Autoantibodies in Liver Disease

Epidemiology PBC

•

Rare disease

–

0.6 - 2 % deaths from cirrhosis worldwide

–

Annual European incidence 7 - 75 per million Europe

•

Disease of civilization

–

Middle class women in industrialized countries

–

Urban > Rural areas

•

Clustering of disease

–

Sweden

Selenium deficiency

–

England

Water supply

–

“

Transmission” from patient to nurse

–

Familial

Public Health Impact of

PBC in the US

•

Epidemiology of PBC in US

–

Prevalence (1995): 400 per million (> 47,000 in US)

–

Incidence: 27 per million (unchanged >2 decades)

•

Treatment options for PBC

–

Liver transplantation (OLT):



survival



and QOL

–

Ursodeoxycholic acid:



time to death or OLT



•

The impact of these interventions on mortality and

health service utilization on a national scale is

unknown.

Kim (Gastro, 2000

in press

)



Markus (NEJM, 1989)

Gross (Hepatology, 1999)



Poupon (Gasto, 1997)

Overall Mortality from PBC

and PSC

0.2

0.4

0.6

0.8

1980

1985

1990

1995

PSC
PBC

NIH

consensus

conference

on OLT

Poupon’s

NEJM paper

on UDCA

PBC Presentation

•

40 - 60 year old middle class woman

–

95% AMA positive

•

Symptomatic

–

Fatigue 70% or pruritus 70%

–

Mild xerostomia or xero-ophthalmia 30 - 40 %

( 72 - 100 % patients undergoing specific

testing )

–

Bili x 2 Alk Phos x 4 AST x 2



cholesterol

•

Asymptomatic

–

Abnormal liver enzyme tests on routine

screening predominantly elevated Alk Phos

–

Referred from Rheumatology or Endocrinology

Laboratory Values PBC

Lab test

Normal

SymptomaticAsymptomatic

Alk Phos

87-250 IU/L

1695 IU/L

1395 IU/L

Tot Bili

< 1 mg/dl

4.4 mg/dl

0.9 mg/dl

AST

8-20 IU/L

85 IU/L

78 IU/L

Albumin

3.5-4.7 g/dl

3.5 g/dl

3.8 g/dl

Cholesterol

218-300 mg/dl

377 mg/dl

285 mg/dl

Triglycerides

101-150 mg/dl

133 mg/dl

96 mg/dl

Ig Total

0.8-1.6 g/dl

1.9 g/dl

2.1 g/dl

IgM

0.2-1.4 mg/ml

6.0 mg/ml

7.1 mg/ml

Pro Time

10-12 sec

11.3 sec

11.1 sec

ESR

< 40 mm/hr

65 mm/hr

56 mm/hr

Associated Diseases

PBC

20%

40%

60%

80%

100%

Breast

cancer

Celiac

disease

UTIs

RTA

Arthropathy

Sjorgrens

Thyroiditis

Scleroderm

4.4 x increase

HLA B8

vs 8 % control

urinary

acidification

100 %

Schrimer’s

CREST

NB Lichen planus

Natural History of PBC

Hepatic

failure

0 - 2 years

AMA

IgM GT SymptomsSigns

2 - 10 years 2 - 20 years 3 - 11 years

florid duct ductular fibrosis

cirrhosis

lesion

proliferation

Age in years 20 30 40 50 60 70

Typical

symptoms none malaise itching jaundice

Pathology

Histologic stage i ii iii iv

fibrosis/cirrhosis

number of bile ducts

PBC Histology

NB nodular hyperplasia

•

Progressive non supporative destructive

cholangitis

100

120

  granulomas
  copper
  cholestasis
  florid lesions
  decreased ducts

100

120

  granulomas
  copper
  cholestasis
  florid lesions
  decreased ducts

florid duct ductular fibrosis

cirrhosis

lesion

proliferation

% histologic features

copper

vanishing ducts

cholestasis

florid duct lesion

granulomas

I II III IV stage

Immune Related Disorders

•

Graft Versus Host Disease

–

Vanishing bile ducts syndrome

–

OGDC peptide isolated as T cell epitope from

allogeneic APC reactive to 2C lymphocyte

clone

•

Scleroderma

–

anti-centromere antibodies with cholestatic

liver disease

–

extrahepatic disease CREST

•

Sarcoidosis

–

AMA positive with cholestatic liver disease

–

extrahepatic disease

Complications of PBC

•

End stage liver disease

–

Portal hypertension and GI hemorrhage

Precirrhotic patients with nodular hyperplasia

–

Hepatocellular Cancer

rare???

