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1.  Pathogenicity of IFN-γ–producing Th17 cells is independent of T-bet 
During the development of experimental autoimmune encephalomyelitis (EAE), the proportion of pathogenic and myelin-specific cells within CNS-infiltrating cytokine producing T helper (Th) cells is unknown. Using an IL-17A-IFN-γ double reporter mouse and I-Ab/MOG38–49 tetramer, we show here that IL-17+ IFN-γ+ Th cells, which are expanded in the CNS during EAE, are highly enriched in MOG-specific T cells. We further demonstrate that IL-23 is essential for the generation and expansion of IFN-γ producing Th17 cells independently of the Th1-associated transcription factors T-bet, STAT1 and STAT4. Furthermore, Th17 and IL-17+ IFN-γ+ Th cells can induce CNS autoimmunity independently of T-bet. While T-bet is crucial for Th1 mediated EAE T-bet is dispensable for Th17 cell-mediated autoimmunity. Our results suggest the existence of different epigenetic programs that regulate IFN-γ expression in Th1 and Th17 cells.
PMCID: PMC3633668  PMID: 23543757
2.  Ultrasound-Guided Creation of a Small Animal Model of Aortic Injury 
To develop a small animal model of controlled aortic intimal injury with ultrasound imaging guidance.
Materials and Methods
Via carotid artery cut down, we advanced a custom-made micro-catheter/angled-metal-device system to damage the intima of the ascending aortas of 20 Sprague Dawley (SD) rats and 10 JCR atherosclerotic rats. This minimally invasive endovascular procedure was monitored by a clinical ultrasound imaging system. Injured aortas were harvested for histologic confirmations using a grading system: Grade I with intima injury, Grade II with injury to media, and Grade III with injury through the entire aortic wall. Neointimal reactions at the injury site were compared by calculating the ratio of intimal to medial thickness among different animal groups at various survival times (week 1, weeks 2–3 and weeks 4–7).
Clear visualization of the architectures of the heart, great vessels and the exact location of the angled-metal-device by ultrasound imaging ensured consistent intimal damage of the aorta. Histopathology confirmed that most of the aortic injures were classified as Grade I. There was no significant difference between the two rat groups. Analysis on pathophysiological reactions at the injury sites revealed increased thickening of neointimal hyperplasia as animal survival times extended from week 1 to weeks 4–7 after the aortic interventions.
This study demonstrates the feasibility of using clinical ultrasound imaging to precisely guide the creation of controlled aortic intimal injury in rats, which may become a useful tool to facilitate research involving the prevention and treatment of atherosclerotic cardiovascular disease.
PMCID: PMC3148304  PMID: 21570869
atherosclerotic cardiovascular disease; rat model; ultrasound; vascular injury
3.  MRI of Auto-Transplantation of Bone Marrow-Derived Stem-Progenitor Cells for Potential Repair of Injured Arteries 
PLoS ONE  2012;7(2):e31137.
This study was to validate the feasibility of using clinical 3.0T MRI to monitor the migration of autotransplanted bone marrow (BM)-derived stem-progenitor cells (SPC) to the injured arteries of near-human sized swine for potential cell-based arterial repair.
The study was divided into two phases. For in vitro evaluation, BM cells were extracted from the iliac crests of 13 domestic pigs and then labeled with a T2 contrast agent, Feridex, and/or a fluorescent tissue marker, PKH26. The viability, the proliferation efficiency and the efficacies of Feridex and/or PKH26 labeling were determined. For in vivo validation, the 13 pigs underwent endovascular balloon-mediated intimal damages of the iliofemoral arteries. The labeled or un-labeled BM cells were autotransplanted back to the same pig from which the BM cells were extracted. Approximately three weeks post-cell transplantation, 3.0T T2-weighted MRI was performed to detect Feridex-created signal voids of the transplanted BM cells in the injured iliofemoral arteries, which was confirmed by subsequent histologic correlation.
Principal Findings
Of the in vitro study, the viability of dual-labeled BM cells was 95–98%. The proliferation efficiencies of dual-labeled BM cells were not significantly different compared to those of non-labeled cells. The efficacies of Feridex- and PKH26 labeling were 90% and 100%, respectively. Of the in vivo study, 3.0T MRI detected the auto-transplanted BM cells migrated to the injured arteries, which was confirmed by histologic examinations.
This study demonstrates the capability of using clinical 3.0T MRI to monitor the auto-transplantation of BM cells that migrate to the injured arteries of large animals, which may provide a useful MRI technique to monitor cell-based arterial repair.
PMCID: PMC3281926  PMID: 22363566
4.  Magnetic Resonance Imaging of Bone Marrow Cell-Mediated Interleukin-10 Gene Therapy of Atherosclerosis 
PLoS ONE  2011;6(9):e24529.
A characteristic feature of atherosclerosis is its diffuse involvement of arteries across the entire human body. Bone marrow cells (BMC) can be simultaneously transferred with therapeutic genes and magnetic resonance (MR) contrast agents prior to their transplantation. Via systemic transplantation, these dual-transferred BMCs can circulate through the entire body and thus function as vehicles to carry genes/contrast agents to multiple atherosclerosis. This study was to evaluate the feasibility of using in vivo MR imaging (MRI) to monitor BMC-mediated interleukin-10 (IL-10) gene therapy of atherosclerosis.
For in vitro confirmation, donor mouse BMCs were transduced by IL-10/lentivirus, and then labeled with a T2-MR contrast agent (Feridex). For in vivo validation, atherosclerotic apoE−/− mice were intravenously transplanted with IL-10/Feridex-BMCs (Group I, n = 5) and Feridex-BMCs (Group II, n = 5), compared to controls without BMC transplantation (Group III, n = 5). The cell migration to aortic atherosclerotic lesions was monitored in vivo using 3.0T MRI with subsequent histology correlation. To evaluate the therapeutic effect of BMC-mediated IL-10 gene therapy, we statistically compared the normalized wall indexes (NWI) of ascending aortas amongst different mouse groups with various treatments.
Principal Findings
Of in vitro experiments, simultaneous IL-10 transduction and Feridex labeling of BMCs were successfully achieved, with high cell viability and cell labeling efficiency, as well as IL-10 expression efficiency (≥90%). Of in vivo experiments, MRI of animal groups I and II showed signal voids within the aortic walls due to Feridex-created artifacts from the migrated BMCs in the atherosclerotic plaques, which were confirmed by histology. Histological quantification showed that the mean NWI of group I was significantly lower than those of group II and group III (P<0.05).
This study has confirmed the possibility of using MRI to track, in vivo, IL-10/Feridex-BMCs recruited to atherosclerotic lesions, where IL-10 genes function to prevent the progression of atherosclerosis.
PMCID: PMC3168522  PMID: 21915349

Results 1-4 (4)