82Rb cardiac PET allows the assessment of myocardial perfusion using a column generator in clinics that lack a cyclotron. We and others have previously shown that quantitation of myocardial blood flow (MBF) and coronary flow reserve (CFR) is feasible using dynamic 82Rb PET and factor and compartment analyses. The aim of the present work was to determine the intra- and inter-observer variability of MBF estimation using 82Rb PET as well as the reproducibility of our generalized factor + compartment analyses methodology to estimate MBF and assess its accuracy by comparing, in the same subjects, 82Rb estimates of MBF to those obtained using 13N-ammonia. Methods Twenty-two subjects were included in the reproducibility and twenty subjects in the validation study. Patients were injected with 60±5mCi of 82Rb and imaged dynamically for 6 minutes at rest and during dipyridamole stress Left and right ventricular (LV+RV) time-activity curves were estimated by GFADS and used as input to a 2-compartment kinetic analysis that estimates parametric maps of myocardial tissue extraction (K1) and egress (k2), as well as LV+RV contributions (fv,rv). Results Our results show excellent reproducibility of the quantitative dynamic approach itself with coefficients of repeatability of 1.7% for estimation of MBF at rest, 1.4% for MBF at peak stress and 2.8% for CFR estimation. The inter-observer reproducibility between the four observers that participated in this study was also very good with correlation coefficients greater than 0.87 between any two given observers when estimating coronary flow reserve. The reproducibility of MBF in repeated 82Rb studies was good at rest and excellent at peak stress (r2=0.835). Furthermore, the slope of the correlation line was very close to 1 when estimating stress MBF and CFR in repeated 82Rb studies. The correlation between myocardial flow estimates obtained at rest and during peak stress in 82Rb and 13N-ammonia studies was very good at rest (r2=0.843) and stress (r2=0.761). The Bland-Altman plots show no significant presence of proportional error at rest or stress, nor a dependence of the variations on the amplitude of the myocardial blood flow at rest or stress. A small systematic overestimation of 13N-ammonia MBF was observed with 82Rb at rest (0.129 ml/g/min) and the opposite, i.e., underestimation, at stress (0.22 ml/g/min). Conclusions Our results show that absolute quantitation of myocardial bloof flow is reproducible and accurate with 82Rb dynamic cardiac PET as compared to 13N-ammonia. The reproducibility of the quantitation approach itself was very good as well as inter-observer reproducibility.

SUMMARY Interleukin 9 (IL-9) is a pleiotropic cytokine that can regulate autoimmune responses by enhancing regulatory CD4+FoxP3+ T regulatory (Treg) cell survival and T helper 17 (Th17) cell proliferation. Here, we analyzed the costimulatory requirements for the induction of Th9 cells, and demonstrated that Notch pathway cooperated with TGF-β signaling to induce IL-9. Conditional ablation of Notch1 and Notch2 receptors inhibited the development of Th9 cells. Notch1 intracellular domain (NICD1) recruited Smad3, downstream of TGF-β cytokine signaling, and together with recombining binding protein (RBP)-Jκ bound the Il9 promoter and induced its transactivation. In experimental autoimmune encephalomyelitis (EAE), Jagged2 ligation regulated clinical disease in an IL-9-dependent fashion. Signaling through Jagged2 expanded Treg cells and suppressed EAE when administered before antigen immunization, but worsened EAE when administered concurrently with immunization by favoring Th17 cell expansion. We propose that Notch and Smad3 cooperate to induce IL-9 and participate in regulating the immune response.

Notch signaling pathway plays an important role in T cell differentiation. Delta-like ligand (Dll)4, one of five known Notch ligands, has been implicated in regulating Th2 cell differentiation in animal models of human diseases. However, the role of Dll4 in Th1/Th17-mediated autoimmune diseases remains largely unknown. Using an anti-Dll4 blocking mAb, we show that neutralizing Dll4 during the induction phase of experimental autoimmune encephalomyelitis in C57BL/6 mice significantly increased the pool of CD4+Foxp3+ regulatory T cells (Treg) in the periphery and in the CNS, and decreased the severity of clinical disease and CNS inflammation. Dll4 blockade promoted induction of myelin-specific Th2/Treg immune responses and impaired Th1/Th17 responses compared with IgG-treated mice. In vitro, we show that signaling with recombinant Dll4 inhibits the TGF-β–induced Treg development, and inhibits Janus kinase 3-induced STAT5 phosphorylation, a transcription factor known to play a key role in Foxp3 expression and maintenance. Depletion of natural Treg using anti-CD25 Ab reversed the protective effects of anti-Dll4 Ab. These findings outline a novel role for Dll4–Notch signaling in regulating Treg development in EAE, making it an encouraging target for Treg-mediated immunotherapy in autoimmune diseases, such as multiple sclerosis.