•

Malabsorption

–

Fat soluble vitamin deficiency

–

Vit A Vit D Vit E

•

Cholestyramine may reduce fat absorption

–

Hepatic osteodystrophy

osteoporosis > osteomalacia

•

Eye: scleral degeneration

•

Mouth: dental decay

Therapy for PBC

•

Management of Complications

–

Pruritis

Cholestyramine H 1 antagonists

–

Hyperlipidemia

Diet Statins

–

Osteodystrophy

Calcium Vit D

•

Immunosuppressive and other agents

–

Low efficacy and high toxicity

corticosteroids azathiaprine D - penicillamine

chlorambucil colchicine cyclosporine A

•

Ursodeoxycholic Acid

–

Improves biochemistry

–

Delays need for transplantation

•

Orthotopic Liver Transplantation

PBC Physical

Examination

•

Abdomen

–

Hepatomegaly 55%

–

Splenomegaly 33%

–

Ascites 5%

•

Musculoskeletal

–

Clubbing 8%

–

Bone tenderness

rare

•

Misc.

–

Jaundice 55%

–

Edema 5%

Pigmentation
Spider Naevi
Excoriation
Xantholasmsa
Xanthoma

Skin Lesions

Follow-Up AMA +ve (IF) Subjects

With Normal Biochemistry

no: pts

no: developed

symptoms

no: developed



ALP / AST

NB: 4/29 biopsies initial not compatible with PBC

5 patients died (non-hepatic)

Adapted from Metcalf et al., Lancet 1996;348:1400

time to develop

symptoms



ALT

24 / 26

1 - 17 yrs

1 - 19 yrs

(mean 5.6)

Autoantibodies in Liver

Disease

Anti-Liver Kidney

Microsomal

cytochrome

P450 IID6

Anti - Nuclear

Antibodies

RNPs

nuclear pore

lamins histones

Anti-Smooth Muscle

Antibody



- actin

Anti-Neutrophil Cytoplasmic

Antibody

lysosomal proteins

Anti-Centromere

kinetochore proteins

Anti-Mitochondrial

Antibody

pyruvate

dehydrogenase E2

Soluble Liver Antigen

glutathione s- transferase

source of

sera

total

no.

indirect

immunofluorescence

(no: positive)

ELISA PDH

(no: positive against)

PBC

Rheum Dis

CAH

ALD

Control

Reactivity of Patient Sera Against

Mitochondrial Dehydrogenase Enzyme

Complexes

Immunoblot PDH-E2

(no: positive against)

PDH, pyruvate dehydrogenase complex; E2, E2 subunit of PDH; PBC, primary billary

cirrhosis; Rheum Dis, rheumatic disease; CAH, chronic active hepatitis; ALD,

alcoholic liver disease;

Yoshida et al., Gastroenterology 1990;99:190

Immunoblotting in 20 AMA Negative

and 20 AMA Positive (IF) PBC Patients

method

Immunofluorescence

ELISA for E2/X immunoblotting for

PDC immunoblotting for OGDC

immunoblotting for BCOADC

AMA-IF -ve pts

testing +ve (n+20*)

AMA-IF +ve pts testing

+ve (n=20*)

PDC=pyruvate dehydrogenase complex; OGDC=2-oxo-glutarate

dehydrogenase complex; BCOADC=branch chain 2-oxo acid dehydrogenase

complex; * for BCOADC, n=14

Michielletti et al., Gut 1994;35:261

PBC and Bacterial

Infections

•

Increased prevalence of bacterial

infections

–

Greater incidence of UTI’s

–

Detection of bacterial R mutants in stool

–

Demonstration of AMA reactivity with R

mutants

absence of cell wall with



PDH E2 reactivity

•

Organisms implicated in disease

–

Mycobacteria Gordonae

•

Mouse model

–

Mycoplasma and biliary disease

Bacterial Endosymbiosis

in Eukaryotic Cell

Evolution

Coccoid cyanobacteria

Chloroxybacteria

photosynthesis

Spiroplasmas

Spirochetes

motility

Thermoplasmas

heat and acid

resistance

Bdellvibrio

Paracocci

respiration

Plant

Animal

Fungus

Is PBC an Infectious

Disorder?

