The recently described tumor necrosis factor (TNF) family member LIGHT (herpes virus entry mediator [HVEM]-L/TNFSF14), a ligand for the lymphotoxin (LT)β receptor, HVEM, and DcR3, was inactivated in the mouse. In contrast to mice deficient in any other member of the LT core family, LIGHT−/− mice develop intact lymphoid organs. Interestingly, a lower percentage of LIGHT−/−LTβ−/− animals contain mesenteric lymph nodes as compared with LTβ−/− mice, whereas the splenic microarchitecture of LIGHT−/−LTβ−/− and LTβ−/− mice shows a comparable state of disruption. This suggests the existance of an additional undiscovered ligand for the LTβ receptor (LTβR) or a weak LTα3–LTβR interaction in vivo involved in the formation of secondary lymphoid organs. LIGHT acts synergistically with CD28 in skin allograft rejection in vivo. The underlying mechanism was identified in in vitro allogeneic MLR studies, showing a reduced cytotoxic T lymphocyte activity and cytokine production. Detailed analyses revealed that proliferative responses specifically of CD8+ T cells are impaired and interleukin 2 secretion of CD4+ T cells is defective in the absence of LIGHT. Furthermore, a reduced 3[H]-thymidine incorporation after T cell receptor stimulation was observed. This for the first time provides in vivo evidence for a cooperative role for LIGHT and LTβ in lymphoid organogenesis and indicates important costimulatory functions for LIGHT in T cell activation.

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Mesenchymal stromal cells (MSC) are used in cell therapies, however cellular senescence increases heterogeneity of cell populations and leads to uncertainty in therapies’ outcomes. The determination of cellular senescence is time consuming and logistically intensive. Here, we propose the use of endogenous autofluorescence as real-time quantification of cellular senescence in human MSC, based on label-free flow cytometry analysis. We correlated cell autofluorescence to senescence using senescence-associated beta-galactosidase assay (SA-β-Gal) with chromogenic (X-GAL) and fluorescent (C12FDG) substrates, gene expression of senescence markers (such as p16INK4A, p18INK4C, CCND2 and CDCA7) and telomere length. Autofluorescence was further correlated to MSC differentiation assays (adipogenesis, chondrogenesis and osteogenesis), MSC stemness markers (CD90/CD106) and cytokine secretion (IL-6 and MCP-1). Increased cell autofluorescence significantly correlated with increased SA-β-Gal signal (both X-GAL and C12FDG substrates), cell volume and cell granularity, IL-6/MCP-1 secretion and with increased p16INK4A and CCND2 gene expression. Increased cell autofluorescence was negatively associated with the expression of the CD90/CD106 markers, osteogenic and chondrogenic differentiation potentials and p18INK4C and CDCA7 gene expression. Cell autofluorescence correlated neither with telomere length nor with adipogenic differentiation potential. We conclude that autofluorescence can be used as fast and non-invasive senescence assay for comparing MSC populations under controlled culture conditions.

Lsc (the murine homolog of human p115 Rho GEF) is a member of the Dbl-homology family of GTP exchange factors and is a specific activator of Rho. Lsc is activated by the G alpha(13) subunit of heterotrimeric G proteins and contains a regulator of G protein signaling domain that downmodulates G alpha(12) and G alpha(13). Lsc is expressed primarily in the hematopoietic system and links the activation of G alpha(12) and G alpha(13)-coupled receptors to actin polymerization in B and T cells. Lsc is essential for marginal zone B (MZB) cell homeostasis and for the generation of immune responses. Although Lsc-deficient lymphocytes show reduced basal motility, MZB cells show enhanced migration after serum activation. Thus, Lsc is a critical regulator of MZB cells and immune functions.

