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Background: Macrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ. Aim of the study: To scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype. Methods: Differentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling. Results: Mreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased. Conclusion: Exposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment. [ABSTRACT FROM AUTHOR]

One of the most important medical interventions for individuals with heart valvular disease is heart valve replacement, which is not without substantial challenges, particularly for pediatric patients. Due to their biological properties and biocompatibility, natural tissue-originated scaffolds derived from human or animal sources are one type of scaffold that is widely used in tissue engineering. However, they are known for their high potential for immunogenicity. Being free of cells and genetic material, decellularized xenografts, consequently, have low immunogenicity and, thus, are expected to be tolerated by the recipient's immune system. The scaffold ultrastructure and ECM composition can be affected by cell removal agents. Therefore, applying an appropriate method that preserves intact the structure of the ECM plays a critical role in the final result. So far, there has not been an effective decellularization technique that preserves the integrity of the heart valve's ultrastructure while securing the least amount of genetic material left. This study demonstrates a new protocol with untraceable cells and residual DNA, thereby maximally reducing any chance of immunogenicity. The mechanical and biochemical properties of the ECM resemble those of native heart valves. Results from this study strongly indicate that different critical factors, such as ionic detergent omission, the substitution of Triton X-100 with Tergitol, and using a lower concentration of trypsin and a higher concentration of DNase and RNase, play a significant role in maintaining intact the ultrastructure and function of the ECM. [ABSTRACT FROM AUTHOR]

Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular vesicles (L-EVMreg) with an average size of 7.5 μm which regulate wound healing and angiogenesis in vitro. The aim of this study was to investigate whether L-EVMreg also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EVMreg were isolated from culture supernatants by differential centrifugation. Activation of human T-cells was induced by CD3/CD28-coated beads in the absence or presence of Mreg or different concentrations of L-EVMreg. Inhibition of T-cell activation was quantified by flow cytometry and antibodies directed against the T-cell marker granzyme B. Phosphatidylserine (PS) exposure on the surface of Mreg and L-EVMreg was analyzed by fluorescence microscopy. Incubation of human lymphocytes with CD3/CD28 beads resulted in an increase of cell size, cell granularity, and number of granzyme B–positive cells (P < 0.05) which is indicative of T-cell activation. The presence of Mreg (0.5 × 106 Mreg/ml) led to a reduction of T-cell activation (number of granzyme B–positive cells; P < 0.001), and a similar but less pronounced effect was also observed when incubating activated T-cells with L-EVMreg (P < 0.05 for 3.2 × 106 L-EVMreg/ml). A differential analysis of the effects of Mreg and L-EVMreg on CD4+ and CD8+ T-cells showed an inhibition of CD4+ T-cells by Mreg (P < 0.01) and L-EVMreg (P < 0.05 for 1.6 × 106 L-EVMreg/ml; P < 0.01 for 3.2 × 106 L-EVMreg/ml). A moderate inhibition of CD8+ T-cells was observed by Mreg (P < 0.05) and by L-EVMreg (P < 0.01 for 1.6 × 106 L-EVMreg/ml and 3.2 × 106 L-EVMreg/ml). PS was restricted to confined regions of the Mreg surface, while L-EVMreg showed strong signals for PS in the exoplasmic leaflet. L-EVMreg attenuate CD3/CD28-mediated activation of CD4+ and CD8+ T-cells. L-EVMreg may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity. Key messages: Mreg release large extracellular vesicles (L-EVMreg) with an average size of 7.5 µm L-EVMreg exhibit phosphatidylserine positivity L-EVMreg suppress CD4+ and CD8+ T-cells L-EVMreg hold clinical potential in T-cell-related diseases [ABSTRACT FROM AUTHOR]

Background Mitral valved stents tend to migrate or to develop paravalvular leakage due to high–left ventricular pressure in this cavity. Thus, this study describes a newly developed mitral valved stent anchoring technology. Methods Based on an existing mitral valved stent, four anchoring units with curved surgical needles were designed and fabricated using three-dimensional (3D) software and print technology. Mitral nitinol stents assembled with four anchoring units were successively fixed on 10 porcine annuli. Mechanical tests were performed with a tensile force test system and recorded the tension forces of the 10 nitinol stents on the annulus. Results The average maximum force was 28.3 ± 5.21 N, the lowest was 21.7 N, and the highest was 38.6 N until the stent lost contact with the annulus; for the break force (zero movement of stent from annulus), the average value was 18.5 ± 6.7 N with a maximum value of 26.9 N and a minimum value of 6.07 N. It was additionally observed that the puncture needles of the anchoring units passed into the mitral annulus in all 10 hearts and further penetrated the myocardium in only one additional heart. The anchoring units enhanced the tightness of the mitral valved stent and did not destroy the circumflex coronary artery, coronary sinus, right atrium, aortic root, or the left ventricular outflow tract. Conclusion The new anchoring units for mitral nitinol stents were produced with 3D software and printing technology; with this new type of anchoring technology, the mitral valved stent can be tightly fixed toward the mitral annulus. [ABSTRACT FROM AUTHOR]