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The role of B- cells in Diabetes

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 284:1183-1187. Rationale.

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The role of B- cells in Diabetes

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  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 284:1183-1187.

  2. 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.

  3. 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.

  4. Diabetogenic T cell proliferation • Scientists test to see what causes T cell proliferation.

  5. 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.

  6. 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.

  7. Suppressing GAD • In order to suppress GAD expression, transgenic NOD mice with an antisense GAD transgene were produced under the control of the rat insulin promoter. • Six lines of antisense GAD65.67 transgenic mice were established. • They were categorized according to the amount of transgenic expression.

  8. 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

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

  10. 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.

  11. Is B cell GAD expression necessary for the development of autoimmune diabetes in NOD mice? • Experimentation is necessary to find the answer. • Here’s how they did it.

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

  13. 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.

  14. 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.

  15. What do these results imply?

  16. 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

  17. 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.

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

  19. Endogenous retroviral env protein • A putative B cell autoantigen • Expressed in the B cells of NOD mice.

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

  21. 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.

  22. Does B cell specific suppression affect B cell specific autoimmunity • The salivary gland was examined to answer this question.

  23. 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.

  24. Indication • Autoimmunity is not affected in other tissues.

  25. How does the suppression of B cell inhibit disease? • Possibly by blocking the development of DG T cells.

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

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

  28. Results • None of the mice with the transfused splenocytes from the H-AS-GAD-NOD mice developed diabetes by the age of ten weeks. • 90% of the mice with the transfused splenocytes from the transgene negative littermates developed diabetes within nine weeks of transfusion.

  29. 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.

  30. 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.

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