The role of B- cells in Diabetes

1
The role of B- cells in Diabetes

• Yoon, J., Yoon, C., Lim, H., Huang, Q., Kang, Y.,
  Pyun, K., Hirasawa, K.,
• Sherwin, R., Jun, H. (1999). Control of
  autoimmune diabetes in NOD mice by GAD
  expression or suppression in B Cells. Science
  2841183-1187.

2
Background

• used to be know as insulin dependent diabetes
• Autoimmune disease
• Bodies white blood cells kill pancreatic cells
  responsible for insulin production
• Patients depend on insulin shots

3
Requirements of a Diabetic

• Daily insulin shots
• Monitoring of blood-sugar levels
• Adjustment of diet

4
Consequences of Diabetes

• Blindness
• Vascular disease
• Kindney failure
• Peripheral vascular disease
• Decreased circulation
• Amputation of limbs

5
Rationale

• T- cell mediated autoimmune destruction of
  pancreatic cells is due to Type 1 diabetes
  mellitus.
• Tests were done using NOD mice because there
  immune system is most closely related to the
  human immune system
• Glutamic acid decarboxylase (GAD) is the most
  likely autoantigen responsible for the triggering
  of B- cell specific autoimmunity.

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Glutamic acid dicarboxylase

• GAD is a pancreatic B cell autoantigen in humans
  and nonobese diabetic mice (NOD mice).
• B cell specific supression of GAD expression in
  two lines of NOD mice prevented autoimmune
  diabetes.
• Persistent GAD in NOD mice resulted in diabetes.
  
• Complete suppression of B cell GAD expression
  resulted in the absence of diabetogenic T cells
  (DG T cells) and prevent injury to autoimmunity.

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Diabetogenic T cell proliferation

• Scientists test to see what causes T cell
  proliferation.

8
Causes of expression of the diabetogenic T cell.

• In comparison with other pancreatic B cell
  autoantigens, GAD showed the most positive effect
  in the production of DG T cells.
• However, there is no conclusive evidence that GAD
  transgenic is absolutely necessary for the
  initiation of diabetes.

9
Purpose behind experiment

• The scientists wanted to find out if GAD is
  necessary for the initiation of diabetes.
• This is the basis for the experiment.
• The suppression of B cell GAD expression is
  necessary to begin the experiment.

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Suppressing GAD

• Transgenic NOD mice with an antisense GAD
  transgene were produced
• Six lines of antisense GAD65.67 transgenic mice
  were established.

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• The mice were categorized according to the amount
  of transgenic expression.

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Categorization

• - First three lines
• - High levels- H-AS-GAD-NOD
• - Medium levels- M-AS-GAD-NOD
• - Low levels- L-AS-GAD-NOD
• Second three lines
• - High levels- Hk-AS-GAD-NOD
• - Medium levels- Mk-AS-GAD-NOD
• - Low levels- Lk-AS-GAD-NOD

13
Testing the manipulation

• The scientists had to check the amount of
  repression of the B cell GAD expression.
• Protein immunoblot analysis was used to check
  repression.

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Results for expression

• H-AS-GAD-NOD showed complete suppression.
• M-AS-GAD-NOD and L-AS-GAD-NOD showed moderate to
  low suppression.
• GAD expression was detected to be the same in the
  brain tissue of the transgene negative mice and
  the three lines of AS-GAD-NOD mice.
• The three lines of AS-GAD-NOD mice were
  indistinguishable from the transgene negative
  littermates concerning pancreatic insulin control
  andplasma insulin concentrations.

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Is B cell GAD expression necessary for the
development of autoimmune diabetes in NOD mice?

• Experimentation is necessary to find the answer.
• Heres how they did it.

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Experiment

• The disease development was monitored in the
  three lines of AS-GAD-NOD mice and transgene
  negative littermates.

17
Results for first three lines

• H-AS-GAD-NOD mice
• -did not develop diabetes after 40 weeks of age
• -over 80 had intact islets at 20 weeks of age
• -less than 20 showed periinsulitis by 20 weeks
  of age.
• (67)M-AS-GAD-NOD, (75)L-AS-GAD-NOD, and the
  (81)transgene negative mice all demonstrated
  diabetes at the same age.
• In contrast with H-AS-GAD-NOD mice, the
  M-AS-GAD-NOD, L-AS-GAD-NOD, and the transgene
  negative littermates showed insulitis by 20 weeks
  of age.

