Assuntos
Relações Interprofissionais , Sexismo , Estágio Clínico , Feminino , Humanos , Sorriso , Estudantes de MedicinaRESUMO
BACKGROUND: Following protein replacement therapy, one-third of severe hemophilia A patients develop antibodies to factor VIII (FVIII), which also hinders the efficacy of gene therapy. Regulatory T cells (Tregs) have a naturally suppressive function that potentially reduces the immune response to FVIII therapy. Furthermore, antigen-specific Tregs are functionally much more potent than polyclonal cells. Adoptive transfer of antigen-specific Tregs can effectively suppress anti-FVIII antibody responses. OBJECTIVE: Develop a clinically feasible protocol to enrich and expand Tregs specific to FVIII for suppressing anti-FVIII immune responses. METHODS: Regulatory T cells are isolated from FVIII-sensitized mice, sorted on CD25high markers, and expanded specifically with FVIII, antigen-presenting cells, and interleukin 2 (IL 2). Subsequently, Tregs are further cultured with anti-CD3/anti-CD28 beads, anti-Crry antibodies, and IL 2 to achieve 10-fold to 20-fold expansion. Expanded Tregs are characterized and tested for their suppressive activity in vitro and in vivo. RESULTS: In vitro FVIII-specific suppressive assays indicate that FVIII specifically expanded Tregs are more suppressive than non-specifically expanded and naive Tregs. Adoptive transfer of expanded Tregs into HemA mice showed that FVIII-specifically expanded Tregs are significantly more potent in suppressing anti-FVIII immune responses in FVIII plasmid-treated HemA mice. Moreover, the FVIII-specific immune tolerance is maintained after a secondary challenge with FVIII plasmid. CONCLUSIONS: Our results demonstrate that the FVIII-specific sensitization and expansion protocol yields more potent Tregs to suppress anti-FVIII antibody responses and induce long-term tolerance to FVIII, increasing the potential for adoptive Treg cell therapy to modulate anti-FVIII immune responses.
Assuntos
Fator VIII , Hemofilia A , Animais , Fator VIII/genética , Terapia Genética , Hemofilia A/terapia , Humanos , Tolerância Imunológica , Camundongos , Linfócitos T ReguladoresRESUMO
While ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2â¯MPa or above, emerging research showed that this may not be a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions close to the MB inertial cavitation threshold and focused towards further increasing gene transfer efficiency and reducing associated cell damage. We created a matrix of 21 conditions (nâ¯=â¯3/cond.) to test in HEK293T cells using pulse durations spanning 18⯵s-36â¯ms and PNPs spanning 0.5-2.5â¯MPa. Longer pulse duration conditions yielded significant increase in transgene expression relative to sham with local maxima between 20â¯J and 100â¯J energy curves. A similar set of 17 conditions (nâ¯=â¯4/cond.) was tested in mice using pulse durations spanning 18⯵s-22â¯ms and PNPs spanning 0.5-2.5â¯MPa. We observed local maxima located between 1â¯J and 10â¯J energy curves in treated mice. Of these, several low pressure conditions showed a decrease in ALT and AST levels while maintaining better or comparable expression to our positive control, indicating a clear benefit to allow for effective transfection with minimized tissue damage versus the high-intensity control. Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo, circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Overall, these results demonstrate the advancement of UMGD technology for achieving efficient gene transfer and potential scalability to larger animal models and human application.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Microbolhas , Ultrassonografia/métodos , Acústica , Animais , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pressão , Fatores de Tempo , TransfecçãoRESUMO
OBJECTIVES: We sought to describe the caliber and vascular health of the subclavian and axillary arteries as related to their potential utilization in complex cardiovascular procedures. BACKGROUND: Patients referred for advanced catheter-based therapies frequently have lower extremity peripheral vascular disease that may prohibit the use of large bore arterial catheters. Utilization of the upper extremity peripheral vasculature is rarely considered as an alternative access strategy. This may be due in part to a lack of familiarity with the thoracic vasculature. METHODS AND RESULTS: 208 consecutive patients undergoing routine CTA prior to transcatheter aortic valve replacement were retrospectively evaluated in a systematic analysis of upper and lower extremity vasculature. Minimal luminal diameters (MLDs) for the axillary arteries and iliofemoral arteries were 6.0 ± 1.1 mm and 6.6 ± 1.8 mm respectively. Compared to the iliofemoral arteries, the axillary arteries demonstrated substantially lower rates of significant stenosis (2% vs. 12%, p < 0.01) and significantly lower rates of moderate to severe calcification disease (9% vs. 64%, p < 0.01). Diabetes and tobacco use were independently associated with smaller axillary artery caliber by MLD (p < 0.01) but not with significant stenotic disease. CONCLUSIONS: The axillary arteries are slightly smaller but less frequently diseased than the corresponding iliofemoral vessels.