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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 modulates phenotype and function of DC.A - Dendritic cells were generated from bone marrow of NOD mice treated either with AG490 or DMSO as described in materials and methods and stained directly with CD11c, MHC II, CD80 and CD86. Data shown represents CD11c gated population of pool fresh purified immature CD11c of AG490 and DMSO treated mice. B- Histograms represent expression of CD11c, CD86, CD80 and CD62L on gated CD11c population of mice treated with AG490 or DMSO in vivo. C- Bone marrow cells were isolated from NOD mice treated with AG490/DMSO for 5 weeks and then differentiated into DC in vitro as described. Total RNA (200 ng) of DC was converted into cDNA and then expression of Tgf-β1 was measured by Real-Time TaqMan Gene expression assays (Applied Biosystems). Data represents the mean (fold change) of two real-time RT-PCR experiments and the bars represent standard deviation of the mean. Fold change of Tgf-β1expression is shown (n = 5 mice/group, * p = 0.02). D- BMDCs were generated from BALB/c mice treated in vitro with 20 µM AG490 or DMSO for 12 hrs and then co-cultured with constant numbers of CD4+CD25− T-cells (1×105) with soluble anti-CD3 (2.5 ng/ml) for 72 hrs. Incorporation of 3H-thymidine was measured in the last 16 hr of cell culture. Experiment was performed in triplicate format and repeated at least twice. Data analysis was performed using the student t-test and p value≤0.05 considered significant. Bars represent deviation of the mean (* p = 0.002, **p = 0.0002; ***p = 0.001). [Figures A & B are intended to be in color online and black and white in print].
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pone-0036079-g006: AG490 modulates phenotype and function of DC.A - Dendritic cells were generated from bone marrow of NOD mice treated either with AG490 or DMSO as described in materials and methods and stained directly with CD11c, MHC II, CD80 and CD86. Data shown represents CD11c gated population of pool fresh purified immature CD11c of AG490 and DMSO treated mice. B- Histograms represent expression of CD11c, CD86, CD80 and CD62L on gated CD11c population of mice treated with AG490 or DMSO in vivo. C- Bone marrow cells were isolated from NOD mice treated with AG490/DMSO for 5 weeks and then differentiated into DC in vitro as described. Total RNA (200 ng) of DC was converted into cDNA and then expression of Tgf-β1 was measured by Real-Time TaqMan Gene expression assays (Applied Biosystems). Data represents the mean (fold change) of two real-time RT-PCR experiments and the bars represent standard deviation of the mean. Fold change of Tgf-β1expression is shown (n = 5 mice/group, * p = 0.02). D- BMDCs were generated from BALB/c mice treated in vitro with 20 µM AG490 or DMSO for 12 hrs and then co-cultured with constant numbers of CD4+CD25− T-cells (1×105) with soluble anti-CD3 (2.5 ng/ml) for 72 hrs. Incorporation of 3H-thymidine was measured in the last 16 hr of cell culture. Experiment was performed in triplicate format and repeated at least twice. Data analysis was performed using the student t-test and p value≤0.05 considered significant. Bars represent deviation of the mean (* p = 0.002, **p = 0.0002; ***p = 0.001). [Figures A & B are intended to be in color online and black and white in print].

Mentions: Bone marrow of AG490 and DMSO treated NOD mice (4 weeks/3x/week, treated for 5 weeks) were harvested at week 10 and yeilded the same number of cells in each group (p = not significant). We did not, however, notice any remarkable difference in morphology, such as the size, shape and clonoy formation, between the two groups in the course of differentiation in cell culture. As shown in figure 6A and B, BMDCs (bone marrow derived dendritic cells)of mice treated by AG490 had markdly lower expression of co-stimualtory moelcules CD80 and CD86 and adhesion molecule CD62L. Additionally, our real-time TaqMan Gene expression Asssays (Assays-on-Demand) (Life technologies, Applied Biosystems) indicates that AG490 significantly increased expression of regulatory cytokine Tgf-β1 in mature and immature DC (dendritic cell) when it was compared with the sham treated control NOD mice (figure 6C, n = 5/group, p = 0.02). Furthermore, BMDCs treated with AG490 (overnight, 20 µM) or DMSO suppressed proliferation of purified responding CD4+CD25− T-cells in vitro (figure 6D).