•

Clustering of disease

–

Sweden

Selenium deficiency

–

England

Water supply

–

Transmission

Patient to nurse

–

Familial

•

False positive HIV ELISA in 15% patients

•

Recurrent disease after OLT

–

Vanishing bile duct syndrome with



AMA

–

PDC-E2 reactivity on biliary epithelium

HIV

gag

Reactivity in Liver

Disease

100

HIV Western blot studies

singular HIV p24 reactivity

Healthy Donors

n = 175

ALD and A-1 AT

n = 24

HCV

n = 13

HBV

n = 15

PSC

n = 19

PBC

n = 77

Sjogrens

n = 47

HIAP Western Blots in Liver

Disease

100

HIAP retrovirus derived from Sjogren’s syndrome pts



2 HIAP bands  1 HIAP band

Healthy Donors

n = 105

ALD and A-1 AT

n = 24

HCV

n = 53

HBV

n = 75

PSC

n = 19

PBC

n = 72

Sjogrens

n = 190

HIAP Immunoblots in Liver Disease

100

HIAP retrovirus derived from Sjogren’s syndrome pts

HIAP Western blot reactivity to  2 HIAP bands

Healthy Donors

n = 105

ALD and A-1 AT

n = 24

HCV

n = 53

HBV

n = 75

PSC

n = 19

PBC

n = 72

Sjogrens

n = 190

HIAP Western Blots in Liver

Disease

HIAP retrovirus derived from Sjogren’s syndrome pts

HIAP Western blot reactivity  1 HIAP band

ALD and A-1 AT

n = 24

HCV

n =

HBV

n =

PSC

n =

PBC

n =

-
--

* Representations PCR fragments < 1kb

Representations PCR fragments < 1kb

complexity BamHI < BglII < HindIII

55 13 8

Restriction digest

GATC

Ligate

GATC

adaptors

Add

primer

and amplify

Ligate

GATC

adaptors

tester

then mix melt Restriction digest

GATC

PCR representations

anneal & fill in ends

MB nuclease digest ssDNA

Tester

Driver

-
--

Amplification

exponential

linear

in excess

Representational Difference

Analysis

hybrid

DNA

ssDNA

Repeat step

2 with new

with new

GATC

adaptors

primers

PBC Serum Reactivity to HIV

Proteins

•

PBC patients assessed for liver

transplantation

•

HIV ELISA

–

15% patients positive

n=46

–

ELISA titres corelated with AMA levels

•

HIV Western blot

–

1 patient with p41 and p24 reactivity

•

Risk factors for HIV

–

1 patient ( WB -ve ) received multiple blood

transfusions prior to tests for HIV

HLA Association with

Autoimmune Liver Disease

Class I Class II Class III Haplotype Amino Acids

AIH

A1 B8 DR3 & DR4 C4A del. A1 B8 DR3 leucine aa 71

RR X 3 RR X 3 DR



95% UK

PSC

A1 B8 DR3 & DR4 A1 B8 DR3

DR52A

RR X 3

PBC

NR DR8 DPB1 C4B DR8 C4A Q0 leucine aa 35

DR2 DR3 DR4 C4A Q0 RR X 184 DR



95% Japan

Summary of the Comfirmed HLA

Associations in Primary Biliary Cirrhosis

(First Studies Only

)

C4B2

DR8/DR11

C4AQ0

DQB1*0402

DPB1*0301

DR8

DPB1*0501

DRB10803

Brigg et al 1987

Gores et al 1987

Manns et al 1991

Underhill et al 1992

Mella et al. 1995

Maeda et al 1992

Seki et al 1993

Ogura et al 1994

S/R

N. Europe

USA

N. EUROPE

Japan

Allele

Author

S/R

Group

Summary of Non-MHC Genes in

Autoimmune Liver Diseases

IL-1B

1L-1RN

CTLA-4

IL-10

TCR C

lg allotype

2q13-q21

2q33

1q31-32

7p14-15

14q32-33

AIH

Unkown

Yes

PSC

Yes

Unkown

Unknown

Gene

Location

Association

PBC

Yes

Unkow

Unkno

Primary Biliary Cirrhosis

•

Granulomatous destruction of

interlobular bile ducts

•

Anti-mitochondrial antibodies

–

PDH-E2

95%

–

OGDH-E2

40-85%

–

BCODH-E2

55%

•

AMA immunohistochemistry



antigens resembling

PDH-E2 on cell surface

specifically in PBC pts

PBC

PSC

Rationale for PBC

Therapy

Hepatic

failure

0 - 2 years

AMA

IgM GT SymptomsSigns

2 - 10 years 2 - 20 years 3 - 11 years

Age in years 20 30 40 50 60 70

Typical

symptoms none malaise itching jaundice

Histologic stage i ii iii iv

Therapy related to disease process

Immunosuppression +++ +

Anti-fibrosis

- ++

+/-

Bile acid

+++ +++

Transplantation - +

+++

Treatments for PBC - Single Agents

Penicillamine

Cyclosporine

Cholorambucil

Tacrolimus

Azathioprine

Cholchicine

Thalidomide

Methotrexate

Urodeoxycholic

acid

Levamisole

Steroids

Malotilate

Ineffective

Equivocol or

Unknown

Effective

Better Pacebo

Better UDCA

1/64

1/16

1/14

1/64

1/16

1/14

Better Pacebo

Better UDCA

Does Ursodeoxycholic Acid Work?

