Review of the research article, Control of Autoimmune Diabetes in NOD Mice by GAD Expression or Supp

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Review of the research article, Control of
Autoimmune Diabetes in NOD Mice by GAD Expression
or Suppression in B Cells

• Articles Authors Yoon, Ji-Won et al.
• Review by Greg Lendzion

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Background

• Type II Diabetes
• Typically caused by obesity
• Usually controlled by diet and weight loss
• Type I Diabetes
• Typically a genetic disorder
• Caused by an autoimmune response directed at the
  pancreatic Beta cells.

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Background

• Pancreatic Beta Cells in Type I Diabetes
• Autoantigens
• Causes autoimmunity
• Several different types
• GAD (glutamic acid decarboxylase)
• Shows the earliest T-cell proliferation in
  autoimmune response.
• Found in both humans and Non-obese Diabetic (NOD)
  mice

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Overall Hypothesis

• GAD expression is required for the development of
  autoimmune diabetes in NOD mice.

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First set of experiments to show

• Suppression of pancreatic beta cell GAD expression

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Creation of Antisense GAD transgene.

• GAD mRNA
• ?
  ?
• 5----RIP----GAD65-----I/A----RIP-----GAD67---
  ---I/A--3

Created Antisense GAD transgene.
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Southern Blot of various mice expressions of
antisense GAD transgene.
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Northern Blot of RNA

• Tg (-)
  Tg (L) Tg (M) Tg (H)

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Protein Blot comparing pancreas islet cells vs.
brain cells
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Visual Examination of Islet Cells
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Will the created antisense GAD transgene work?

• Southern Blot demonstrates binding of cDNA
  antisense GAD mRNA
• Northern Blot demonstrates GAD transgene binds
  with the antisense GAD mRNA
• Western Blot shows that antisense GAD transgene
  is specific to pancreatic beta cells.

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Second series of experiments to support

• GAD expression causes diabetes in NOD mice.

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Observation of mice over 40 week period.
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Analysis of Islet Cells

• Legend
• 0 Normal islet cells
• 1 Mononuclear infiltration largely around
  periphery, lt25 infiltrated
• 2 25-50 of islet showing mononuclear
  infiltration
• 3 gt50 of islet showing mononuclear
  infiltration
• 4 Small, retracted islet with few mononuclear
  cells

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Observation of 7th Generation of mice over a 40
week period
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Analysis of 7th Generation Islet Cells

• Legend
• 0 Normal islet cells
• 1 Mononuclear infiltration largely around
  periphery, lt25 infiltrated
• 2 25-50 of islet showing mononuclear
  infiltration
• 3 gt50 of islet showing mononuclear
  infiltration
• 4 Small, retracted islet with few mononuclear
  cells

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Visual Inspection of Pancreatic Cells
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Visual Inspection of GAD salivary Cells
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Conclusion of second set of experiments

• GAD expression leads to a high incidence of
  diabetes within NOD mice.

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Third Set of Experiments to determine

• Does GAD expression inhibit T-Cell Proliferation

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Observation of NOD.scid mice injected with three
types of splenocytes.
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Examination of all autoantigens
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Conclusion of third set of experiments

• T-cell generation that is specific for beta cells
  is reduced with GAD suppression.

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Fourth Set of Experiments to explore

• Does GAD suppressed beta cells show immunity to
  attack by diabetogenic T-cells derived from
  acutely diabetic NOD mice?

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Analysis of blood glucose levels in NOD mice with
grafted islet cells
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Examination of Insulinitis in grafted Islet Cells

• Legend
• 0 Normal islet cells
• 1 Mononuclear infiltration largely around
  periphery, lt25 infiltrated
• 2 25-50 of islet showing mononuclear
  infiltration
• 3 gt50 of islet showing mononuclear
  infiltration
• 4 Small, retracted islet with few mononuclear
  cells

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Visual examination of grafted islet cells

• H-AS-GAD-NOD grafted islet cells
• Transgene negative grafted islet cells

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Analysis of transplanted splenocytes into acutely
diabetic mice.
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Conclusion of fourth set of experiments

• GAD is specifically the cause for T-cell
  proliferation and destruction of the beta cells
  in the pancreas.

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Article Conclusion

• Type I diabetes could be possibly prevented with
  the suppresion of GAD specific pancreas beta
  cells.
• GAD expression is essential for T-cell mediated
  autoimmunity leading to the destruction of
  pancreatic beta cells.

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References

• Yoon, Ji-Won et al., Control of Autoimmune
  Diabetes in NOD mice by GAD Expression or
  Suppression in ß Cells. Science 284, pp.
  1183-1186, (1999).
• Porterfield, Susan. Endocrine Physiology, (2001)
  Mosby Publishing Co.
• Dr Lins Lecture.