation

ALL FEES OR HONORIA SUPPORT MY FELLOWS’S RESEARCH INITIATIVES/CONFERENCE TRAVEL EXPENSES

slide3

I have received diagnostic reagents free of charge and/

or participated in collaborative projects

EUROPE

AID

Biorad

CyBio

Diarect

Euclone

EUROIMMUN

Generic Assays

InnoVision

Invitrogen-

MabTech

Mardx

Meridian LS

Menarini

Miltenyi

Molecular ProbesPeproTech

Pharmacia

Roche

Disclosure statement II

AMERICA

Gilead

INOVA

IMCCO

Virusys

JAPAN

MBL

slide4

Inflammatory Bowel Diseases (IBD)

Immunology of IBD

Autoimmunity in IBD

slide5

IBD: EPIDEMIOLOGY & STATASTICS

  • Estimated prevalence – Active cases 100/100,000 of general population
  • Estimated approx 1 million cases in US split equally among CD and UC
  • More Prevalent in developed/ developing countries
  • Equal distribution among Male:Female
  • etiopathogenesis not resolved yet
    • autoimmunitymayplay a role
  • subsets
  • Crohn’s disease
    • Ulcerative colitis
    • Colitis indeterminate
slide6

Ulcerative Colitis

  • Autoimmune Process ?????????????????
  • Inflammation confined to colon
  • Bimodal Incidence (Ages 15-40 yrs OR 50-80 yrs)
  • Signs and symptoms: Rectal bleeding, loose bloody stools, passage of mucus from rectum, abdominal pain
  • Complications: perforation, stricture, megacolon, cancer
slide7

Ulcerative Colitis

  • Inflammation confined to Treatment:
    • Medical:
      • Mild/moderate disease—5-ASA, corticosteroids
      • Severe disease—IV steroids or immunosuppressants for refractory disease
    • Surgical: Proctocolectomy (curative)
      • Indications: Failure of medical therapy, increasing risk of cancer with long standing disease, bleeding, perforation
  • Prognosis: Approximately 1-2% risk of cancer at 10 years, 1%/year thereafter
imaging ulcerative colitis
Imaging Ulcerative Colitis
  • Barium Enema vs. CT
    • Barium Enema is no longer the test of choice
  • Findings
    • Continuous lesions from rectum proximally with circumferential involvement
  • Lead Pipe Sign
    • Repeated episodes of mucosal ulceration and marked muscularis hypertrophy results in shortening, narrowing and smoothing out of the normal haustral markings.
    • “Lead pipe” appearance of colon due to chronic scarring and retraction/loss of haustra

Weinstein A et al. A super ‘lead pipe’ colon: radio-pathological correlation of long-standing ulcerative colitis. SA Journal of Radiology;2008 Oct:70-72

imaging crohn s disease
Imaging Crohn’s Disease
  • Small bowel contrast study vs CT
    • SBFT useful for characterizing length of involvement and areas of stricture
  • Characteristic Findings
    • Mucosal nodularity
    • Narrowed lumen
    • Ulceration
    • String sign
    • Abscesses or fistula
  • String Sign
    • Term often applied to the appearance of any marked narrowing of the lumen, but originated as descriptor of reversible narrowing in Crohn disease.
    • Narrowing caused by incomplete filling as result of irritability/spasms associated with ulceration.

String Sign

Masselli G. The gastrointestinal string sign. Radiology. 2007 Feb;242(2):632-3.

extraintestinal manifestations
Extraintestinal Manifestations
  • Dermatologic features: erythema nodosum, pyoderma gangrenosum
extraintestinal manifestations1
Extraintestinal Manifestations
  • Ocular: episcleritis, anterior uveitis
  • Rheumatic: arthritis, ankylosing spondylitis, sacroiliitis
  • Hepatobiliary: steatohepatitis, cholelithiasis, primary sclerosing cholangitis
slide12

Features of UC versus CD

Feature UCCD

Depth of inflamation Mucosal Transmural

Pattern of disease Contiguous Skip areas

Location Colorectal Mouth-Anus

Rectal involvement Usual less common

Ileal disease Backwash 10-15% Common

Fistulas Rare Common

Perianal Disease Rare Common

Granulomas Unlikely 10-30% pts

Overt Bleeding Usual less common

Malnutrition Unlikely more common

Cancer Risk CRC, CholangioCRC,SmBwl

Tobacco use Protective Harmful

laboratory testing
Laboratory testing
  • CBC (high rate of anemia, due to chronic inflamm., blood loss, B12 malabsorption)
  • ESR, CRP often elevated
  • Albumin (often low due to chronic inflamm., blood loss, malabsorption)
  • Stool studies to rule out infection
  • Noncaseating granulomas on biopsy suggest CD
immunopathogenesis of uc
ImmunoPathogenesis of UC

