New hemophilia insights could minimize or eliminate side effects in the future frontiersin
If not otherwise indicated, whole splenocytes were used as APCs in most experiments.
To evaluate the composition of APCs, splenocytes were stained for CD3e PerCP-eFluor 710 , CD19 FITC , CD11c APC , CD11b PE-Cy7 and HLA-DR BV605 and analyzed using a BD FACS Aria III and FlowJo software . Nonspecific binding through Fc gamma receptors was blocked by a mixture of anti-CD16 and anti-CD32 antibodies .Splenocytes were prepared as described before.
The FVIII peptide repertoire presented by APCs was monitored by analyzing the peptide specificities of subsequently activated FVIII specific CD4+ T cell clones. In detail, APCs from naïve HLA-DRB1*1501 mice were incubated with human recombinant FVIII, thrombin activated FVIII or FVIII in complex with human purified plasma-derived VWF. Equal units of human FVIII and human VWF were co-incubated in serum-free medium at room temperature for one hour to facilitate complex building.
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Frontiers | Identification of a naturally-occurring canine model for early detection and intervention research in high grade urothelial carcinomaBackground: Early detection and intervention research is expected to improve the outcomes for patients with invasive urinary bladder cancer, specifically muscle invasive urothelial carcinoma (InvUC). With limited patients in suitable high-risk study cohorts, identifying relevant animal models for this research is critical. Experimental animal modes, while essential in InvUC research, often fail to adequately represent human cancer, and fail to predict drug responses. The purpose of this study was to define a naturally-occurring canine model for early intervention research by studying dogs with high breed-associated risk for InvUC, e.g. Scottish Terriers (STs). Methods: STs (n=120) ≥ 6 years old with no outward evidence of urinary disease were screened at 6-month intervals for 3 years. Screening included physical exam with digital rectal exam, ultrasonography, and urinalysis with sediment exam. Cystoscopic biopsy was performed in dogs with positive screening tests. The pathological, clinical, and molecular characteristics of the “early” cancer detected by screening were determined. Transcriptomic signatures were compared between the early tumors and published findings in human InvUC, and to more advanced “later” canine tumors from STs who had the typical clinical presentation of hematuria and urinary dysfunction. An early intervention trial of an oral cyclooxygenase inhibitor, deracoxib, was conducted in dogs with cancer detected through screening. Results: Biopsy-confirmed bladder cancer was detected in 32 (27%) of 120 STs including InvUC (n=29, three starting as dysplasia), grade 1 noninvasive cancer (n=2), and carcinoma in situ (n=1). Transcriptomic signatures including druggable targets such as EGFR and the PI3K-AKT-mTOR pathway, were very similar between canine and human InvUC, especially within luminal and basal molecular subtypes. Marked transcriptomic differences were noted between the early and later canine tumors, particularly within luminal subtype tumors.
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