De novo Vessel Formation Through Cross-Talk of Blood-Derived Cells and Mesenchymal Stromal Cells in the Absence of Pre-existing Vascular Structures

Background: The generation of functional blood vessels remains a key challenge for regenerative medicine. Optimized in vitro culture set-ups mimicking the in vivo perivascular niche environment during tissue repair may provide information about the biological function and contribution of progenitor cells to postnatal vasculogenesis, thereby enhancing their therapeutic potential. Aim: We established a fibrin-based xeno-free human 3D in vitro vascular niche model to study the interaction of mesenchymal stromal cells (MSC) with peripheral blood mononuclear cells (PBMC) including circulating progenitor cells in the absence of endothelial cells (EC), and to investigate the contribution of this cross-talk to neo-vessel formation. Materials and Methods: Bone marrow-derived MSC were co-cultured with whole PBMC, enriched monocytes (Mo), enriched T cells, and Mo together with T cells, respectively, obtained from leukocyte reduction chambers generated during the process of single-donor platelet apheresis. Cells were embedded in 3D fibrin matrices, using exclusively human-derived culture components without external growth factors. Cytokine secretion was analyzed in supernatants of 3D cultures by cytokine array, vascular endothelial growth factor (VEGF) secretion was quantified by ELISA. Cellular and structural re-arrangements were characterized by immunofluorescence and confocal laser-scanning microscopy of topographically intact 3D fibrin gels. Results: 3D co-cultures of MSC with PBMC, and enriched Mo together with enriched T cells, respectively, generated, within 2 weeks, complex CD31C /CD34C vascular structures, surrounded by basement membrane collagen type-IVC cells and matrix, in association with increased VEGF secretion. PBMC contained CD31C CD34CCD45dimCD14 progenitor-type cells, and EC of neo-vessels were PBMC-derived. Vascular structures showed intraluminal CD45C cells that underwent apoptosis thereby creating a lumen. Cross-talk of MSC with enriched Mo provided a proangiogenic paracrine environment. MSC co-cultured with enriched T cells formed "cellin-cell" structures generated through internalization of T cells by CD31C CD45dim = cells. No vascular structures were detected in co-cultures of MSC with either Mo or T cells. Conclusion: Our xeno-free 3D in vitro vascular niche model demonstrates that a complex synergistic network of cellular, extracellular and paracrine cross-talk can contribute to de novo vascular development through self-organization via co-operation of immune cells with blood-derived progenitor cells and MSC, and thereby may open a new perspective for advanced vascular tissue engineering in regenerative medicine

Increasing test specificity without impairing sensitivity: lessons learned from SARS-CoV-2 serology

Background: Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the € sensitivity improved two-test' or € SIT²' algorithm. Methods: SIT² involves confirmatory retesting of samples with results falling in a predefined retesting zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT² to single tests and orthogonal testing (OTA) in an Austrian cohort (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives). Results: The specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with single testing or OTA, SIT² significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. Conclusion: For SARS-CoV-2 serology, SIT² proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases

Surface disinfection of packed red blood cells with 70% ethanol

AbstractNo data or recommendations are available on feasibility of surface disinfection of blood bags, but some circumstances can make such procedures inevitable. Impact of immersion of blood bags in 70% ethanol for 30min was investigated with respect to alcohol penetration and changes of hemolysis parameters in the product, and bag material changes influencing material stability and composition. After immersion ethanol concentration in blood bags was below detection limit. Hemolysis parameters did not differ between blood products that had been exposed to ethanol and a control group. Inner surface of the bag material was unchanged according to our infrared spectrometry results. Also endurance testing showed no altered results. We conclude that immersion of blood bags in 70% ethanol for surface disinfection is a safe procedure for the quality of the blood product and the bag material

PLOS ONE / Transfusion of standard-issue packed red blood cells induces pulmonary vasoconstriction in critically ill patients after cardiac surgeryA randomized, double-blinded, clinical trial

Background Experimental and volunteer studies have reported pulmonary vasoconstriction during transfusion of packed red blood cells (PRBCs) stored for prolonged periods. The primary aim of this study was to evaluate whether transfusion of PRBCs stored over 21 days (standard-issue, siPRBCs) increases pulmonary artery pressure (PAP) to a greater extent than transfusion of PRBCs stored for less then 14 days (fresh, fPRBCs) in critically ill patients following cardiac surgery. The key secondary aim was to assess whether the pulmonary vascular resistance index (PVRI) increases after transfusion of siPRBCs to a greater extent than after transfusion of fPRBCs. Methods The study was performed as a single-center, double-blinded, parallel-group, randomized clinical trial. Leukoreduced PRBCs were transfused while continuously measuring hemodynamic parameters. Systemic concentrations of syndecan-1 were measured to assess glycocalyx injury. After randomizing 19 patients between January 2014 and June 2016, the study was stopped due to protracted patient recruitment. Results Of 19 randomized patients, 11 patients were transfused and included in statistical analyses. Eight patients were excluded prior to transfusion, 6 patients received fPRBCs (103 storage days), whereas 5 patients received siPRBCs (334 storage days). The increase in PAP (73 vs. 22 mmHg, P = 0.012) was greater during transfusion of siPRBCs than during transfusion of fPRBCs. In addition, the change in PVRI (15089 vs. -437 dyn·s·cm·m, P = 0.018) was greater after transfusion of siPRBCs than after transfusion of fPRBCs. The increase in PAP correlated with the change of systemic syndecan-1 concentrations at the end of transfusion (R = 0.64,P = 0.034). Conclusion Although this study is underpowered and results require verification in larger clinical trials, our findings suggest that transfusion of siPRBCs increases PAP and PVRI to a greater extent than transfusion of fPRBCs in critically ill patients following cardiac surgery. Glycocalyx injury might contribute to pulmonary vasoconstriction associated with transfusion of stored blood.(VLID)495023

INTERCEPT Pathogen Reduction in Platelet Concentrates, in Contrast to Gamma Irradiation, Induces the Formation of trans-Arachidonic Acids and Affects Eicosanoid Release during Storage

Pathogen inactivation techniques for blood products have been implemented to optimize clinically safe blood components supply. The INTERCEPT system uses amotosalen together with ultraviolet light wavelength A (UVA) irradiation. Irradiation-induced inactivation of nucleic acids may actually be accompanied by modifications of chemically reactive polyunsaturated fatty acids known to be important mediators of platelet functions. Thus, here, we investigated eicosanoids and the related fatty acids released upon treatment and during storage of platelet concentrates for 7 days, complemented by the analysis of functional and metabolic consequences of these treatments. Metabolic and functional issues like glucose consumption, lactate formation, platelet aggregation, and clot firmness hardly differed between the two treatment groups. In contrast to gamma irradiation, here, we demonstrated that INTERCEPT treatment immediately caused new formation of trans-arachidonic acid isoforms, while 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-HETE were increased and two hydroperoxyoctadecadienoic acid (HpODE) isoforms decreased. During further storage, these alterations remained stable, while the release of 12-lipoxygenase (12-LOX) products such as 12-HETE and 12-hydroxyeicosapentaenoic acid (12-HEPE) was further attenuated. In vitro synthesis of trans-arachidonic acid isoforms suggested that thiol radicals formed by UVA treatment may be responsible for the INTERCEPT-specific effects observed in platelet concentrates. It is reasonable to assume that UVA-induced molecules may have specific biological effects which need to be further investigated