Bogdanos and Polymeros Gastroentrol 2004

Sartor Nat Clin Pract Gastroenterol Hepatol 2006,

Stephen Gastr Hepatol 2009

Bamias Cur Opin Gastroenterol 2013

immunopathogenesis of uc1
ImmunoPathogenesis of UC

Strobe and Fuss Gastroenterol 2013

slide18

Autoantibodies in Crohn‘s disease

  • (Auto)antibodies to glycans specific for Crohn’s disease
    • ASCA, Main et al., 1988
    • anti-chitobioside carbohydrate ab (ACCA)
    • anti-laminaribioside carbohydrate ab (ALCA)
    • anti-mannobioside carbohydrate ab (AMCA)
    • ELISA, Altstock et al., 2005
  • Antibodies to bacterial antigens
    • Outer-membrane porin of E.coli (OmpC),
    • Flagellin CBir1
    • Pseudomonas fluorescens ass. Sequence I2
  • Pancreatic autoantibodies - autoantibodies to exocrine pancreas
    • 30% Crohn’s disease patients
    • indirect immunofluorescence, Stöcker et al., 1984
slide19

Clumpy staining

in the lumen of

pancreatic acinar

type 1

Speckled cytoplasmic staining in pancreatic acinar cells, type 2

Pancreatic autoantibodies, type 1 and type 2

Stöcker W et al., 1987 Scand J Gastroenterol

BogdanosAutoimmun Rev 2011

slide20

PAB, type 1 and type 2

Pancreatic acinus

type I staining

type II staining

Roggenbuck D et al., 2013 Adv ClinChem

Komorowski L et al., 2012 JCC

BogdanosAutoimmun Rev 2011

PavlidisClin Dev Immun 2013

slide21

Is there any connection between Pancreas and Colon in IBD?

Pavlidis and Bogdanos Clin Dev Immun in press

Roggenbuck Adv Clin Chem 2013

Bogdanos and Forbes Clin Dev Immun 2013

slide22

Identification of PAB target

Two-dimensional electrophoresis and immunoblot

Roggenbuck D et al., 2009 Gut

slide23

IFT huGP2 in HEK293

GP2 specific IgG and IgA in patients with PAB-positive and PAB-negative CD, UC, and blood donors detected by IIF using GP2 transfected HEK293 cells

Roggenbuck D et al., 2009 Gut

slide24

Identification of PAB target

MALDI-TOF mass spectrometry:GP2, zymogen granule glycoprotein 2

Roggenbuck D et al., 2009 Gut

slide25

GP2 in human intestine

  • Physiological roleof GP2
    • not fully understood yet
    • homology to Tamm-Horsfall protein (uromodulin)
    • first line defense against microbial agents
    • Interaction with type 1 fimbriae of E.coli(FimH)
    • Transcytotic receptor in M cells – regulation of innate and acquired immunity
slide26

GP2 – M cellreceptor

Hase K et al., 2009 Nature

slide27

Peyer‘s patches

Ohno and Hase., 2010 Gut Microbes

slide29

Pancreatic autoantigen: GP2 in human intestine

First confirmation of GP2 in human intestine, the side of inflammation in IBD

*

*

A CD, n=4B CU, n=4D controls, n=5

* p<0.02

Roggenbuck et al., 2009 Gut

Pavlidis Gut 2012

slide30

Thus the pancreatic GP2 autoantigen is also an intestinal protein

Roggenbuck et al., 2009 Gut

Pavlidis Gut 2012

LiaskosClin Dev Immunol 2013

slide31

Peyer‘s patches

Hase K et al., 2009 Nature

slide32

Scavengerreceptorbinding

Hölzlet al., 2010 Cell Immunol

slide33

Putative physiological function

GP2

antimicrobial IgG

P

FAE

Fim H +

B

Fim H +

D

M

T

D

intestinal lumen

mucosa associated lymphoid tissue

Roggenbuck D et al., 2013 Adv ClinChem

slide34

Expression of recombinant GP2

Purification of recombinant GP2 (baculovirus expression system)

A reducing SDS-PAGE

B immunoblot - anti-HIS

C immunoblot using anti-human GP2

1 cell culture supernatant of transfected SF9 cells

2 Ni-chelate chromatography

3 anion exchange chromatography on Mono Q

Roggenbuck et al., 2011 ClinChimActa

slide35

Anti-GP2 IgG ELISA

A: PAB-positive CD patients (n = 72)

B: PAB-negative CD patients (n = 106)

C: UC patients (n = 100)

D: BD (n = 162)

Roggenbuck and Bogdanos 2011 ClinChimActa

slide36

Anti-GP2 IgA ELISA

A: PAB-positive CD patients (n = 72)

B: PAB-negative CD patients (n = 106)

C: UC patients (n = 100)

D: BD (n = 162)

