AG490 Prevents and Reverses Diabetes Figure 4. 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 (36 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 mm 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-b1 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.] doi:10.1371/journal.pone.0036079.g004 destruction was more or less completed at this stage; therefore, improvement in blood glucose level is the result of increased insulin sensitivity by AG490. Rapid recovery from diabetes in response to AG490 therapy, inefficiency of AG490 on the C- peptide levels, the lack of any AG490 effect on leukocyte islet infiltration- are all in agreement with improved insulin sensitivity in peripheral target tissues. On the other hand, Real-time RT- PCR data consistently indicates that expression of Foxp3 in infiltrating lymphocytes in the pancreas of AG490 treated NOD mice was detectable while it was undetectable in DMSO treated mice (see figure 4C). This data suggests that AG490 treatment changed composition of infiltrating leukocyte of the islet in NOD mice. Our previous reports regarding autoactivation and impaired function of the Jak-Stat pathway [37,45] and our data in this study-all may suggest that the Jak-Stat signaling pathway may be PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e36079