AUTOANTIBODIES PRECEDING AUTOIMMUNE DISEASES STUDY GROUP - - - PowerPoint PPT Presentation

AUTOANTIBODIES PRECEDING AUTOIMMUNE DISEASES

Luis Eduardo Coelho Andrade, M.D, Ph.D.

Associate Professor Rheumatology Division UNIFESP luis.andrade@unifesp.br Consultant in Immunology Fleury Medicine & Health luis.andrade@fleury.com.br

STUDY GROUP - AUTOANTIBODY STANDARDIZING COMMITTEE Washington, November 12th 2012

Conflict of interest

Immunology consultant to Fleury Medicine & Health Laboratories (private clinical laboratory )

Autoantibodies precede the clinical onset of various autoimmune diseases

• Anti-perinuclear factor (APF) & RA (Aho et al, 1985)
• Anti-insulin & type I diabetes (Dean et al, 1986)
• Anti-thyroid peroxidase & Hashimoto`s disease (Watanabe et al, 1995)
• Anti-dsDNA & SLE (Arbuckle et al, 2003)
• Anti-CCP e RA (Rantapää-Dahlqvist et al, 2003)
• AMA & PBC (Crosignani et al, 2008)

Time frame

Detectable autoantibody Tissue injury Autoantibodies precede clinical manifestations of autoimmune diseases

Horizon of events timeline

Autoantibody detection Clinical onset

Clinical-immunological temporal dissociation

Horizon of events timeline

Autoantibody detection Clinical onset

Clinical-immunological temporal dissociation

“Clinical stage”

Horizon of events timeline

Autoantibody detection Clinical onset

Clinical-immunological temporal dissociation

“Immunological stage”

Horizon of events timeline

Autoantibody detection Clinical onset

Clinical-immunological temporal dissociation

What is going on here? Is an evolving process going on?

Horizon of events timeline

Autoantibody detection Clinical onset

Clinical-immunological temporal dissociation

Progression on the underlying immunological disorder?

• Titer increase?

Avidity increase? Ig isotype?

• Spreading of targets?
• epitope?

molecules?

AMA AMA + Sp-100 gp-210 Centromere

Autoantibodies precede Primary Biliary Cirrhosis for decades

Ascitis Portal hypertension Ictericia

Biliary tract lymphocyte accumulation

 Autoimmune liver disease with inflammation and injury to epithelial cells of intra- hepatic biliary ducts;   Th17 cells and  Treg cells;  Female preponderance;  Peak of incidence – fifth decade;  Prevalence – women above 40 years old (1:1000);  Heterogeneous disease evolution;  Anti-mitochondria antibodies precede the disease for years or decades. Facts about Primary Biliary Cirrhosis

Concept of pre-biochemical stage of PBC

• PBC –

Primary biliary cirrhosis AMA & normal liver enzymes Biochemically normal - BN

Earliest PBC-specific manifestation ==> increase in serum alkalyne phosphatase

Horizon of events timeline

Autoantibody detection Clinical onset

How different is the humoral autoimmune response before and after PBC ecclosion?

Biochemically normal - BN Primary biliary cirrhosis - PBC

The bait....

 Serum concentration of autoantibodies;  Avidity of anti-PDC-E2 antibodies (“fraction M2”);  Isotype class of AMA (IgG, IgM, IGA);  Number of targeted autoantigens.

AMA individuals with normal serum alkaline phosphatase PBC patients

X

n = 82 n = 130

Dellavance A, Andrade LEC et al, Hepatol Int, in press

Other autoantibodies associated with PBC

 anti-gp-210  Anti-centromere Anti-Sp-100

Methods for evaluation of autoantibodies

• a. Indirect immunofluorescence on rodent tissue
• b. ELISA for antibodies to PDC-E2 antibodies (“M2 fraction”),

gp210, Sp-100, CENP-B

• c. Relative avidity of anti-PDC-E2 IgG  urea 8M

Autoantibodies in pre-biochemical stage of PBC Dellavance A, Cançado E, Andrade LEC et al, Hepatol Int, in press

p <0.001 GROUPS

Anti-PDC-E2 (arbitrary units)

PBC had higher anti-PDC-E2 serum levels than AMA BN

Dellavance A, Cançado E, Andrade LEC et al, Hepatol Int, in press

p <0.001

Relative avidity

GROUPS

PBC had higher anti-PDC-E2 avidity than AMA  BN

Dellavance A, Cançado E, Andrade LEC et al, Hepatol Int, in press

p =0.011 GROUPS

Anti-gp210 (arbitrary units)

PBC had higher anti-gp210 serum levels than AMA BN

Dellavance A, Cançado E, Andrade LEC et al, Hepatol Int, in press

10 20 30 40 50 60 1 isotype 2 isotypes 3 isotypes

CBP BN

p =0.006 PBC BN

PBC had higher anti-PDC-E2 avidity than AMA  BN PBC (triple and double isotype AMA) & BN (mono-isotype AMA)

Number of Ig Classes (IgA, IgG, IgM) in Rat Tissue IIF

Mitochondria Nuclear envelope PML Centromere Tissue - IFI ELISA

Cell domains targeted by autoantibodies

ELISA & ANA-HEp-2

Dellavance A, Cançado E, Andrade LEC et al, Hepatol Int, in press

10 20 30 40 50 60 1 domain 2 domains 3 domains 4 domains

CBP BN

p =0.002 Number of Cell Domains Targeted in HEp-2 IIF PBC BN

PBC serum recognized more cell domains than BN

High titer anti-PDC-E2 High affinity anti-PDC-E2 IgG High titer anti-gp210 Triple and double AMA isotype ≥2 cell domains targeted Low titer anti-PDC-E2 Low affinity anti-PDC-E2 IgG Low titer anti-gp210 Mono AMA isotype Only 1 cell domain targeted

Established PBC AMA - BN individuals with normal liver enzymes

Horizon of events timeline

Autoantibody detection Clinical onset

Progression in humoral autoimmune response?

