Download Present - Harlem Children Society

Antibody Mediated Rejection
and
Gene Expression Profiles
Anatasia Gangadin
Dr. Mario C Deng
Columbia University
College of Physicians and Surgeons
Key Ideas
• Allograft
• Endomyocardial
Biopsy
• Rejection
• CARGO
• Functional Genomics
• Patient Care
Patient Care
Endomyocardial biopsy
• Currently Only way
to test for rejection
• Risk Factors
associated
Rejection
overall survival
of heart
transplant is
85%
Incidence
Mortality
(Of those
affected)
Humoral
17.6%
17%
Mild
Acute
Cellular
1.4%
23%
Chronic
13%
18%
Severe
CARGO Study
• To Reduce the Number
of Biopsies by taking
samples of blood as
opposed to heart Tissue
• Being able to predict
rejection after a
transplant
• Establishing a pattern of
Genes which could
ultimately predict
Rejection
CARGO Clinical Study
Design

Prospective, multicenter, 4 year observational study on 629 patients
Hypothesis
Gene expression profiling of peripheral blood mononuclear cells
discriminate quiescence (ISHLT Grade 0) from moderate/severe
rejection (Grade ≥ 3A/2R)
Method

7370 Gene microarray and PCR analysis taken at time of biopsies

Validated 20 gene classifier to distinguish rejection from quiescence
Result
Deng/Eisen/Mehra et al. Am J Transplant 2006;6:150
Basic Forms Of Rejection
Chronic Rejection
Cellular Rejection
Inflammatory Response
due to T cell
infiltration in tissue
The movement of Smooth
Muscle Tissue into
the Coronary Arteries
Causes Overtime
narrowing of the
Coronary Arteries
Cardio Myocyte
Damage
Most
Common Form
Of rejection
Leads to lack of
blood flow, Tissue
Death, Heart Failure
Antibody Mediated Rejection
Humoral Rejection
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Accounts for 20-30%
of rejections in
allografts.
Common Risk Factors
Causes hemodynamic
dysfunction (shock,
hypertension, decreased
cardiac output, and rise
in pulmonary artery
pressure).
Antibody Mediated Rejection
Introduction
• Antibodies produced by B lymphocytes recognize the
allograft and attempt to destroy it.
• Characterized by IgM, IgG, IgA and IgE.
• Increased secretion of cytokines and up-regulation of
HLA molecules in the arteries and capillaries makes the
allograft hypersensitive and antibodies bind to the graft
endothelium more frequently.
• Either May Occur:
– Lysis occurs when the membrane is attacked and the cell lyses.
– Activation occurs when the complement components trigger
growth factors, such as extra tissue production.
Diagnostic Criteria
For Humoral Rejection
Clinical
Any Clinical Evidence of Graft
Dysfunction
Serological
HLA specific antibodies or Donor specific
antibodies
Histological
Fibrinoid Necrosis, Injury, Infiltrates
Immunopathological C3 and C4d deposits, IG staining
Colvin RB, Smith RN, 2005
How Humoral Rejection Works
HLA
Antigen Displayed
T Cell receptor
CD4
CD8
Gene Expression
When Lymphocytes release specific cytokines and
plasma cells, They are doing so because of the
patient’s gene Expression, which is why some patients
experience rejection or no Rejection.
Proposed Genes
GENE
NAME
FUNCTION
CD28
CD28 molecule
Essential for CD4 T cell proliferation
CD40
CD40 molecule
Mediates CD40 responses, inflammation and other Immune
responses
C4B
Complement
component 4B
Provides interaction between antigen and complement
components
CD8
Complement
Component 8D
Present on surface of T lymphocytes, Helps recognize
Antigens displayed by Antigen Presenting Cells
CCR6
Chemokine receptor 6
Helps B cell differentiation , and maturation. Expressed by
CD4 and CD8 (natural killer cells)
CCR4
Chemokine (C-C
motif) receptor 4
Development, homeostasis and function of Immune System
CCr7
Chemokine receptor 7
Activation of B and T lymphocytes
CCL2
(C-C motif) ligand
Inflammatory response
IL2
Interleukin 2
B and T cell proliferation
IL10
Interleukin 10
Immunoregulation, inflammation and B cell survival
Experiment
Aim: To find genes related to the entity “Antibody Mediated