Roggenbuck, Bogdanoset al., 2011 ClinChimActa

slide37

Disease phenotype in CD

Bogdanos et al., 2012 BMC Gastroenterol

slide40

Disease phenotype in CD

Association with disease location

*

*

*

*

*

Bogdanos et al., 2012 BMC Gastroenterol

slide41

Disease phenotype in CD

Association with disease location

P = 0.0128

Pavlidiset al., 2012 Clin Dev Immunol 2012

slide42

Disease phenotype in CD

Association with disease behavior

*

*

*

Bogdanos D et al., 2012 BMC Gastroenterol

Roggenbuck D et al., 2012 JPGN

Rieder F et al., 2012 Gastroenterol

slide43

At diagnosis

Association with disease activity

2 months on

immunosuppressive Tx

60 mo

on Tx

3 mo

on Tx

36 mo

on Tx

Correlation with disease activity?

Similarity to anti-ASGPR in autoimmune hepatitis serology

12 mo

on Tx

Anti-ASGPR (BI)

Rigopoulouet al., 2012 Autoimmun Rev

Roggenbuck et al., 2012 Autoimmun Highlights

slide47

GP2 expression on PBMCs

unstimulated

CD3 activated

GP2 expression

50

*

GP2

40

30

Percent expression

20

β actin

10

unstimulated

CD3 activated

Werner et al., 2012 J Immunol

slide48

GP2 effect on epithelium

Apoptosis

50

40

Percent AnV+PI-

positive cells

*

30

T84

**

hIECs

20

10

0

10ug/ml

20ug/ml

GP2

Werner et al., 2012 J Immunol

slide49

GP2 effect on epithelium

Proliferation

1

*

0.8

*

O.D.

**

T84

0.6

hIECs

0.4

0.2

0

10ug/ml

20ug/ml

GP2

Werner et al., 2012 J Immunol

slide50

GP2 – phagocytosis of E-coli

**

180

*

170

Epithelial T84

Monocytes

160

150

140

Percent effect

130

120

110

100

90

GP2:

0

5ug/ml

10ug/ml

Werner et al., 2012 J Immunol

slide51

Depletion of Tregs - GP2 effect

Activation

80

*

PBMC

60

Percent CD3+CD25+

positive cells

PBMC-Treg

*

40

PBMC-Treg+Treg

20

0

10 ug/ml

GP2

Werner et al., 2012 J Immunol

Pavlidis JCC 2013

LiaskosClin Dev Immunol 2013

slide52

Depletion of Tregs - GP2 effect

Apoptosis

40

30

Percent AnV+PI-

positive cells

PBMC

20

*

*

PBMC-Treg

10

PBMC-Treg+Treg

0

10 ug/ml

GP2

Werner et al., 2012 J Immunol

slide53

antiTNFa modulates GP2

Apoptosis

20

*

Percent AnV+PI-

positive cells

10

*

Activation

50

*

40

*

Percent CD3+CD25+

positive cells

*

30

20

*

10

Werner et al., 2012 J Immunol

slide54

antiTNFa modulates GP2

Untreated

CD3

CD3 +IFX

CD3 +ADA

4

4

4

4

0

1

2

3

0

0

1

1

2

2

3

3

0

1

2

3

GP2

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

β-Actin

120

120

120

120

30.1%

20.9%

13.6%

21.7%

Counts

60

60

60

60

Untreated

0

0

0

0

GP2/SA-FITC

PBMCs (N=3) were stimulated with anti-CD3 and Caco2 cells (N=3) with 10 µg/ml LPS. Cells were incubated either with or without 10 µg/ml IFX or ADA. RNA levels were determined using PCR. Surface expression was determined using flow cytometry.

slide55

Putative pathophysiology

anti-GP2 IgA

anti-GP2 IgG

P

M

Fim H +

B

D

FAE

M

Fim H +

T

M

D

Fim H +

mucosa associated lymphoid tissue

intestinal lumen

Roggenbuck D et al., 2013 Adv ClinChem

slide56

Take-home message

  • Summary
    • GP2 is a target of PAB in CD
    • GP2 is expressed in human intestine, transcytotic receptor of M cells
    • IgA and IgG anti-GP2 detected by novel ELISA are specific for CD
    • Anti-GP2 detection may improve serological diagnosis of CD
slide57

Take-home message

  • Summary
    • Anti-GP2 antibodies are associated with clinical phenotype in Crohn’s disease
    • Anti-GP2 IgA and IgG were more prevalent in CD:
    • at a younger age (A1),
    • with ileocolonic location (L3),
    • stricturing behaviour (B2)
    • GP2 modulates innate and adaptive immune mechanisms
slide58

Is there any connection between Pancreas and Colon in IBD?

YES THERE IS

Pavlidis and Bogdanos Clin Dev Immun in press

Roggenbuck Adv Clin Chem 2013

Bogdanos and Forbes Clin Dev Immun 2013

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