Developing Immunotherapy for Autoimmune Diseases
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Developing Immunotherapy for Autoimmune Diseases. Premkumar Christadoss, M.B.B.S. Department of Microbiology and Immunology University of Texas Medical Branch 301 University Blvd. Galveston, Texas 77555-1070 [email protected] Generalized Myasthenia Gravis. MG.

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Developing Immunotherapy for Autoimmune Diseases

Premkumar Christadoss, M.B.B.S.

Department of Microbiology and Immunology

University of Texas Medical Branch

301 University Blvd.

Galveston, Texas 77555-1070

[email protected]




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Neuromuscular Junction (NMJ)

Conti-Fine, B.M. et al. J. Clin. Invest. (116) 2843-22854, 2006


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Normal

MG

NMJ in MG


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Electronmicroscopy Study of NMJ of an MG Patient

Engel et al.

Mayo clinic proc. 52:267, 1977


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AChR is a transmembrane glycoprotein formed by five homologous subunits in thestoichiometrya2bgdora2bed. The molecular weights of the subunits range between 45 and 55 kDa.

Theasubunit is Considered to be the highly immunogenic region.


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EAMG induction

AChR Source

Primary immunization: 20 microgram AChR/CFA

28-30 days

Boost: 20 microgram AChR/CFA

28-30 days

Monitor for clinical EAMG

Immunopathological evaluation


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MG in Mice

Normal

MG


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Molecular Mechanisms of EAMG

AChR-Ab

Plasma cells

Complement activation

Class II

Peptide

(a146-162)

CD4

TCR

IFN-g

IL-18

NK

B7

CD 28

Proliferation and Differentiation

IL-10, TNF-a, IL-6,

IL-12, IL-18

APC

CD4

CD40L/CD40

AChR

IL-1, IL-12

AChR-specific memory B cells

AChR-specific memory T cell

C’

C’

Damage to the neuromuscular junction

B


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COMPLEMENT PATHWAYS

CLASSICAL PATHWAY

C1

C4b

C2a

MASP1

MASP2

MBL

C3

C5

MAC

(C5bC9)

MBL PATHWAY

C3b

C3bBb

Bb

ALTERNATIVE PATHWAY


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AChR-immunized C3-/- and C4-/- mice

are resistant to clinical EAMG

Tuzun -Christadoss.

J. Immunol. 171:3847, 2003


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Serum anti-AChR antibody levels of

AChR-immunized mice

Tuzun- Christadoss.

J. Immunol. 171:3847, 2003


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IF Studies Reveal IgG Deposits But Not C3 or MAC Deposits at the NMJs of Mice with C3 or C4 Deficiency

C4+/+

C4-/-

C3-/-

C3

MAC

IgG

Tuzun –Christadoss

J. Immunol. 171:3847, 2003

RED -bungarotoxin binding (NMJ)

GREEN C3, MAC or IgG deposits


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Antigen/organ specific the NMJs of Mice with C3 or C4 Deficiency

Disease specific

I. AChR T cell epitope tolerance

II. AChR B cell epitope tolerance

I. Anti-Proinflammatory Cytokine

a. Soluble TNFR (etanercept)

b. IL1-Ra

c. Anti-IL-6

II. Blocking classical complement pathway

a. Anti C1q/C2/C4

Immune Intervention


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Targeting Classical Complement Pathway the NMJs of Mice with C3 or C4 Deficiency


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Anti-C1q Administration Suppresses EAMG the NMJs of Mice with C3 or C4 Deficiency

Tuzun-Christadoss, J.Neuroimmunol.174:157-167, 2006


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Dual Effect of Anti-C1q the NMJs of Mice with C3 or C4 Deficiency

Tuzun-Christadoss, J.Neuroimmunol.174:157-167, 2006


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Anti-C1q Ab Treats EAMG the NMJs of Mice with C3 or C4 Deficiency

B6

RIII

Tuzun-Christadoss, J.Neuroimmunol, 182: 167-176, 2007


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Anti-C1q Ab Treatment Suppresses AChR the NMJs of Mice with C3 or C4 Deficiency

and Dominant Peptide Specific

IL-6 Production


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Effect of Cytokine Deficiencies in the NMJs of Mice with C3 or C4 Deficiency

Clinical EAMG

Normal

IL-4

IL-10

Gene Depletion

IFN-g

Anti-AChR Ab

Disease

IL-12

IL-18

IL-6

TNF-a p55p75

0

20

40

60

80

100

120

% clinical disease and anti-AChR antibodies


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IL-6 and TNF in EAMG the NMJs of Mice with C3 or C4 Deficiency

TNF

TNF

AChR

specific

IL6

IL-6

Th

Th

GC formation

Activation of B cells and generation of effector B cells.

