RESUMEN
Study of disease-relevant immune cells, namely monocytes and macrophages, is limited based on availability of primary tissue, a limitation that can be remedied using human induced pluripotent stem cell (hiPSC) technology. Here, we present a protocol for differentiation of monocytes and macrophages from hiPSCs. We describe steps for hiPSC maintenance, mesoderm lineage induction, hematopoietic progenitor cells (HPCs) commitment and expansion, and myeloid lineage induction. We then detail procedures for monocyte formation and functional macrophage formation and polarization. For complete details on the use and execution of this protocol, please refer to Chen et al.1.
RESUMEN
Breast implants are extensively employed for both reconstructive and esthetic purposes. However, the safety of breast implants with textured surfaces has been questioned, owing to a potential correlation with anaplastic large-cell lymphoma and the recurrence of breast cancer. This study investigates the immune response elicited by different prosthetic surfaces, focusing on the comparison between macrotextured and microtextured breast implants. Through the analysis of intraoperatively harvested periprosthetic fluids and cell culture experiments on surface replicas, we demonstrate that macrotextured surfaces elicit a more pronounced chronic-like activation of leucocytes and an increased release of inflammatory cytokines, in contrast to microtextured surfaces. In addition, in vitro fluorescent imaging of leucocytes revealed an accumulation of lymphocytes within the cavities of the macrotextured surfaces, indicating that the physical entrapment of these cells may contribute to their activation. These findings suggest that the topography of implant surfaces plays a significant role in promoting a chronic-like inflammatory environment, which could be a contributing factor in the development of lymphomas associated with a wide range of implantable devices.
Asunto(s)
Implantación de Mama , Implantes de Mama , Neoplasias de la Mama , Linfoma Anaplásico de Células Grandes , Humanos , Femenino , Implantes de Mama/efectos adversos , Linfoma Anaplásico de Células Grandes/etiología , Linfoma Anaplásico de Células Grandes/cirugíaRESUMEN
The study of endothelial dysfunction (ED) is crucial to identify the pathogenetic mechanism(s) and provide indications for patient management in cardiovascular diseases. It is currently hindered by the limited availability of patient-specific primary endothelial cells (ECs). Endothelial colony-forming cells (ECFCs) represent an optimal non-invasive tool to overcome this issue. Therefore, we investigated the use of ECFCs as a substrate in thrombogenesis and thrombin generation assay (TGA) to assess ED. Both assays were set up on human umbilical vein endothelial cells (HUVECs) and then tested on ECFCs obtained from healthy donors. To prove the ability of the assays to detect endothelial activation, ECs stimulated with TNFα were compared with unstimulated ECs. EC activation was confirmed by the upregulation of VCAM-1 and Tissue Factor expression. Both assays discriminated between unstimulated and activated HUVECs and ECFCs, as significantly higher platelet deposition and fibrin formation in thrombogenesis assay, and thrombin generation in TGA, were observed when TNFα-activated ECs were used as a substrate. The amount of fibrin and thrombin measured in the two assays were directly correlated. Our results support the combined use of a thrombogenesis assay and TGA performed on patient-derived ECFCs to provide a personalized global assessment of ED relevant to the patient's hemostatic profile.
RESUMEN
The incidence of periprosthetic joint infections (PJIs) is ~2% of total procedures and it is expected to rise due to an ageing population. Despite the large burden PJI has on both the individual and society, the immune response to the most commonly isolated pathogens, i.e., Staphylococcus aureus and Staphylococcus epidermidis, remains incompletely understood. In this work, we integrate the analysis of synovial fluids from patients undergoing hip and knee replacement surgery with in-vitro experimental data obtained using a newly developed platform, mimicking the environment of periprosthetic implants. We found that the presence of an implant, even in patients undergoing aseptic revisions, is sufficient to induce an immune response, which is significantly different between septic and aseptic revisions. This difference is confirmed by the presence of pro- and anti-inflammatory cytokines in synovial fluids. Moreover, we discovered that the immune response is also dependent on the type of bacteria and the topography of the implant surface. While S. epidermidis seems to be able to hide better from the attack of the immune system when cultured on rough surfaces (indicative of uncemented prostheses), S. aureus reacts differently depending on the contact surface it is exposed to. The experiments we performed in-vitro also showed a higher biofilm formation on rough surfaces compared to flat ones for both species, suggesting that the topography of the implant could influence both biofilm formation and the consequent immune response.