The reconstruction of critical size bone defects is still clinically challenging. Even though the transplantation of autologous bone is used as gold standard, this therapy is accompanied by donor site morbidities as well as tissue limitations. The alternatively used allografts, which are devitalized due to thermal, chemical or physical processing, often lose their matrix integrity and have diminished biomechanical properties. High Hydrostatic Pressure (HHP) may represent a gentle alternative to already existing methods since HHP treated human osteoblasts undergo cell death and HHP treated bone cylinders maintain their mechanical properties. The aim of this study was to determine the biological effects caused by HHP treatment regarding protein/matrix integrity and type of cell death in trabecular bone cylinders. Therefore, different pressure protocols (250 and 300 MPa for 10, 20 and 30 min) and end point analysis such as quantification of DNA-fragmentation, gene expression, SDS-PAGE, FESEM analysis and histological staining were performed. While both protein and matrix integrity was preserved, molecular biological methods showed an apoptotic differentiation of cell death for lower pressures and shorter applications (250 MPa for 10 and 20 min) and necrotic differentiation for higher pressures and longer applications (300 MPa for 30 min). This study serves as a basis for further investigation as it shows that HHP successfully devitalizes trabecular bone cylinders.

Macrophages and immuno-modulation play a dominant role in the pathology of pancreatic cancer. Gas plasma is a technology recently suggested to demonstrate anticancer efficacy. To this end, two murine cell lines were employed to analyze the inflammatory consequences of plasma-treated pancreatic cancer cells (PDA) on macrophages using the kINPen plasma jet. Plasma treatment decreased the metabolic activity, viability, and migratory activity in an ROS- and treatment time-dependent manner in PDA cells in vitro. These results were confirmed in pancreatic tumors grown on chicken embryos in the TUM-CAM model (in ovo). PDA cells promote tumor-supporting M2 macrophage polarization and cluster formation. Plasma treatment of PDA cells abrogated this cluster formation with a mixed M1/M2 phenotype observed in such co-cultured macrophages. Multiplex chemokine and cytokine quantification showed a marked decrease of the neutrophil chemoattractant CXCL1, IL6, and the tumor growth supporting TGFβ and VEGF in plasma-treated compared to untreated co-culture settings. At the same time, macrophage-attractant CCL4 and MCP1 release were profoundly enhanced. These cellular and secretome data suggest that the plasma-inactivated PDA6606 cells modulate the inflammatory profile of murine RAW 264.7 macrophages favorably, which may support plasma cancer therapy.

Background: Gram-negative infections of the peritoneal cavity result in profound modifications of peritoneal B cell populations and induce the migration of peritoneal B cells to distant secondary lymphoid organs. However, mechanisms controlling the egress of peritoneal B cells from the peritoneal cavity and their subsequent trafficking remain incompletely understood. Sphingosine-1-phosphate (S1P)-mediated signaling controls migratory processes in numerous immune cells. The present work investigates the role of S1P-mediated signaling in peritoneal B cell trafficking under inflammatory conditions. Methods: Differential S1P receptor expression after peritoneal B cell activation was assessed semi‑quantitatively using RT-PCR in vitro. The functional implications of differential S1P1 and S1P4 expression were assessed by transwell migration in vitro, by adoptive peritoneal B cell transfer in a model of sterile lipopolysaccharide (LPS)‑induced peritonitis and in the polymicrobial colon ascendens stent peritonitis (CASP) model. Results: The two sphingosine-1-phosphate receptors (S1PRs) expressed in peritoneal B cell subsets S1P1 and S1P4 are differentially regulated upon stimulation with the TLR4 agonist LPS, but not upon PMA/ionomycin or B cell receptor (BCR) crosslinking. S1P4 deficiency affects both the trafficking of activated peritoneal B cells to secondary lymphoid organs and the positioning of these cells within the functional compartments of the targeted organ. S1P4 deficiency in LPS-activated peritoneal B cells results in significantly reduced numbers of splenic innate response activator B cells. Conclusions: The S1P-S1PR system is implicated in the trafficking of LPS-activated peritoneal B cells. Given the protective role of peritoneal B1a B cells in peritoneal sepsis, further experiments to investigate the impact of S1P4-mediated signaling on the severity and mortality of peritoneal sepsis are warranted.

IntroductionSplenic B cells exhibit a high expression of the G protein-coupled sphingosine-1-phosphate (S1P) receptor type 4 (S1PR4). Little is known about the functional relevance of S1PR4 expression on those cells.MethodsIn this study, S1PR4-deficient mice were used to study the role of S1PR4-mediated S1P signaling in B cell motility in vitro and for the maintenance of the splenic architecture under steady state conditions as well as in polymicrobial abdominal sepsis in vivo. Finally, the impact of S1PR4 deficiency on antibody production after immunization with T cell dependent antigens was assessed.ResultsLoss of S1PR4 resulted in minor alterations of the splenic architecture concerning the presence of B cell follicles. After sepsis induction, the germinal center response was severely impaired in S1PR4-deficient animals. Splenic B cells showed reduced motility in the absence of S1PR4. However, titres of specific antibodies showed only minor reductions in S1PR4-deficient animals.DiscussionThese observations suggest that S1P signaling mediated by S1PR4 modifies chemokine-induced splenic B cell chemotaxis, thus modulating splenic microarchitecture, GC formation and T-cell dependent antibody production.