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 modulates phenotype and function of DC.A - Dendritic cells were generated from bone marrow of NOD mice treated either with AG490 or DMSO as described in materials and methods and stained directly with CD11c, MHC II, CD80 and CD86. Data shown represents CD11c gated population of pool fresh purified immature CD11c of AG490 and DMSO treated mice. B- Histograms represent expression of CD11c, CD86, CD80 and CD62L on gated CD11c population of mice treated with AG490 or DMSO in vivo. C- Bone marrow cells were isolated from NOD mice treated with AG490/DMSO for 5 weeks and then differentiated into DC in vitro as described. Total RNA (200 ng) of DC was converted into cDNA and then expression of Tgf-β1 was measured by Real-Time TaqMan Gene expression assays (Applied Biosystems). Data represents the mean (fold change) of two real-time RT-PCR experiments and the bars represent standard deviation of the mean. Fold change of Tgf-β1expression is shown (n = 5 mice/group, * p = 0.02). D- BMDCs were generated from BALB/c mice treated in vitro with 20 µM AG490 or DMSO for 12 hrs and then co-cultured with constant numbers of CD4+CD25− T-cells (1×105) with soluble anti-CD3 (2.5 ng/ml) for 72 hrs. Incorporation of 3H-thymidine was measured in the last 16 hr of cell culture. Experiment was performed in triplicate format and repeated at least twice. Data analysis was performed using the student t-test and p value≤0.05 considered significant. Bars represent deviation of the mean (* p = 0.002, **p = 0.0002; ***p = 0.001). [Figures A & B are 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-g006: AG490 modulates phenotype and function of DC.A - Dendritic cells were generated from bone marrow of NOD mice treated either with AG490 or DMSO as described in materials and methods and stained directly with CD11c, MHC II, CD80 and CD86. Data shown represents CD11c gated population of pool fresh purified immature CD11c of AG490 and DMSO treated mice. B- Histograms represent expression of CD11c, CD86, CD80 and CD62L on gated CD11c population of mice treated with AG490 or DMSO in vivo. C- Bone marrow cells were isolated from NOD mice treated with AG490/DMSO for 5 weeks and then differentiated into DC in vitro as described. Total RNA (200 ng) of DC was converted into cDNA and then expression of Tgf-β1 was measured by Real-Time TaqMan Gene expression assays (Applied Biosystems). Data represents the mean (fold change) of two real-time RT-PCR experiments and the bars represent standard deviation of the mean. Fold change of Tgf-β1expression is shown (n = 5 mice/group, * p = 0.02). D- BMDCs were generated from BALB/c mice treated in vitro with 20 µM AG490 or DMSO for 12 hrs and then co-cultured with constant numbers of CD4+CD25− T-cells (1×105) with soluble anti-CD3 (2.5 ng/ml) for 72 hrs. Incorporation of 3H-thymidine was measured in the last 16 hr of cell culture. Experiment was performed in triplicate format and repeated at least twice. Data analysis was performed using the student t-test and p value≤0.05 considered significant. Bars represent deviation of the mean (* p = 0.002, **p = 0.0002; ***p = 0.001). [Figures A & B are intended to be in color online and black and white in print].
Mentions: Bone marrow of AG490 and DMSO treated NOD mice (4 weeks/3x/week, treated for 5 weeks) were harvested at week 10 and yeilded the same number of cells in each group (p = not significant). We did not, however, notice any remarkable difference in morphology, such as the size, shape and clonoy formation, between the two groups in the course of differentiation in cell culture. As shown in figure 6A and B, BMDCs (bone marrow derived dendritic cells)of mice treated by AG490 had markdly lower expression of co-stimualtory moelcules CD80 and CD86 and adhesion molecule CD62L. Additionally, our real-time TaqMan Gene expression Asssays (Assays-on-Demand) (Life technologies, Applied Biosystems) indicates that AG490 significantly increased expression of regulatory cytokine Tgf-β1 in mature and immature DC (dendritic cell) when it was compared with the sham treated control NOD mice (figure 6C, n = 5/group, p = 0.02). Furthermore, BMDCs treated with AG490 (overnight, 20 µM) or DMSO suppressed proliferation of purified responding CD4+CD25− T-cells in vitro (figure 6D).

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