High titer anti-PDC-E2 High affinity anti-PDC-E2 IgG High titer anti-gp210 Triple and double AMA isotype ≥2 cell domains targeted

Low titer anti-PDC-E2 Low affinity anti-PDC-E2 IgG Low titer anti-gp210 Mono AMA isotype Only 1 cell domain targeted

Dellavance A, Cançado E, Andrade LEC et al, Hepatol Int, in press

?

Auto-antibody parameter 2003 2008 2009 IIF-AMA 1/40 Non-reactive 1/640 Anti-PDC-E2 IgG Non-reactive Non-reactive 5.7 Anti-PDC-E2 IgG avidity (%) Non-reactive Non-reactive 65 WB-AMA bands (kDa) 52 74, 52 74, 52

Preliminary evaluation of longitudinal follow-up

AMA BN Serial samples available for 13 of 82 cases (16%)

Preliminary evaluation of longitudinal follow-up

1/13 (7.6%)

 STABLE

NO DECLINE 12/13 (92.3%) EXACERBATION BN & BN/AID (n = 13) 12/26

(46.2%)

14/26

(53.8%)

 STABLE  DECLINE

NO Exacerbation PBC & PBC/AID (n = 26)

Horizon of events timeline

Autoantibody detection Clinical onset

Clinical-immunological temporal dissociation

Is there any tissue inflammation or injury at this stage?

Dellavance A, Baldo D, Andrade LEC et al, in submission

Per-cutaneous liver biopsy in an asymptomatic individual?

Biochemical biomarkers of liver fibrosis & inflammation

Hyaluronic acid (HA) Type III Procollagen N-terminal Propeptide (PIIINP) Tissue inhibitor of metalloproteinase 1 (TIMP-1) Structural tissue organization, Water distribution in the extracellular matrix

Synthesis and degradation of type 3

collagen Liver tissue remodelling

Rosenberg et al, Gastroenterol 127: 1704, 2004

Enhanced Liver fibrosis (ELF) SCORE

ELF score in PBC and AMA BN

PBC (n = 30) BN (n = 45)

Dellavance A, Baldo D, Cançado E, Andrade LEC et al, unpublished

Time frame

Detectable autoantibody Tissue injury

Changing paradigm

“Clinical stage” “Immunological stage”

Which one is easier to fight?

Is it possible to treat asymptomatic autoimmune diseases or to prevent autoimmune diseases?

Preventive treatment of autoimmune diseases?

• Ursodeoxycholic acid is beneficial when administered

early in the natural history of PBC (Ishibashi H et al, Intern

Med 50: 1, 2011)

• Antimalarial treatment is associated with later onset of

systemic lupus erythematosus (James JA et al, Lupus.

16:401, 2007)

• Primary prevention in anti-phospholipid antibody carriers

(Bertero MT, Lupus 21: 751, 2012)

• Hyperbaric oxygen therapy (HOT) in pre-diabetic NOD

mice decreases the incidence and severity of type I DM

(Faleo et al, Diabetes 61:1769–1778, 2012)

. Patients with established PBC presented a more vigorous humoral autoimmune response than asymptomatic individuals with normal alkaline phosphatase levels, characterized by: Higher serum levels of anti-mitochondrial and anti-gp210 antibodies Higher relative avidity of anti-mitochondrial antibodies Triple or double isotype autoantibodies Autoantibodies targeting a higher number of cell domains .

Concluding remarks

. Patients with established PBC presented a more vigorous humoral autoimmune response than asymptomatic individuals with normal alkaline phosphatase levels, characterized by: Higher serum levels of anti-mitochondrial and anti-gp210 antibodies Higher relative avidity of anti-mitochondrial antibodies Triple or double isotype autoantibodies Autoantibodies targeting a higher number of cell domains . Preliminary longitudinal follow-up indicates that asymptomatic individuals with normal alkaline phosphatase tended to present exacerbation of the autoimmune humoral response

Concluding remarks

. The immunological disorder in pre-clinical stages of autoimmune diseases exhibit a dynamic behavior with progressive intensification and amplification of the humoral autoimmune response

Concluding remarks

. The immunological disorder in pre-clinical stages of autoimmune diseases exhibit a dynamic behavior with progressive intensification and amplification of the humoral autoimmune response The pre-clinical stages of autoimmune diseases offer a potential opportunity for early intervention and prevention of instatement of full-blown disease

Concluding remarks

ACKNOWLEDGMENTS

Alessandra Dellavance. Danielle Baldo & Cristiane Gallindo Research and Development Department, Fleury Medicine and Health Laboratories. Eduardo Luiz Rachid Cançado, Michelle Harriz, & Clarice Abrantes- Lemos – Hepatology Division – Universidade de São Paulo Support: São Paulo State Research Support Agency (FAPESP) & Brazilian National Council for Research (CNPq)

Alessandra Danielle Eduardo