Mahirah Binti Mohd Norazinan22352821

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INTRODUCTION What is Adenosine Deaminase? ADA deficiency

ADA-DEFICIENT IN MOUSE MODEL PEG-ADA REPLACEMENT THERAPY CONCLUSION REFERENCES

What is Adenosine Deaminase? An enzyme involve in purine metabolism

Aminohydrolases, catalyze conversion of adenosine to inosine and 2’-deoxyadenosine to 2’-deoxyinosine

Mostly important in human for lymphocyte development, survival and function

Source: Sauer et al. 2012

ADA Deficiency Caused by mutation in a gene that encodes for ADA

enzyme on chromosome 20 Autosomal recessive disease Very rare, but very dangerous Main features ADA deficiency: Causes accumulation

of ADA cytotoxic metabolites (Adenosine & 2’-Deoxyadenosine) in plasma, lymphoid tissues, and red blood cells

Associated with severe depletion of three major categories of lymphocytes, T-cells, B-cells and NK cells that play vital role in immune system (Aldrich et al. 2003)

Could result in severe combined immunodeficiency (SCID)

ADA-Deficient Mouse Model

Using two-stage genetic engineering strategy

ADA expression in trophoblast cells of placenta critical for fetal development in mouse (Blackburn et al. 1998)

In order to generate completely ADA-deficient postnatal mice, ADA minigene which targeted expression specifically to the trophoblast lineage was introduced onto ADA-deficient background

Accomplished by intercrossing mice carrying the trophoblast-specific ADA minigene with mice heterozygous for the null ADA allele

Source : Apasov et al. 2001

Figure 1. Comparison between ADA +/+ and ADA -/- phenotype

Using histological analysis Tissues of interest were dissected from control and ADA-

deficient mice on postnatal day 15 Tissues were rinsed in ice-cold phosphate-buffered saline

and then fixed overnight in 4% paraformaldehyde in phosphate-buffered saline at 4 °C

Stained with hematoxylin and eosin using a Rapid Chrome staining kit

Source: Blackburn et al. 1998

Figure 2. Reduction in the size of thymus and spleen of ADA deficient mice

Immunology For xamination of peripheral lymphocytes, blood was

collected from the tail vein of mice directly into EDTA-coated microcentrifuge tubes

Complete blood counts were determined using an H1 analyzer (Technicon Instruments Corp.)

For determination of serum immunoglobulins, whole blood was collected by cardiac puncture, allowed to set on ice for 15 min and then centrifuged

Serum immunoglobulin levels were determined using a single radioimmunodiffusion assay

Source: Blackburn et al. 1998

Figure 3. ADA deficient mice exhibit lymphopenia and immunodeficiency

The level of ADA substrates, Adenosine and 2’-deoxyadenosine in ADA deficient mice detection by nucleoside analysis accumulation of adenosine and 2’-deoxyadenosine found in

the serum of ADA deficient mice Adenosine levels were found to be elevated in all tissues

examined with the greatest accumulation occurring in the liver, kidney, and lung

2’-Deoxyadenosine was also detected in all ADA deficient tissues examined with the greatest accumulation occurring in the lymphoid organs

Source: Blackburn et al. 1998

Figure 4. Levels of Adenosine and 2’-deoxyadenosine in ADA deficient mice

In situ analysis of apoptosis in thymocytes Frozen tissue preparations and apoptosis analysis in the

spleen, thymus,and lymph nodes of ADA+/+ and ADA–/– mice were performed

Side scatter versus forward scatter in thymi from ADA–/– and ADA+/+ littermates revealed an increase in the number of dead cells among immature T cells from ADA–/– thymi

The proportions of dead cells among mature T cells found in the peripheral lymphoid organs (spleen and lymph nodes) were similar between ADA+/+ and ADA–/– mice

Source : Apasov et al. 2001Figure 5. Increased apoptosis in ADA -/- mice

Pegylated-Bovine ADA (PEG-ADA) Replacement Therapy Livesaving, but not curative treatment Modified enzyme remains in the extracellular

fluid where it can degrade toxic purines Almost normalizes the metabolic

abnormalities in purine metabolism Does not completely correct immune

function, but immunoprotection is restored, dramatic clinical improvements occurs

Require weekly intramuscular injections with PEG-ADA

Source: Sauer et al. 2012

Source: Sauer et al. 2012

Figure 6. Immune reconstitution and development of autoimmunity after PEG-ADA treatment

Conclusions ADA deficiency cause by mutation in ADA

gene at chromosome number 20 Lack of ADA enzyme affect the development

of lymphocytes in immune system Accumulation of ADA metabolites:

Adenosine & 2’-deoxyadenosine due to ADA deficiency

PEG-ADA replacement therapy act as an alternative treatment for ADA deficiency

Further research involving the mechanisms of ADA metabolites with the development of lymphocytes in immune system

ReferencesAldrich, M.B., Chen, W., Blackburn, M.R., Valdez, H.M., Datta, S.K. & Kellems, R.E. (2003), ‘Impaired germinal center maturation in adenosine deaminase deficency’, The Journal of Immunology, vol. 171, no. 10, pp. 5562-5570.

Apasov, S.G., Blackburn, M.R., Kellems, R.E., Smith, P.T. & Sitkovsky, M.V. (2001). ‘Adenosine deaminase deficiency increases thymic apoptosis and causes defective T cell receptor signaling’, The Journal of Clinical Investigation, vol. 108, no.1, pp. 131-141.

Blackburn, M.R., Datta, S.K. & Kellems, R.E. (1998). ‘Adenosine Deaminase-deficient Mice Generated Using a Two-stage Genetic Engineering Strategy Exhibit a Combined Immunodeficiency’, The Journal of Biological Chemistry, vol. 273, no.9, pp. 5093-5100.

Sauer, A.V., Brigida, I., Carriglio, N. & Aiuti, A. (2012), ‘Adenosine Deaminase-deficient Mice Generated Using a Two-stage Genetic Engineering Strategy Exhibit a Combined Immunodeficiency’, Frontiers in Immunology, vol. 3, no. 265, pp. 1-19.

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