specific Th1 cells in vivo [36]- all suggesting that this drug possess immunomodulating properties. Previously, we reported several defects in the Jak-Stat5 signaling pathway in NOD mice including autoactivation of Stat5, germ- line mutation in Stat5B and impaired DNA binding of Stat5B in NOD mice [37–39]. Given the influence of AG490 on the Jak-Stat pathway, we sought to block the Jak-Stat signaling pathway and investigate the effects of this compound on early (4weeks) and late model of T1D development (8 weeks), insulitis and initiation of remission of newly diagnosed and established diabetic NOD mice. Results AG490 prevents development of autoimmune diabetes in NOD Prediabetic female NOD mice were treated with AG490 (i.p, 1 mg/mouse) and control mice received the same volume of vehicle (DMSO, solvent of AG490, and sterile PBS) three times/ week for 5 consecutive week and once a week for five more week starting at 4 (preinsulitis) or 8 week of age (established insulitis). Our data suggests that AG490 prevents development of autoim- mune diabetes in NOD mice when applied at 4 or 8 week of age (p = 0.02 and p = 0.005, respectively at week 30) (figure 1A & B). Blood glucose and body weights (figure 1C and D) were monitored in both groups as early as 6 week of age and continued for the entire period of investigation. There was no statistically significant differences in terms of body weights between AG490 and DMSO treated mice suggesting administration of AG490 at the given dose is safe in mice. Additionally, we also did not observe any adverse effect of AG490 treated mice by gross examination suggesting that efficacy and safety of multiple injections of AG490. This data suggests that AG490 is an efficient and safe drug in prevention of diabetes in NOD mice. AG490 induces remission of autoimmune diabetes in NOD mice We sought to investigate whether AG490 would reverse pathogenesis of T1D in newly diagnosed diabetic NOD mice. Newly dignosed and established diabetic NOD mice were treated with AG490 (i.p, 1 mg/mouse; no insulin was given to mice at any time) and control group was treated with vehicle and blood glucose was measured. Our data indicate that AG490 led significantly to remission of diabetic mice (p = 0.003, Log-rank test) while DMSO treated control mice led to remission of 0% (n = 10) (figure 2A & B). Once euglycemia was established (Table S1), treatment was ceased and normaglycemia lasts for several weeks and sustainable euglycemia was maintained after drug withdrawal in majority of the cured mice. These data suggest that NOD mice did not develop resistance to AG490 treatment and additionally suggests that AG490 has a durable effect on restoration of blood glucose metabolism/regulation. However, diabetic NOD mice treated with DMSO or left untreated expired within 2–4 weeks (figure 2B). Average of blood glucose in the cured NOD mice at the disease onset was 314.43 mg/dl (Max = 374; Min = 277 mg/dl) versus 384.58 mg/dl (Max = 478, Min = 335 mg/dl) (p,0.0014) in mice that AG490 failed to restore euglycemia while an average blood glucose in cure NOD mice was 169.82623.21 mg/dl versus 555.13663.79 (p = 1.15E-11) in uncured NOD mice. This data suggest that AG490 was more effective in diabetic mice with blood glucose level ,314.43 mg/dl. A combination therapy, e.g AG490+ insulin, may improve efficacy of AG490 treatment in mice with blood glucose level .314.43 mg/dl. AG490 Prevents and Reverses Diabetes AG490 treatment did not cause clinical signs of toxicity and behavior Treatment-related mortality in the course of i.p (intraperitoneal) injection of AG490 and thereafter did not observe. Clinical signs of toxicity related to treatment such as loss of body weight, strange behavior such as aggressiveness, hypermobility, hunchback, and other signs such as discoloration of the fur or fur loss, urine and stool discoloration did not observed.Treatment did not change apetite and food/water consumption as judged by body weight gain/loss as measured 1–2 times per week and continued throughtout the study and no differences between AG490 and DMSO treated mice were observed (see figure 1C and D) owning to the good general condition of animls. Laboratory examination such as hematology, clinical chemistry and urine analysis did not perform. AG490 induces remission of diabetes in STZ induced diabetic mice To futher explore whether AG490 can reverse diabetes in STZ- induced diabetic mice, BLAB/c mice were treated with STZ and then were treated one week ater the last STZ injection either with AG490 or DMSO (n = 5/group). Our data indicate that the mean blood glucose in DMSO treated mice was significantly higher than AG490 treated mice after 2-weeks of treatment (DMSO: 498.6629.3 mg/dl; AG490: 240.4695.2 mg/dl; p,0.004) (figure 2C & D). This data further suggests that AG490 is an effective and safe drug to reverse onset of diabetes and further is suggesting that the Jak-Stat signaling pathway may have additional role(s) in pathogenesis of diabetes and on glucose metabolism than currently appreciated. Adoptive transfer of AG490 treated splenocytes did not transfer diabetes in NOD.Scid mice In order to understand how AG490 reversed and blocked the onset of autoimmune diabetes in vivo, we treated prediabetic NOD mice (4-week of age) with AG490 or with DMSO for 5 week. Mice were sacrificied at week 10 and splenocyte cell suspensions were prepared with Histopaque gradient separation. The dead cells and reticulocytes were removed by the Histopaque gradient separa- tion, therefore, dead cells were excluded without magnetic purification and without activation of T-cells and cell viability was greater than 99%. Five million freshely prepared of pooled splenocytes of NOD mice treated with AG490 (n = 5) or DMSO (n = 5) were transferred by i.p injection into recipient NOD.Scid mice and blood glucose and body weight was closely monitored. Our data as shown in figure 3 suggests that prevention of autoimmune diabetes by AG490 could be due to inactivation of atuoreactive T-cells and/or induction/generation of regulatory T- cell or other regulatory population. Administration of AG490 does not diminish leukocyte infiltration within the pancreas 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). PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36079