Jabari Capp: Assignment

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Jabari Capp Assignment 2

• Presentation based on the journal article
• Control of Autoimmune Diabetes in NOD Mice by
  GAD Expression or Suppression in ß Cells by Yoon
  et al.

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GAD What Is It Why Do We Care?

• GAD (Glutamic Acid Decarboxylase) is an
  auto-antigen found on the surface of pancreatic ß
  cells (Insulin-producing cells) brain cells in
  humans mice.
• The presence of GAD has become strongly
  implicated in the development of type 1
  (Insulin-dependent) diabetes.

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Suppression of GAD

• According to the research conducted in this
  study, the suppression of GAD expression in the ß
  cells of the pancreas will prevent the
  development of autoimmune diabetes.
• Through the use of transgenic NOD (Non-obese
  Diabetic) mice displaying various degrees of GAD
  expression suppression, substantial data was
  accumulated which supports the importance GAD in
  Type 1 diabetes.

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The Participants

• The study utilized the following types of mice
• Non-transgenic NOD mice (control mice)
• H1-AS-GAD-NOD H2-AS-GAD-NOD mice
• M1-AS-GAD-NOD M2-AS-GAD-NOD mice
• L1-AS-GAD-NOD L2-AS-GAD-NOD mice

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Non-transgenic NOD mice

• These mice were not genetically altered.

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H1-AS-GAD-NOD H2-AS-GAD-NOD mice

• These mice were genetically-altered to show a
  high level of expression of the antisense GAD
  transgene.
• Both sets of these mice are genetically identical.

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M1-AS-GAD-NOD M2-AS-GAD-NOD mice

• These mice were genetically-altered to show a
  medium level of expression of the antisense GAD
  transgene.
• Both sets of these mice are genetically
  identical.

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L1-AS-GAD-NOD L2-AS-GAD-NOD mice

• These mice were genetically-altered to show a low
  level of expression of the antisense GAD
  transgene.
• Both sets of these mice are genetically
  identical.

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AS-GAD Transgene

• This transgene was developed to manipulate
  pancreatic ß cells to not express GAD as a cell
  surface antigen in either of its two isoforms
  (GAD65 GAD 67). The extent of this lack of
  expression is inversely proportionate to the
  development of autoimmune diabetes (type 1).
• ?AS transgene expression? ?Diabetes

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Experiment 1 Rationale

• The rationale is simple Monitor the growth
  development of normal mice (non-transgenic NOD
  mice), along with that of the genetically-altered
  mice and look for trends in the development of
  type 1 diabetes.

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Experiment 1 Data

• Researchers monitored specimens in this group
  (group 1) at 40 weeks of age, their incidence of
  diabetes was noted.
• Results
• H1-AS-GAD NOD Mice- 0/150
• M1-AS-GAD NOD Mice- 12/1867
• L1-AS-GAD NOD Mice- 12/1675
• Non-Transgenic Mice1 - 17/2181

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Experiment 1Data (continued)

• Researchers monitored specimens in this group
  (group 2) at 40 weeks of age, their incidence of
  diabetes was noted.
• H2-AS-GAD NOD Mice- 1/362.8
• M2-AS-GAD NOD Mice- 15/1883.3
• L2-AS-GAD NOD Mice-21/2680.8
• Non-Transgenic Mice2 - 18/21 85.7

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Conclusion

• The numerical data derived from both experiments
  displays that a increasing level of anti-sense
  transgene expression correlates to a decreased
  occurrence of diabetes.
• Group 1 showed a lower overall incidence of
  diabetes compared to group 2, although the
  constituents of both groups were engineered to
  genetically identical. The study attributes this
  to gene leakiness, or a relatively lower level
  of transgene expression across the board within
  group 2.

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Experiment 2 Rationale

• The rationale here is similar to that of the
  previous experiment, just more in depth in the
  examination of the mice their development of
  diabetes.

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Experiment 2 Data

• At 20 weeks of age, islets were extracted from
  group 1 and examined for histological damage
  consistent with that caused by diabetogenic T
  cells and resulting in insulitis.
• Results
• H1-AS-GAD-NOD Mice- lt20
• M1-AS-GAD-NOD Mice- gt90
• L1-AS-GAD-NOD Mice- Approximately 95
• Non-Transgenic Mice1- Approximately 95

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Conclusion

• As expected, the mice with the higher levels of
  antisense transgene expression displayed a much
  lower incidence of islet damage, with a higher
  incidence of islet damage accompanying a
  decreasing level of transgene expression.
• Similar results were obtained when using groups 2.

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Timeout Question?

• Could the decrease in diabetes development be the
  consequence of some other effect of the antisense
  transgene, other than GAD suppression?

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Answer Probably not!

• It is very unlikely that some other non-specific
  phenomena associated with the antisense transgene
  is responsible for the prevention of diabetes. If
  this were not so, all of the transgenic mice
  would show extremely low levels of diabetes
  development, regardless of the level of GAD
  expression. To exhibit the necessity of GAD
  suppression, the following experiment was
  conducted.

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Experiment 3 Rationale

• To show that GAD suppression is the key proponent
  involved in the decreased incidence of diabetes,
  NOD mice were engineered transgenically for the
  murine leukemia proviral env gene. If some other
  effect brought about by the transgene was
  responsible for decrease in diabetes, it would be
  shown in this experiment.

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Data

• Unlike the GAD-suppressed mice, these mice
  developed diabetes at a rate (15/19-79) similar
  to that of the non-transgenic mice (9/11-82).

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Conclusion

• The data strongly suggests that in order to
  decrease the occurrence of diabetes, GAD must be
  suppressed.

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Experiment 4 Rationale

• To test the role of T-cells in the development of
  autoimmune diabetes, splenocytes (T-cell
  producers) taken from H-AS-GADNOD mice and
  non-transgenic NOD mice were transplanted into
  NOD scid (severe combined immune deficient) mice.
  Theoretically, splenocytes taken from
  H-AS-GAD-NOD mice will produce T-cells that will
  be

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Experiment 4 Rationale (Cont.)

• non-specific towards GAD, since these mice would
  have never expressed GAD as an pancreatic ß cell
  antigen. However, splenocytes from the
  non-transgenic mice will produce T-cells specific
  to GAD, since expression would be normal. This
  should cause the mice to eventually develop
  diabetes.

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Experiment 4 Data

• The experiment yielded results consistent with
  expectations based on the theory behind the
  study.
• None of the NOD scid mice (0/8-0) given
  splenocytes from the H-AS-GAD-NOD mice developed
  diabetes.
• However 90 (9/10) of the mice given splenocytes
  from the non-transgenic NOD mice developed
  diabetes.

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Conclusion

• The data further supports current evidence that
  GAD has an extremely strong effect on autoimmune
  diabetes development. Although more research must
  be conducted, the uncovering of such an
  association has provided substantial promise in
  establishing a treatment for type 1 diabetes.