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We previously demonstrated that VEGF and its receptors were expressed in human herniated discs (HD). TNF-a induced VEGF, resulting in neovascularization of disc tissues in a model of HD. The goal of the current research was to investigate the precise role of TNF-a-induced VEGF and the mechanism of angiogenesis in disc tissues. We performed ELISAs, Western blots, and immunohistological examinations to assess the role of TNF-a-induced VEGF using organ disc cultures with wild type, TNF receptor 1-null (TNF-RI null ), or TNF receptor 2-null (TNF-RII null ) mice. VEGF induction was inhibited when we used TNF-RI null -derived disc tissues. NF-kB pathway inhibitors also strongly suppressed VEGF induction. Thus, TNF-a induced VEGF expression in disc cells primarily through the NF-kB pathway. In addition, VEGF immunoreactivity was detected predominantly in annulus fibrosus cells and increased after TNF-a stimulation. TNF-a treatment also resulted in CD31 expression on endothelial cells and formation of an anastomosing network. In contrast, angiogenic activity was strongly inhibited in the presence of NF-kB inhibitors or anti-VEGF antibody. Our data show angiogenesis activity in disc tissues is regulated by VEGF and the NF-kB pathway, both of which are induced by TNF-a. The level of angiogenic activity in disc tissues was closely related to aging. Because neovascularization of HD is indispensable for HD resorption, the prognosis of HD and the rate of the resorption process in patients may vary as a function of the patient's age. ß

Introduction: (1) To evaluate the influence of pedicle screw loosening on clinical outcomes; (2) to clarify the association between the pull-out length and screw loosening 1 year after surgery; and (3) to determine radiographically which screw parameters predominantly influence the pull-out resistance of screws. Methods: We analyzed 32 consecutive patients who underwent minimally invasive lumbar or thoracic spinal stabilization by intraoperative three-dimensional computed tomography (CT)-guided navigation without anterior reconstruction and were followed up for 1 year. The screw pull-out length was measured on axial CT images obtained both immediately after screw insertion and postoperatively. Loosening of screws and clinical outcomes were evaluated radiographically, clinically, and by CT 1 year after surgery. Results: There were no significant differences in the mean age, sex, bone mineral density, mean stabilized length, and smoking habits of patients with (+) or without (−) loosening. The Oswestry Disability Index and the lumbar visual analog scale 1 year after surgery were significantly higher in patients with loosening (+) than in those without (−). The overall pedicle screw pull-out rate was 16.2% (47/290) of screws and the overall screw loosening rate was 15.2% (44/290) of screws. Screws with loosening (+) had significantly lower (axial) trajectory angles and higher screw pull-out lengths than those without (−). Approximately 82% of loosened screws had been pulled out during rod connection. Conclusions: A lower axial trajectory and an increased screw pull-out length after rod reduction are crucial risk factors for screw loosening.

Objective. To determine whether thymic stromal lymphopoietin (TSLP) plays a role in the resorption of herniated disc tissue.Methods. The expression of TSLP messenger RNA (mRNA) and protein in mouse intervertebral disc cells was assessed by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical analysis. The ability of mouse intervertebral disc cells to respond to TSLP stimulation was examined by Western blot analysis, ELISA, and protein array analysis. Intracellular signaling pathways involved in TSLP signaling in mouse intervertebral disc cells were investigated using several chemical inhibitors. The role of TSLP in macrophage migration into the intervertebral disc was assessed by in vitro migration assay. Finally, TSLP expression in clinical specimens derived from patients with a herniated disc was examined by immunohistochemistry.Results. Mouse intervertebral disc cells expressed TSLP mRNA and protein upon stimulation with NF-B-activating ligands such as tumor necrosis factor ␣. In addition, the mouse intervertebral disc cells expressed the TSLP receptor and produced monocyte chemotactic protein 1 (MCP-1; CCL2) and macrophage colony-stimulating factor in response to TSLP stimulation. Both anulus fibrosus and nucleus pulposus intervertebral disc cells expressed MCP-1 upon TSLP stimulation, which was mediated via the phosphatidylinositol 3-kinase/Akt pathway. Consistently, the supernatants of TSLP-activated intervertebral disc cultures had the capacity to induce macrophage migration in an MCP-1-dependent manner. Finally, TSLP and MCP-1 were coexpressed in human herniated disc specimens in which macrophage infiltration into the tissue was observed.Conclusion. TSLP induced by NF-B-activating ligands in intervertebral discs may contribute to the recruitment of macrophages to the intervertebral disc by stimulating MCP-1 production and may be involved in the resorption of herniated disc tissue.

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Subtilase cytotoxin (SubAB) is the prototype of a newly identified family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. SubAB specifically cleaves the essential endoplasmic reticulum (ER) chaperone BiP (GRP78), resulting in the activation of ER stress-induced unfolded protein response (UPR). We have recently shown that the UPR following ER stress can suppress cellular responses to inflammatory stimuli during the later phase, in association with inhibition of NF-κB activation. These findings prompted us to hypothesize that SubAB, as a selective UPR inducer, might have beneficial effects on inflammation-associated pathology via a UPR-dependent inhibition of NF-κB activation. The pretreatment of a mouse macrophage cell line, RAW264.7, with a subcytotoxic dose of SubAB-triggered UPR and inhibited LPS-induced MCP-1 and TNF-α production associated with inhibition of NF-κB activation. SubAA272B, a SubAB active site mutant that cannot induce UPR, did not show such effects. In addition, pretreatment with a sublethal dose of SubAB, but not SubAA272B, protected the mice from LPS-induced endotoxic lethality associated with reduced serum MCP-1 and TNF-α levels and also prevented the development of experimental arthritis induced by LPS in mice. Collectively, although SubAB has been identified originally as a toxin associated with the pathogenesis of hemolytic uremic syndrome, the unique ability of SubAB to selectively induce the UPR may have the potential to prevent LPS-associated inflammatory pathology under subcytotoxic conditions.

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine produced by epithelial cells and triggers dendritic cell-mediated Th2 type allergic inflammatory responses. This study investigated whether Toll-like receptor (TLR) ligands, lipopolysaccharide (LPS) and poly-IC affect TSLP production in synovial fibroblasts. Enzyme-linked immunosorbent assay showed that LPS and poly-IC upregulated TSLP production in synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). In addition, we found that nuclear factor (NF)-kappaB inhibitor IMD-0354, dexamethasone, and interferon (IFN)-gamma inhibited the LPS- and poly-IC-induced TSLP production in RA and OA synovial fibroblasts. Thus, LPS and poly-IC can upregulate TSLP via a NF-kappaB pathway in synovial fibroblasts, which is downregulated by dexamethasone and interferon (IFN)-gamma. The current findings suggest that TSLP may be involved in the pathophysiology of inflammatory arthritis as well as allergic disease.