[Mechanisms of human glutamic acid decarboxylase 65 DNA vaccine preventing diabetes in non-obese diabetic mice].
OBJECTIVE
To investigate the mechanisms of human GAD65 DNA vaccine preventing insulitis and diabetes in NOD mice.


METHODS
Female NOD mice at 4 weeks of age were randomly divided into PBS (n = 21), pcDNA (n = 20), and hGAD65 (n = 21) groups. Mice in each group received two intramuscular injections of 0.05 ml PBS alone, 50 microg pcDNA3.1 and 50 microg DNA vaccine emulsified in 0.05 ml PBS 7 days apart respectively. The accumulative diabetes incidence was followed-up to 30 weeks of age in each group of NOD mice. Pancreas was removed from NOD mice at 12 weeks of age in each group (n = 10) to score insulitis severity by routine H-E staining. The apoptotic beta cells in islets were observed with double-labeling technique of TUNEL in situ combined standard sensitive avdin-biotin complex (sABC) immunohistochemical method. Their spleens were for cell culture and total RNA extraction. Spleen IL-4, IFN-gamma, NF-ATc and NF-ATp mRNA levels were tested by RT-PCR. IL-4 and IFN-gamma levels in sera and supernatants of spleen cells were measured by ELISA.


RESULTS
(1) At 30 weeks of age, the diabetes incidence was 95.2%, 80.0% and 61.9% in PBS, pcDNA and hGAD65 group respectively. The diabetes incidence in the PBS group was higher than that in hGAD65 group (P = 0.008). (2) At 12 weeks of age, the insulitis scores in hGAD65 group was lower than that in PBS group (P = 0.001) and pcDNA group (P = 0.027) respectively. (3) The apoptotic beta cell rates in hGAD65 group was lower than that in PBS group (P = 0.014) and pcDNA group (P = 0.023). (4) IL-4 levels in sera, spleen IL-4 and NF-ATc mRNA level in hGAD65 group were higher than those in PBS group (all P < 0.05) and pcDNA group (all P < 0.05) respectively, NF-ATp mRNA level in hGAD65 group was lower than that in PBS group (P < 0.05).


CONCLUSIONS
Human GAD65 DNA vaccine via downregulating NF-ATp and upregulating NF-ATc and IL-4, makes Th cells deviate to Th2, and sequently prevents insulitis, beta-cell apoptosis and diabetes onset in NOD mice.
Luo
Jian-hua
JH
Institute of Metabolism and Endocrinology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhou
Zhi-guang
ZG
Jiang
Tie-jian
TJ
Pei
Jian-hao
JH
Li
Xia
X
He
Ling
L
Sun
Shu-guang
SG
D000818:Animals; D017209:Apoptosis; D003921:Diabetes Mellitus, Experimental / Q000517:prevention & control; D003922:Diabetes Mellitus, Type 1 / Q000517:prevention & control*; D005260:Female; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007515:Islets of Langerhans / Q000276:immunology / Q000473:pathology; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology*; D051379:Mice; D016688:Mice, Inbred NOD; D016176:T-Lymphocyte Subsets / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D019444:Vaccines, DNA / Q000276:immunology*
D007527:Isoenzymes; D019444:Vaccines, DNA; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2