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The tyrphostin agent AG490 prevents and reverses type 1 diabetes in NOD mice.

Davoodi-Semiromi A, Wasserfall CH, Xia CQ, Cooper-DeHoff RM, Wabitsch M, Clare-Salzler M, Atkinson M - PLoS ONE (2012)

Bottom Line: However, the therapeutic effects of AG490 on the development of T1D are unknown.AG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points).The use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacotherapy & Translational Research, College of Pharmacy, University of Florida, Gainesville, Florida, United States of America. dsemiromi@cop.ufl.edu

ABSTRACT

Background: Recent studies in the NOD (non-obese diabetic) mouse model of type 1 diabetes (T1D) support the notion that tyrosine kinase inhibitors have the potential for modulating disease development. However, the therapeutic effects of AG490 on the development of T1D are unknown.

Materials and methods: Female NOD mice were treated with AG490 (i.p, 1 mg/mouse) or DMSO starting at either 4 or 8 week of age, for five consecutive week, then once per week for 5 additional week. Analyses for the development and/or reversal of diabetes, insulitis, adoptive transfer, and other mechanistic studies were performed.

Results: AG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points). Monotherapy of newly diagnosed diabetic NOD mice with AG490 markedly resulted in disease remission in treated animals (n = 23) in comparision to the absolute inability (0%; 0/10, p = 0.003, Log-rank test) of DMSO and sustained eugluycemia was maintained for several months following drug withdrawal. Interestingly, adoptive transfer of splenocytes from AG490 treated NOD mice failed to transfer diabetes to recipient NOD.Scid mice. CD4 T-cells as well as bone marrow derived dendritic cells (BMDCs) from AG490 treated mice, showed higher expression of Foxp3 (p<0.004) and lower expression of co-stimulatory molecules, respectively. Screening of the mouse immune response gene arrary indicates that expression of costimulaotry molecule Ctla4 was upregulated in CD4+ T-cell in NOD mice treated with AG490, suggesting that AG490 is not a negative regulator of the immune system.

Conclusion: The use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease.

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Related in: MedlinePlus

AG490 does not block infiltration of leukocytes in the islets.A- Prediabetic NOD mice at 4 weeks of age were treated with AG490 or DMSO (n = 5 per group) for 5 consecutive weeks (3× per week) and mice were sacrificed one week after the last injection at week 10. Insulitis was scored by two different investigators and an average of two different scores is shown (p = n.s). At least 150 islets per group in two interrupted sections, 100 µm apart, was scored. B- The C-peptide level was measured as described in materials and methods and no significant differences were found. C- Five prediabetic NOD mice at 4- weeks of age were treated with AG490 or DMSO (n = 5 per group) as described above for 5 consecutive weeks and then sacrificed at week 10, C- real-time RT-PCR was performed on enriched infiltrated lymphocyte from the pancreas of 5 mice/group and expression level of Foxp3 and TGF-β1 was measured in three independent RT-PCR by TaqMan Gene expression (Life Technologies, Applied Biosystems, Assays-on-Demand) (* = expression was very low and/or undetectable). [Figure 4A is intended to be in color online and black and white in print.]
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pone-0036079-g004: AG490 does not block infiltration of leukocytes in the islets.A- Prediabetic NOD mice at 4 weeks of age were treated with AG490 or DMSO (n = 5 per group) for 5 consecutive weeks (3× per week) and mice were sacrificed one week after the last injection at week 10. Insulitis was scored by two different investigators and an average of two different scores is shown (p = n.s). At least 150 islets per group in two interrupted sections, 100 µm apart, was scored. B- The C-peptide level was measured as described in materials and methods and no significant differences were found. C- Five prediabetic NOD mice at 4- weeks of age were treated with AG490 or DMSO (n = 5 per group) as described above for 5 consecutive weeks and then sacrificed at week 10, C- real-time RT-PCR was performed on enriched infiltrated lymphocyte from the pancreas of 5 mice/group and expression level of Foxp3 and TGF-β1 was measured in three independent RT-PCR by TaqMan Gene expression (Life Technologies, Applied Biosystems, Assays-on-Demand) (* = expression was very low and/or undetectable). [Figure 4A is intended to be in color online and black and white in print.]

Mentions: In another separate experiment, NOD mice at 4 week of age were treated with AG490 or with DMSO (n = 5/gorup), three times per week for 5 consecutive week. Mice were sacrificed at week 10 and microscopic sections were prepared and stained with H & E and scoring of insulitis was performed as described in materials and methods (figure 4A). There was no statistically significance differences regarding insulitis between the two groups. The C-peptide concentration was significantly different in AG490 treated versus DMSO treated mice (4 week group); however, no significant differences in serum C-peptide concentration of AG490 or DMSO treated NOD mice (8 week group) was found (figure 4B). The C-peptide concentration of AG490 treated NOD (8 week group) was significatly higher than the C-peptide level of mice when treatment was initiated at 4 weeks of age. In a separate experiment, female NOD mice were treated either with AG490 or DMSO for 5 weeks and then were sacrificied at week 10. Total RNA extracted from enriched infiltrating leukocytes from pancrease of AG490 and DMSO treated mice (n = 5 mice/group) was subjected to TaqMan Real-time RT-PCR. Level of Foxp3 and TGF-β1 expression were very low but detectable in AG940 treated NOD mice but we failed to detect expression of Foxp3 in three independent RT-PCR in DMSO treated NOD mice (figure 4C). This data suggests that although administration of AG490 was unable to block infiltration of the leukocytes in the islets or improve the C-peptide concentration; however, it changed composition of the infiltrated leukocytes in the islets.


The tyrphostin agent AG490 prevents and reverses type 1 diabetes in NOD mice.

