Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5 % (non-injured) or 30 % (injured) of disc height. 30 % strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured-IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROα, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.

Development of the axial skeleton is a complex, stepwise process that relies on intricate signaling and coordinated cellular differentiation. Disruptions to this process can result in a myriad of skeletal malformations that range in severity. The notochord and the sclerotome are embryonic tissues that give rise to the major components of the intervertebral discs and the vertebral bodies of the spinal column. Through a number of mouse models and characterization of congenital abnormalities in human patients, various growth factors, transcription factors, and other signaling proteins have been demonstrated to have critical roles in the development of the axial skeleton. Balance between opposing growth factors as well as other environmental cues allows for cell fate specification and divergence of tissue types during development. Furthermore, characterization of progenitor cells for specific cell lineages has furthered the understanding of specific spatiotemporal cues that cells need in order to initiate and complete development of distinct tissues. Identifying specific marker genes that can distinguish between the various embryonic and mature cell types is also of importance. Clinically, understanding developmental clues can aid in the generation of therapeutics for musculoskeletal disease through the process of developmental engineering. Studies into potential stem cell therapies are based on knowledge of the normal processes that occur in the embryo, which can then be applied to stepwise tissue engineering strategies.

Purpose of review Intervertebral discs (IVD) are derived from embryonic notochord and sclerotome. The nucleus pulposus is derived from notochord while other connective tissues of the spine are derived from sclerotome. This manuscript will review the past 5 years of research into IVD development. Recent findings Over the past several years, advances in understanding the step-wise process that govern development of the nucleus pulposus and the annulus fibrosus have been made. Generation of tissues from induced or embryonic stem cells into nucleus pulposus and paraxial mesoderm derived tissues has been accomplished in vitro using pathways identified in normal development. A balance between BMP and TGF-β signaling as well as transcription factors including Pax1/Pax9, Mkx and Nkx3.2 appear to be very important for cell fate decisions generating tissues of the IVD. Summary Understanding how the IVD develops will provide the foundation for future repair, regeneration, and tissue engineering strategies for IVD disease.

We previously demonstrated that the naturally occurring splice variant stromal cell-derived factor 1␥/ CXCL12␥ is the most potent CXCL12 isoform in blocking X4 HIV-1, with weak chemotactic activity. A conserved BBXB domain (B for basic and X for any residue) located in the N terminus ( 24 KHLK 27 ) is found in all six isoforms of CXCL12. To determine whether the potent antiviral activity of CXCL12␥ is due to the presence of the extra C-terminal BBXB domains, we mutated each domain individually as well as in combination. Although binding of CXCL12␥ to heparan sulfate proteoglycan (HSPG) was 10-fold higher than that observed with CXCL12␣, the results did not demonstrate a direct correlation between HSPG binding and the potent antiviral activity. CXCL12␥ mutants lacking the conserved BBXB domain (designated ␥B1) showed increased binding to HSPG but reduced anti-HIV activity. In contrast, the mutants lacking the C-terminal second and/or third BBXB domain but retaining the conserved domain (designated B2, B3, and B23) showed decreased binding to HSPG but increased anti-HIV activity. The B2, B3, and B23 mutants were associated with enhanced CXCR4 binding, receptor internalization, and restored chemotaxis. Internalization of CXCR4 was more potent with CXCL12␥ than with CXCL12␣ and was significantly reduced when the conserved BBXB domain was mutated. We concluded that the observed potent anti-HIV-1 activity of CXCL12␥ is due to increased affinity for CXCR4 and to efficient receptor internalization.Chemokines are small, structurally related chemoattractant cytokines characterized by conserved cysteine residues. Based on the positions of the first N-terminal cysteines, chemokines fall into four subfamilies. The CC and CXC subfamilies have been well characterized. The CC subfamily includes the following: regulated on activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory peptides 1 (MIP-1). The prototype of the CXC subfamily is interleukin-8 (IL-8)/ CXCL8. The C chemokine (lymphotactine) and CX3C chemokine (fractalkine) subfamilies were recently identified (reviewed in reference 30). The physiological activities of chemokines are mediated by the selective recognition and activation of chemokine receptors belonging to the seven-membrane-domain G-protein-coupled receptor superfamily (GPCRs). In addition, chemokines also bind to glycosaminoglycans (GAGs) through distinct binding sites. Chemokine binding to GAGs on cells, particularly endothelial cells, results in chemotactic chemokine gradients that allow correct presentation of chemokines to leukocytes, therefore enabling target cells to cross the endothelial barrier and migrate into tissues (reviewed in reference 10).Stromal cell-derived factor 1 (SDF-1)/CXCL12 is a member of the CXC chemokine family and is a key regulator of B-cell lymphopoiesis, hematopoietic stem cell mobilization, and leukocyte migration (reviewed in reference 10). CXCL12 was originally thought to mediate these processes through...