Rejection” which can predict Present/Future Humoral Rejection
and give light to pathophysiological mechanisms.
Inclusion:
• Patients included in the CARGO study that have been tested
with gene microarrays.
Exclusion:
• Patients with Oversensitive Immune Systems
• Patients > 1 transplant
Methods
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•
•
•
Peripheral Blood Mononuclear cells
Gene Microarray Analysis
C4d Staining of endomyocardial biopsies
Statistical Analysis of Clinical data
– SPSS 11.5
– T-Test, Chi-square test, Kaplan Meier
• Statistical Analysis of gene expression data
– Significance analysis of microarrays (SAM)
• Gene Ontology analysis
Gene Microarray Analysis
• RNA of a patient is added to a solution of
hybridization buffer and fragmentation buffer,
along with cyanine 3 and 5.
• This solution is put into a gasket slide and into a
hybridization chamber.
• After washing, these are now gene chips, which
are read by a microarray scanner, which detects
fluorescent molecules (by the cyanine 3 and 5).
Gene Microarray Analysis
C4D Staining
• Paraffin embedded sections.
• Antigens retrieval by a 10
minute bath at 95-100o C in
a pH 6.0 buffer.
• Biotin blockage by Biotin
Blocking kit.
• Incubation 30 minutes with
C4dpAb and anti-C4d
antibody.
• Prevention of peroxidase
activity: by washing with
buffer and hydrogen
peroxide or methanol
C4D Staining
Microarray Analysis
45 patients tested with gene microarrays (CUMC)
105 total samples obtained (CUMC)
ImmunoSerological
pathological
Control
No
(N= 12)
Humoral
Yes
(N= 5)
Any criteria y/n
(N= 28)
Histological Clinical
No
No
No
Yes
Yes
Yes
y/n
y/n
y/n
Humoral SAM
Humoral Clustered Genes (FDR 5%)
Gene Ontology Analysis
• The Gene Ontology project classify genes into
specific categories according to their:
– Cellular component
– Biological process
– Molecular function
• High-Throughput GoMiner organizes lists of
'interesting' genes for biological interpretation in
the context of the Gene Ontology.
• http://discover.nci.nih.gov/gominer/htgm.jsp
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•
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Apoptosis
Programmed cell death
Cell death
Regulation of cell death
Regulation of apoptosis
Response to other organism
Humoral immune response
Antimicrobial humoral response
Humoral defense mechanism
mRNA metabolism
RNA metabolism
•Negative regulation of apoptosis
•Negative regulation of programmed cell
death
•Regulation of cell differentiation
•Lymphocyte differentiation
•T cell differentiation
•Positive regulation of T cell activation
•Regulation of immune response
•Immune cell activation
•Lymphocyte activation
These clusters of genes are
very up-regulated or down
regulated in the humoral
patients
The Genes in these clusters
relate to the gene categories
previously
Humoral HTGM results
References
• Marboe C, Deng MC, Billingham M. Nodular Endocardial Infiltrates (Quilty
Lesions) Cause A Significant Variability in Diagnosis of ISHLT Grade 2 and
3A Rejection in Cardiac Allograft Recipients. Journal of Heart and Lung
Transplantation July 2005; 24:s219-s226.
• Evans R, Williams G, Deng MC. The Economic Implications of Noninvasive
Molecular Testing for Cardiac Allograft Rejection. American Journal of
Transplantation 2005; 5:1553-1558
• Deng MC, Eisen HJ, Mehra MR. Noninvasive Discrimination of Rejection in
Cardiac Allograft Recipients Using gene Expression Profiling. American
Journal of Transplantation 2006; 6:150-160
• Deng MC. Cardiac Transplantation. Heart 2002; 87:177-184
• Michaels PJ, Fishbein MC, Colvin RB. Humoral rejection of Human organ
Transplants. Springer Seminars in Immunopathology 2003 119-140.
Acknowledgements
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Dr. Mario Deng
Martin Cadeiras
Manuel Prinz von Bayern
Sarfaraz Memon
Dr. Sat Bhattacharya
Columbia College of Physicians and Surgeons
Harlem Children Society