Potentiates production of IgG anti-AChR antibodies (pathogenic)

B

B

Activates C3

Promotes EAMG pathology


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Targeting ProinflammatorCytokines the NMJs of Mice with C3 or C4 Deficiency

A. Soluble Recombinant HumanTNFR (Etanercept)


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Soluble TNFR (Etanercept) Treats EAMG the NMJs of Mice with C3 or C4 Deficiency

Christadoss and Goluszko

J. Neuroimmunol, 122:186, 2002


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Etanercept Treatment Fails to Suppress Serum Anti-AChR Ab the NMJs of Mice with C3 or C4 Deficiency

Christadoss and Goluszko, J. Neuro. Immunol. 122:186, 2002.


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A pilot Trial of Etanercept in the Treatment of Steroid-Dependent MG *+

  • Mean change in QMG score from basline at 6 months was - 2.9 (p=0.041).

  • Mean change in MMT at 6 months was - 8.4 (p=0.020).

  • Mean decrease in prednisone dose from baseline to end of study was 17.5 mg/48 hr dose (p=0.0084).

  • Etanercept was well tolerated, and no severe adverse reaction observed

  • + 11 patients enrolled; 8 completed, and 2 patients withdrawn due to disease worsening.


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Immunological Effect of Etanercept Steroid-Dependent MG *+

  • No reduction in plasma anti-AChR antibody.

  • Peripheral blood CD4 and B cell (CD19+) counts rose steadily during the 24 week study.

  • Patients who had higher increases in their cytokine levels had a worse outcome.


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Targeting ProinflammatorCytokines Steroid-Dependent MG *+

b. Recombinant Human IL1-Ra



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IL-1Ra treatment Steroid-Dependent MG *+

stopped

IL-1Ra treatment

stopped

IL-1Ra Treatment Prevents Clinical EAMG

Yang –Christadoss, J. Immunol.

175:2018, 2005


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IL-1Ra Treats EAMG Steroid-Dependent MG *+

Yang –Christadoss, J. Immunol.

175:2018, 2005

P<0.05

P<0.05

P<0.05


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Possible Consequence of Down Regulating IL-1 by IL-1Ra in Mice with Clinical EAMG

IL-1

IL-1Ra

CD40L, OX40 Expression on T cells

Anti-AChR IgG , IgG1 and C3

Inflammatory cytokines

IFN-g, IL-2, IL-1, IL-6, TNF-a

Anti-AChR antibodies and complement

mediated NMJ pathology


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IL-6 in MG: Multiple Hit Mice with Clinical EAMG

AChR Specific

NMJ

CD4

IL-6

B

IgG2b -C1q

C4-C3-

C5-9

C3


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IL-6 Mice with Clinical EAMG–ADanger Molecule in EAMG !

1. IL-6 deficient mice are resistant to EAMG and produce less C3

2. C3 and FCγRIII deficient mice are resistant to EAMG and produce less IL-6

3. Amelioration of EAMG following anti-C1q treatment is associated with reduced IL-6


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Anti-IL-6 Ab Treatment Reduces the Incidence of EAMG Mice with Clinical EAMG

Days after second immunization


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Anti-IL-6 Ab Treatment Suppresses Serum Anti-AChR IgG Mice with Clinical EAMG

and IgG2b Ab


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Anti-IL-6 Ab Treatment Suppresses AChR Specific Mice with Clinical EAMG

Cytokine Production


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Classical Complement Pathway and IL-6 in EAMG Pathogenesis Mice with Clinical EAMG

anti-AChR antibody production

T helper

B cell

Antigen presenting cell

AChR

IL-6

C3

Immune complex formation

AChR

C1q

C1r

C1s

C1q

FcγRIII activation

Classical complement pathway activation

Stimulation of

IL-6, C1q and C3 production

Membrane attack complex formation


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Balancing the Immune System to Treat Autoimmune Disease (MG)

Disease

IL-6, TNF

C3-C5b-C9

Anti-AChR IgG

IL-6, TNF-normal level

Healthy

Suppress anti-AChR

and C5b-C9


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Targeting IL-6 and Classical Complement Pathway (MG)

Suppressed anti-AChR antibody production

T helper

B cell

Antigen presenting cell

AChR

IL-6

C3

Immune complex formation

AChR

C1q

C1r

C1s

C1q

Classical complement pathway activation

FcγRIII activation

Stimulation of

IL-6, C1q and C3 production

Membrane attack complex formation



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MG Lab - Galveston (MG)

Erdem Tuzun1,2

Shamsher Saini

Andrey Bednov

Ben Scott 3

Jing Li1

Iris Wingrow3

Huibin Qi

Xiarong Wu

Collaborators

Huan Yang

Bo Wu

Stephen Higgs

Tian Lin Xio

Galen Kaufmann

Mat Merigioli

Juli Rowin

1MG foundation Osserman/Sosin/McClure Post doctoral Fellows

1,2MDA Research Career Award Recipients

3 MG Foundation Henry Viets Fellow

Supported by NIH,MDA, AFM,and MG Foundation


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