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Results for second three lines

• Hk-AS-GAD-NOD
• - 2.8 of mice showed diabetes by the age of
  40 weeks
• Mk-AS-GAD-NOD
• - 83.3 had diabetes by the age of 40 weeks
• Lk-AS-GAD-NOD
• - 80.8 had diabetes by the age of 40 weeks
• Transgene negative littermates
• - 85.7 had diabetes
• - There was no signigicant difference between
  the samples concerning insulitis at 19 and 20
  weeks of age.

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• What do these results imply?

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Indications

• B cell GAD expression is an ablsolute
  requirement for the development of diabetes in
  NOD mice.
• - The H-AS-GAD-NOD mice did not develop
  diabetes

21
Indications

• The complete and near complete prevention of
  diabetes is not due to the nonspecific effect of
  antisense transgene incorporated into DNA.
• - The low and moderate suppression of B cell
  GAD expression in the first and second lines of
  transgene mice did not prevent diabetes.

22
Further examination of experiment

• Further examination required the use of new
  control.
• Mice carrying the antisense endogenous murine
  leukemia proviral env region DNA

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• Endogenous retroviral env protein

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Results

• 79 of the antisense transgenic mice carrying the
  endogenous retroviral env protein developed
  diabetes.
• 82 of the transgene negative littermates
  developed diabetes.

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What does this mean?

• The prevention of diabetes in antisense GAD
  transgenic NOD mice is not due to the nonspecific
  effect of an antisense transgene incorporated
  into the chromosomal DNA.

26
Does B cell specific suppression affect B cell
specific autoimmunity

• The salivary gland was examined to answer this
  question.

27
The salivary gland

• The salivary gland show lymphocytic infiltration
  in diabetes prone NOD mice.
• - Lymphocytic infiltration was not prevented in
  the H-AS-GAD-NOD mice.
• Sialitis was similar to that of the transgene
  negative littermates.

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Indication

• Autoimmunity is not affected in other tissues.

29
How does the suppression of B cell inhibit
disease?

• Possibly by blocking the development of DG T
  cells.

30
Samples for experimenation

• Splenocytes were taken from
• -20 week old, female
• H-AS-GAD-NOD nondiabetic mice. - Age
  matched transgene negative nondiabetic
  littermates.

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Experiment

• Splenocytes from the two samples were transfused
  into 6 to 8 week old NOD severe combined
  deficiency disease mice.

32
The effect of B cell-specific suppression of GAD
expression on the development of B cell-cytotoxic
T cells and immune responses to autoantigens
33
Results for A

• H-AS-GAD-NOD mice did not develop diabetes within
  10 weeks of the transplanted splenocytes
• Diabetic NOD mice began to develop diabetes at 1
  week
• The transgene negative began to develop diabetes
  at 2 weeks

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Results for B, C, and D

• No T cell proliferation was detected for any of
  the three ages of H-AS-GAD-NOD mice
• With autoantigen HSP60 and insulin, those mice at
  15 weeks of age showed a small proliferative
  response

35
The effect of B cell-specific suppression of GAD
expression on the development of diabetes and
insulitis
36
Results for A

• Little variation between the control and the low
  and medium AS-GAD-NOD mice
• None of the mice developed diabetes before 10
  weeks of age
• H-AS-GAD-NOD mice did not develop diabetes before
  40 weeks of age

37
Results for B

• The transgene negative littermates, and the low
  and medium AS-GAD-NOD mice all showed moderate to
  severe insulitis
• The H-AS-GAD-NOD mice showed 80 normal islets
• Less than 20 showed periinsulitis

38
Results for C

• There was a little more variation between the
  development of diabetes in the second three lines
  of AS-GAD-NOD mice
• The control developed diabetes by 5 weeks
• Low and medium AS-GAD-NOD mice developed diabetes
  at 10 weeks
• The H-AS-GAD-NOD mice began to develop deiabetes
  by 25 weeks but less than 10

39
Results for D

• There was no significant difference in the extent
  of insulitis between the high, medium, and low
  AS-GAD-NOD mice.

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Indication

• The generation of T cells that cause diabetes is
  blocked without the presence of B cell GAD.
• With this the DG T cells were also blocked.

41
Conclusion

• Diabetes can be successfully prevented with the
  complete suppression of the B cell GAD
  expression.
• B cell GAD expression enhances the development of
  the disease causing diabetogenic T cells.
• The diabetogenic T cells are responsible for the
  development of diabetes.