month

Kisand et al, J Molec Med 1996; 74:271

months

Effect of Therapy on AMA in PBC

0.2

0.4

0.6

0.8

1.2

1.4

Ursodeoxycolic acid

Colchicine

Placeb

p<0.1

p>0.5

ELISA

Index

Frequencies of the Variant

Syndromes in Autoimmune

Chronic Liver Disease

Variant

types

clinical diagnoses

AIH

(n = 162)

PBC

(n = 37)

PSC

(n = 26)

Total patients

(N = 225)

AIH + PBC

AIH + PSC

autoimmune

cholangitis

total variants

8 (5)

0 (0)

11 (7)

19 (12)*

7 (19)

0 (0)

7 (19)†

0 (0)

14 (54)

0 (0)

14 (54)*†

15 (7)

14 (6)

11 (5) 40

(18)

Note: numbers in parentheses are percentages

* p = .000004

† p = .006

Czaja, Hepatology 1998; 28:362

alkaline phosphatase (ALP)

asparate aminotransferase (AST)

Prednisone

UDCA 750 mg/d

20 mg d/l

450

400

350

300

250

200

150

100

-6

-3

biopsey

AMA

ANA

1:320

neg

1:80

neg

1:40

neg

>1:1280

neg

>1:1280

neg

>1:1280

neg

>1:1280

Colombato et al, Gastroenterology 1994; 107:1840

Consecutive PBC

and AIH

Time(months)

Overlap/Variant

Syndromes

Classic

AIH

HCV

PSC

Cryptogenic

hepatitis

Autoimmune

cholangitis

2-8%

6-25%

1-2%

11%

10%

Granulomatous

Hepatitis

PBC

Vanishing Bile

Duct Syndrome

Giant Cell Hepatitis

Mild

idiopathic

ductopenia

of adulthood

AMA neg

PBC

with

normal ERCP

Autoantibody

negative

idiopathic

hepatitis

Fatty Liver

with

autoantibodies

Central Venulitis

Eosinophilic Hepatitis

Overlap/Variant

Syndromes Simplified

AIH

HCV infection

extrahepatic

syndromes

ALKM ANA ASMA

cryptogenic

hepatitis

PBC

phenotype

hepatitis

predominant

AMA neg

PBC

AIC

AIH

PSC

phenotype

hepatitis

predominant



children

AIH

PSC

HCV

AIH

Liver Transplantation for

PBC

•

Superior outcome for patients transplanted

earlier in course of disease

–

Morbidity Mortality and Cost

•

Consider transplantation if....

–

Bilirubin > 7.0 mg/dl

–

Major complications with bilirubin < 7.0 mg/dl

Uncontrolled ascities

Variceal hemorrage

Severe osteodystrophy

Intolerable pruritis

Invalidating malaise

Improved Prognosis in PBC

Patients

with Liver Transplantation

100

120

% Survival 161 PBC patients following Transplantation

Liver Transplantation

group 1 OLT

group 2 OLT

group 3 OLT

Mayo score prediction

group 1 Mayo

group 2 Mayo

group 3 Mayo

Years

•

Mayo Prognostic index for PBC

Bilirubin Albumin Age Prothrombin time Edema

OLT Utilization for Liver

Failure from PBC

•

Logistic model to assess the likelihood of OLT among

those with liver failure

Variable

Odds ratio

95% CI

Age > 65

0.04

(0.03-0.05)

<.01

Diabetes

0.63

(0.46-0.85)

<.01

Year 89-91

Year 92-94

1.29

(1.08-1.54)

<.01

Year 95-97

1.28

(1.05-1.55)

.01

•

Decrease in utilization was due to

–

patients’ age and comorbidity

–

reduction in need, not availability

Conclusions

•

Mortality and hospital service utilization decreased

in patients with PBC 65 years or younger during the

last 10-15 years.

•

While the early reduction in PBC mortality in the

1980s were likely due to liver transplantation,

ursodeoxycholic acid may be responsible for more

recent changes.

–

Reduction in need for liver transplantation (a

true reduction in the incidence of end-stage PBC)

–

No changes in mortality from PSC

Document Outline