Davoodi-Semiromi A, Wasserfall CH, Xia CQ, Cooper-DeHoff RM, Wabitsch M, Clare-Salzler M, Atkinson M - PLoS ONE (2012)

AG490 does not block infiltration of leukocytes in the islets.A- Prediabetic NOD mice at 4 weeks of age were treated with AG490 or DMSO (n = 5 per group) for 5 consecutive weeks (3× per week) and mice were sacrificed one week after the last injection at week 10. Insulitis was scored by two different investigators and an average of two different scores is shown (p = n.s). At least 150 islets per group in two interrupted sections, 100 µm apart, was scored. B- The C-peptide level was measured as described in materials and methods and no significant differences were found. C- Five prediabetic NOD mice at 4- weeks of age were treated with AG490 or DMSO (n = 5 per group) as described above for 5 consecutive weeks and then sacrificed at week 10, C- real-time RT-PCR was performed on enriched infiltrated lymphocyte from the pancreas of 5 mice/group and expression level of Foxp3 and TGF-β1 was measured in three independent RT-PCR by TaqMan Gene expression (Life Technologies, Applied Biosystems, Assays-on-Demand) (* = expression was very low and/or undetectable). [Figure 4A is intended to be in color online and black and white in print.]
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Related In: Results  -  Collection

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pone-0036079-g004: AG490 does not block infiltration of leukocytes in the islets.A- Prediabetic NOD mice at 4 weeks of age were treated with AG490 or DMSO (n = 5 per group) for 5 consecutive weeks (3× per week) and mice were sacrificed one week after the last injection at week 10. Insulitis was scored by two different investigators and an average of two different scores is shown (p = n.s). At least 150 islets per group in two interrupted sections, 100 µm apart, was scored. B- The C-peptide level was measured as described in materials and methods and no significant differences were found. C- Five prediabetic NOD mice at 4- weeks of age were treated with AG490 or DMSO (n = 5 per group) as described above for 5 consecutive weeks and then sacrificed at week 10, C- real-time RT-PCR was performed on enriched infiltrated lymphocyte from the pancreas of 5 mice/group and expression level of Foxp3 and TGF-β1 was measured in three independent RT-PCR by TaqMan Gene expression (Life Technologies, Applied Biosystems, Assays-on-Demand) (* = expression was very low and/or undetectable). [Figure 4A is intended to be in color online and black and white in print.]
Mentions: In another separate experiment, NOD mice at 4 week of age were treated with AG490 or with DMSO (n = 5/gorup), three times per week for 5 consecutive week. Mice were sacrificed at week 10 and microscopic sections were prepared and stained with H & E and scoring of insulitis was performed as described in materials and methods (figure 4A). There was no statistically significance differences regarding insulitis between the two groups. The C-peptide concentration was significantly different in AG490 treated versus DMSO treated mice (4 week group); however, no significant differences in serum C-peptide concentration of AG490 or DMSO treated NOD mice (8 week group) was found (figure 4B). The C-peptide concentration of AG490 treated NOD (8 week group) was significatly higher than the C-peptide level of mice when treatment was initiated at 4 weeks of age. In a separate experiment, female NOD mice were treated either with AG490 or DMSO for 5 weeks and then were sacrificied at week 10. Total RNA extracted from enriched infiltrating leukocytes from pancrease of AG490 and DMSO treated mice (n = 5 mice/group) was subjected to TaqMan Real-time RT-PCR. Level of Foxp3 and TGF-β1 expression were very low but detectable in AG940 treated NOD mice but we failed to detect expression of Foxp3 in three independent RT-PCR in DMSO treated NOD mice (figure 4C). This data suggests that although administration of AG490 was unable to block infiltration of the leukocytes in the islets or improve the C-peptide concentration; however, it changed composition of the infiltrated leukocytes in the islets.

Bottom Line: However, the therapeutic effects of AG490 on the development of T1D are unknown.AG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points).The use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacotherapy & Translational Research, College of Pharmacy, University of Florida, Gainesville, Florida, United States of America. dsemiromi@cop.ufl.edu

ABSTRACT

Background: Recent studies in the NOD (non-obese diabetic) mouse model of type 1 diabetes (T1D) support the notion that tyrosine kinase inhibitors have the potential for modulating disease development. However, the therapeutic effects of AG490 on the development of T1D are unknown.

Materials and methods: Female NOD mice were treated with AG490 (i.p, 1 mg/mouse) or DMSO starting at either 4 or 8 week of age, for five consecutive week, then once per week for 5 additional week. Analyses for the development and/or reversal of diabetes, insulitis, adoptive transfer, and other mechanistic studies were performed.

Results: AG490 significantly inhibited the development of T1D (p = 0.02, p = 0.005; at two different time points). Monotherapy of newly diagnosed diabetic NOD mice with AG490 markedly resulted in disease remission in treated animals (n = 23) in comparision to the absolute inability (0%; 0/10, p = 0.003, Log-rank test) of DMSO and sustained eugluycemia was maintained for several months following drug withdrawal. Interestingly, adoptive transfer of splenocytes from AG490 treated NOD mice failed to transfer diabetes to recipient NOD.Scid mice. CD4 T-cells as well as bone marrow derived dendritic cells (BMDCs) from AG490 treated mice, showed higher expression of Foxp3 (p<0.004) and lower expression of co-stimulatory molecules, respectively. Screening of the mouse immune response gene arrary indicates that expression of costimulaotry molecule Ctla4 was upregulated in CD4+ T-cell in NOD mice treated with AG490, suggesting that AG490 is not a negative regulator of the immune system.

Conclusion: The use of such agents, given their extensive safety profiles, provides a strong foundation for their translation to humans with or at increased risk for the disease.

Show MeSH
Related in: MedlinePlus