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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 upregulates expression of Foxp3 regulatory CD25+ T-cells in vivo.A- Prediabetic NOD mice (4 week) were treated either with AG490 or DMSO three times per week for 5 consecutive weeks and they were sacrificed one week after the last injection at week 10. Data represents pool cell suspensions of 5 NOD mice per group and is gated on live CD4+ T-cells. AG490 treatment significantly increases number of Foxp3 regulatory T-cells (p<0.004) (A–D). E- CD25+Foxp3+ gated population on CD4+ T-cell of several lymph nodes in AG490 and the sham treated control NOD mice. Bars represent standard deviation of the mean. F- Whole blood of NOD mouse (pool of 5 mice/group) treated as described above was stained directly with antibodies indicated as described in materials and methods. Data of gated CD4+ T-cells are shown. G- Total RNA from purified CD4+ T-cells of splenocytes of NOD mice (10 weeks old) treated either with AG490 or DMSO from 5 weeks were subjected to real-time RT-PCR and expression of the immune response genes was screened as described in materials and methods. Data shown is the mean of fold change expression of 18 genes after they were normalized based on expression of two housekeeping genes and then compared with the expression of the same gene in DMSO treated control NOD mice. Spl = spleen, PLN = pancreatic lymph node, MLN = mesenteric lymph node, PP = Peyer's patches, PB = peripheral blood.
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pone-0036079-g005: AG490 upregulates expression of Foxp3 regulatory CD25+ T-cells in vivo.A- Prediabetic NOD mice (4 week) were treated either with AG490 or DMSO three times per week for 5 consecutive weeks and they were sacrificed one week after the last injection at week 10. Data represents pool cell suspensions of 5 NOD mice per group and is gated on live CD4+ T-cells. AG490 treatment significantly increases number of Foxp3 regulatory T-cells (p<0.004) (A–D). E- CD25+Foxp3+ gated population on CD4+ T-cell of several lymph nodes in AG490 and the sham treated control NOD mice. Bars represent standard deviation of the mean. F- Whole blood of NOD mouse (pool of 5 mice/group) treated as described above was stained directly with antibodies indicated as described in materials and methods. Data of gated CD4+ T-cells are shown. G- Total RNA from purified CD4+ T-cells of splenocytes of NOD mice (10 weeks old) treated either with AG490 or DMSO from 5 weeks were subjected to real-time RT-PCR and expression of the immune response genes was screened as described in materials and methods. Data shown is the mean of fold change expression of 18 genes after they were normalized based on expression of two housekeeping genes and then compared with the expression of the same gene in DMSO treated control NOD mice. Spl = spleen, PLN = pancreatic lymph node, MLN = mesenteric lymph node, PP = Peyer's patches, PB = peripheral blood.

Mentions: To further understand as to whether AG490 prevented and reversed diabetes in treated NOD mice, we measured expression of Foxp3 and other regulatory markers by flow cytometry analysis in different organs of the immune system in NOD mice that were treated with either AG490 or DMSO. As shown in figure 5A–D significant higher expression of CD25+Foxp3+ T-cells (p<0.004) is evident in all organs tested in AG490 treated mice. Furthermore, AG490 treatment also increased expression of Foxp3 in CD25+ T-cells in peripheral blood of the same groups of NOD (Fig. 5E).


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 upregulates expression of Foxp3 regulatory CD25+ T-cells in vivo.A- Prediabetic NOD mice (4 week) were treated either with AG490 or DMSO three times per week for 5 consecutive weeks and they were sacrificed one week after the last injection at week 10. Data represents pool cell suspensions of 5 NOD mice per group and is gated on live CD4+ T-cells. AG490 treatment significantly increases number of Foxp3 regulatory T-cells (p<0.004) (A–D). E- CD25+Foxp3+ gated population on CD4+ T-cell of several lymph nodes in AG490 and the sham treated control NOD mice. Bars represent standard deviation of the mean. F- Whole blood of NOD mouse (pool of 5 mice/group) treated as described above was stained directly with antibodies indicated as described in materials and methods. Data of gated CD4+ T-cells are shown. G- Total RNA from purified CD4+ T-cells of splenocytes of NOD mice (10 weeks old) treated either with AG490 or DMSO from 5 weeks were subjected to real-time RT-PCR and expression of the immune response genes was screened as described in materials and methods. Data shown is the mean of fold change expression of 18 genes after they were normalized based on expression of two housekeeping genes and then compared with the expression of the same gene in DMSO treated control NOD mice. Spl = spleen, PLN = pancreatic lymph node, MLN = mesenteric lymph node, PP = Peyer's patches, PB = peripheral blood.
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pone-0036079-g005: AG490 upregulates expression of Foxp3 regulatory CD25+ T-cells in vivo.A- Prediabetic NOD mice (4 week) were treated either with AG490 or DMSO three times per week for 5 consecutive weeks and they were sacrificed one week after the last injection at week 10. Data represents pool cell suspensions of 5 NOD mice per group and is gated on live CD4+ T-cells. AG490 treatment significantly increases number of Foxp3 regulatory T-cells (p<0.004) (A–D). E- CD25+Foxp3+ gated population on CD4+ T-cell of several lymph nodes in AG490 and the sham treated control NOD mice. Bars represent standard deviation of the mean. F- Whole blood of NOD mouse (pool of 5 mice/group) treated as described above was stained directly with antibodies indicated as described in materials and methods. Data of gated CD4+ T-cells are shown. G- Total RNA from purified CD4+ T-cells of splenocytes of NOD mice (10 weeks old) treated either with AG490 or DMSO from 5 weeks were subjected to real-time RT-PCR and expression of the immune response genes was screened as described in materials and methods. Data shown is the mean of fold change expression of 18 genes after they were normalized based on expression of two housekeeping genes and then compared with the expression of the same gene in DMSO treated control NOD mice. Spl = spleen, PLN = pancreatic lymph node, MLN = mesenteric lymph node, PP = Peyer's patches, PB = peripheral blood.
Mentions: To further understand as to whether AG490 prevented and reversed diabetes in treated NOD mice, we measured expression of Foxp3 and other regulatory markers by flow cytometry analysis in different organs of the immune system in NOD mice that were treated with either AG490 or DMSO. As shown in figure 5A–D significant higher expression of CD25+Foxp3+ T-cells (p<0.004) is evident in all organs tested in AG490 treated mice. Furthermore, AG490 treatment also increased expression of Foxp3 in CD25+ T-cells in peripheral blood of the same groups of NOD (Fig. 5E).

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