Background:Two major therapeutic principles can be employed for the treatment of distal femoral fractures: retrograde intramedullary (IM) nailing (RN) or less invasive stabilization on system (LISS). Both operative stabilizing systems follow the principle of biological osteosynthesis. IM nailing protects the soft-tissue envelope due to its minimally invasive approach and closed reduction techniques better than distal femoral locked plating. The purpose of this study was to evaluate and compare outcome of distal femur fracture stabilization using RN or LISS techniques.Materials and Methods:In a retrospective study from 2003 to 2008, we analyzed 115 patients with distal femur fracture who had been treated by retrograde IM nailing (59 patients) or LISS plating (56 patients). In the two cohort groups, mean age was 54 years (17–89 years). Mechanism of injury was high energy impact in 57% (53% RN, 67% LISS) and low-energy injury in 43% (47% RN, 33% LISS), respectively. Fractures were classified according to AO classification: there were 52 type A fractures (RN 31, LISS 21) and 63 type C fractures (RN 28, LISS 35); 32% (RN) and 56% (LISS) were open and 68% (RN) and 44% (LISS) were closed fractures, respectively. Functional and radiological outcome was assessed.Results:Clinical and radiographic evaluation demonstrated osseous healing within 6 months following RN and following LISS plating in over 90% of patients. However, no statistically significant differences were found for the parameters time to osseous healing, rate of nonunion, and postoperative complications. The following complications were treated: hematoma formation (one patient RN and three patients LISS), superficial infection (one patient RN and three patients LISS), deep infection (2 patients LISS). Additional secondary bone grafting for successful healing 3 months after the primary operation was required in four patients in the RN (7% of patients) and six in the LISS group (10% of patients). Accumulative result of functional outcome using the Knee and Osteoarthritis Outcome (KOOS) score demonstrated in type A fractures a score of 263 (RN) and 260 (LISS), and in type C fractures 257 (RN) and 218 (LISS). Differences between groups for type A were statistically insignificant, statistical analysis for type C fractures between the two groups are not possible, since in type C2 and C3 fractures only LISS plating was performed.Conclusion:Both retrograde IM nailing and angular stable plating are adequate treatment options for distal femur fractures. Locked plating can be used for all distal femur fractures including complex type C fractures, periprosthetic fractures, as well as osteoporotic fractures. IM nailing provides favorable stability and can be successfully implanted in bilateral or multisegmental fractures of the lower extremity as well as in extra-articular fractures. However, both systems require precise preoperative planning and advanced surgical experience to reduce the risk of revision surgery. Clinical outcome largely depends on surgical technique rat...

Degeneration of intervertebral discs (IVDs) is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP) to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and posttraumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapymonitoring markers for degenerative disc diseases.

BackgroundHuman bone marrow-derived mesenchymal stem cells (MSC) are adult progenitor cells with great potential for application in cell-based therapies. From a cell-based therapy perspective, there are two limitations to MSC use: (1) these therapies require large numbers of cells, and long-term expansion of MSC in vitro promotes replicative senescence; and (2) patient variability is a challenge for defining MSC quality standards for transplantation. This study aimed to determine whether low or high oxidative status of MSC correlate with changes in cell expansion and differentiation potentials.MethodsWe investigated functional aspects of mitochondria, such as cell metabolic activity indicators and expression of antioxidant enzymes. Furthermore, we tested if senescence-induced changes in oxidative status of MSC could be counteracted by methylene blue (MB), an alternative mitochondrial electron transfer known to enhance cell bioenergetics.ResultsMSC isolated from donors of the same age showed distinctive behavior in culture and were grouped as weak (low colony-forming units (CFU) and a short life in vitro) and vigorous MSC (high CFU and a long life in vitro). In comparison to weak MSC, vigorous MSC had oxidative status characterized by lower mitochondrial membrane potential, lower mitochondrial activity, and fewer reactive oxygen species production, as well as reduced mitochondrial biogenesis. Vigorous MSC had a significantly higher expansion potential compared to weak MSC, while no differences were observed during differentiation. MB treatment significantly improved expansion and differentiation potential, however only in vigorous MSC.ConclusionsTogether, these results demonstrate the importance of mitochondrial function in MSC in vitro, and that cells with low oxidative status levels are better candidates for cell-based therapies.

Mesenchymal stem cells (MSCs) are expected to have a fundamental role in future cell-based therapies because of their high proliferative ability, multilineage potential, and immunomodulatory properties. Autologous transplantations have the "elephant in the room" problem of wide donor variability, reflected by variability in MSC quality and characteristics, leading to uncertain outcomes in the use of these cells. We propose life imaging as a tool to characterize populations of human MSCs. Bone marrow MSCs from various donors and in vitro passages were evaluated for their in vitro motility, and the distances were correlated to the adipogenic, chondrogenic, and osteogenic differentiation potentials and the levels of senescence and cell size. Using life-image measuring of track lengths of 70 cells per population for a period of 24 hours, we observed that slow-moving cells had the higher proportion of senescent cells compared with fast ones. Larger cells moved less than smaller ones, and spindleshaped cells had an average speed. Both fast cells and slow cells were characterized by a low differentiation potential, and average-moving cells were more effective in undergoing all three lineage differentiations. Furthermore, heterogeneity in single cell motility within a population correlated with the average-moving cells, and fast-and slow-moving cells tended toward homogeneity (i.e., a monotonous moving pattern). In conclusion, in vitro cell motility might be a useful tool to quickly characterize and distinguish the MSC population's differentiation potential before additional use. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:84-90