Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression.

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin-producing beta cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA-HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA-HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA-HSP65-injected mice. The protective effect of DNA-HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4(+) and CD8(+) T cells infiltration, appearance of CD25(+) cells influx and an increased staining for interleukin (IL)-10 in the islets. These results show that DNA-HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

Kinetics of TNF-alpha and IFN-gamma mRNA expression in islets and spleen of NOD mice.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop diabetes similar to the human disease. Cytokines produced by islet-infiltrating mononuclear cells may be directly cytotoxic and can be involved in islet destruction coordinated by CD4+ and CD8+ cells. We utilized a semiquantitative RT-PCR assay to analyze in vitro the mRNA expression of TNF-alpha and IFN-gamma cytokine genes in isolated islets (N = 100) and spleen cells (5 x 10(5) cells) from female NOD mice during the development of diabetes and from female CBA-j mice as a related control strain that does not develop diabetes. Cytokine mRNAs were measured at 2, 4, 8, 14 and 28 weeks of age from the onset of insulitis to the development of overt diabetes. An increase in IFN-gamma expression in islets was observed for females aged 28 weeks (149 +/- 29 arbitrary units (AU), P<0.05, Student t-test) with advanced destructive insulitis when compared with CBA-j mice, while TNF-alpha was expressed in both NOD and CBA-j female islets at the same level at all ages studied. In contrast, TNF-alpha in spleen was expressed at higher levels in NOD females at 14 weeks (99 +/- 8 AU, P<0.05) and 28 weeks (144 +/- 17 AU, P<0.05) of age when compared to CBA-j mice. The data suggest that IFN-gamma and TNF-alpha expression in pancreatic islets of female NOD mice is associated with beta cell destruction and overt diabetes.

Kinetics of TNF-alpha and IFN-gamma mRNA expression in islets and spleen of NOD mice

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of pancreatic ß cells. Non-obese diabetic (NOD) mice spontaneously develop diabetes similar to the human disease. Cytokines produced by islet-infiltrating mononuclear cells may be directly cytotoxic and can be involved in islet destruction coordinated by CD4+ and CD8+ cells. We utilized a semiquantitative RT-PCR assay to analyze in vitro the mRNA expression of TNF-alpha and IFN-gamma cytokine genes in isolated islets (N = 100) and spleen cells (5 x 10(5) cells) from female NOD mice during the development of diabetes and from female CBA-j mice as a related control strain that does not develop diabetes. Cytokine mRNAs were measured at 2, 4, 8, 14 and 28 weeks of age from the onset of insulitis to the development of overt diabetes. An increase in IFN-gamma expression in islets was observed for females aged 28 weeks (149 ± 29 arbitrary units (AU), P<0.05, Student t-test) with advanced destructive insulitis when compared with CBA-j mice, while TNF-alpha was expressed in both NOD and CBA-j female islets at the same level at all ages studied. In contrast, TNF-alpha in spleen was expressed at higher levels in NOD females at 14 weeks (99 ± 8 AU, P<0.05) and 28 weeks (144 ± 17 AU, P<0.05) of age when compared to CBA-j mice. The data suggest that IFN-gamma and TNF-alpha expression in pancreatic islets of female NOD mice is associated with ß cell destruction and overt diabetes

Effect of iodide on Fas, Fas-ligand and Bcl-w mRNA expression in thyroid of NOD mice pretreated with methimazole.

Nonobese diabetic (NOD) mice and a derived strain, NOD.H.2h4, have been used as a model for experimental spontaneous thyroiditis and thyroiditis induced by iodide excess after a goiter-inducing period. Some authors have proposed that iodide, given after methimazole or propylthiouracil, is capable of inducing apoptosis in thyroid cells and that anti-thyroid drugs can modulate the expression of apoptosis components such as Fas and its ligand (Fas-L). Here we evaluated the effect of potassium iodide (20 microg/animal for 4 days, i.p.) given to NOD mice at the 10th week of life after exposure to methimazole (1 mg/ml) in drinking water from the 4th to the 10th week of life. Fas, Fas-L and Bcl-w expression were analyzed semiquantitatively by RT-PCR immediately after potassium iodide administration (group MI44D) or at week 32 (MI32S). Control groups were added at 10 (C10) and 32 weeks (C32), as well as a group that received only methimazole (CM10). An increase in the expression of Fas-L and Bcl-w (P<0.01, ANOVA) was observed in animals of group MI44D, while Fas was expressed at higher levels (P = 0.02) in group C32 (72.89 +/- 47.09 arbitrary units) when compared to group C10 (10.8 +/- 8.55 arbitrary units). Thus, the analysis of Fas-L and Bcl-w expression in the MI44D group and Fas in group C32 allowed us to detect two different patterns of expression of these apoptosis components in thyroid tissue of NOD mice.

Effect of iodide on Fas, Fas-ligand and Bcl-w mRNA expression in thyroid of NOD mice pretreated with methimazole

Nonobese diabetic (NOD) mice and a derived strain, NOD.H.2h4, have been used as a model for experimental spontaneous thyroiditis and thyroiditis induced by iodide excess after a goiter-inducing period. Some authors have proposed that iodide, given after methimazole or propylthiouracil, is capable of inducing apoptosis in thyroid cells and that anti-thyroid drugs can modulate the expression of apoptosis components such as Fas and its ligand (Fas-L). Here we evaluated the effect of potassium iodide (20 æg/animal for 4 days, ip) given to NOD mice at the 10th week of life after exposure to methimazole (1 mg/ml) in drinking water from the 4th to the 10th week of life. Fas, Fas-L and Bcl-w expression were analyzed semiquantitatively by RT-PCR immediately after potassium iodide administration (group MI44D) or at week 32 (MI32S). Control groups were added at 10 (C10) and 32 weeks (C32), as well as a group that received only methimazole (CM10). An increase in the expression of Fas-L and Bcl-w (P<0.01, ANOVA) was observed in animals of group MI44D, while Fas was expressed at higher levels (P = 0.02) in group C32 (72.89 ± 47.09 arbitrary units) when compared to group C10 (10.8 ± 8.55 arbitrary units). Thus, the analysis of Fas-L and Bcl-w expression in the MI44D group and Fas in group C32 allowed us to detect two different patterns of expression of these apoptosis components in thyroid tissue of NOD mice