Recent studies have demonstrated that neuropilin 1 (NP-1) is involved in HTLV-1 entry; however, the role NP-1 plays in this process is not understood. We demonstrated that ectopic expression of human NP-1 but not NP-2 cDNA increased susceptibility to HTLV-1. SiRNA-mediated inhibition of NP-1 expression correlated with significant reduction of HTLV-1 Env-mediated fusion. The vascular endothelial growth factor (VEGF165) caused downmodulation of surface NP-1 and inhibited HTLV-1 infection of U87 cells. In contrast, VEGF165 partially inhibited infection of primary astrocytes and had no significant effect on infection of HeLa cells. VEGF165 and antibodies to the glucose transporter protein 1 (anti-GLUT-1) were both needed to block infection of primary astrocytes, however, only anti-GLUT-1 antibodies were sufficient to block infection of HeLa cells. HTLV-1 Env forms complexes with both NP-1 and GLUT-1 in primary human astrocytes. The alternate usage of these two cellular receptors may have important implications regarding HTLV-1 neuro-tropism.

Background Members of the transforming growth factor beta (TGF‐β) family are secreted proteins that regulate skeletal development. TGF‐β signaling is critical in embryonic development of the annulus fibrosus (AF) of the intervertebral disc (IVD). To address the question of the role of TGF‐β signaling in postnatal development and maintenance of the skeleton, we generated mice in which Tgfbr2 was deleted at 2‐weeks of age in Aggrecan (Acan)‐expressing cells using inducible Cre/LoxP recombination. Methods Localization of Cre recombination was visualized by crossing Acan tm1(cre/ERT2)Crm mice to fluorescent mTmG reporter mice. Acan tm1(cre/ERT2)Crm mice were mated to Tgfbr2 LoxP/LoxP mice and Cre recombinase was activated by tamoxifen injection at 2‐weeks postnatally. Following tamoxifen injection, mice were aged to 3, 6, and 12‐months and control mice were compared to the experimental (cKO) group. Mice were initially analyzed using X‐ray and skeletal preparations. Sternocostal joints and IVD tissues were further analyzed histologically by hematoxylin and eosin (H&E), Safranin O, and Picrosirius Red staining as well as Col10 immunostaining. Results Cre recombination was observed in the IVD and sternocostal joints. X‐ray analysis revealed osteophyte formation within the disc space of 12‐month‐old cKO mice. Skeletal preparations confirmed calcification within the IVD and the sternocostal joints in cKO mice. H&E staining of cKO IVD revealed disorganized growth plates, delay in the formation of the bony endplate, and Col10 staining in the AF indicative of ectopic endochondral bone formation. Furthermore, proteoglycan loss was observed and collagen bundles within the inner AF were thinner and less organized. Alterations in the IVD were apparent beginning at 3 months and were progressively more visible at 6 and 12 months. Similarly, histological analysis of cKO sternocostal joints revealed joint calcification, proteoglycan loss, and disorganization of the collagen architecture at 12 months of age. Conclusions TGF‐β signaling is important for postnatal development and maintenance of fibrocartilaginous IVD and sternocostal joints.

Presently, there are no established treatments to prevent, stop or even retard back pain arising from disc degeneration. Previous studies have shown that Link N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens, in IVD. However, the sequences in Link N involved in modulating cellular activity are not well understood. To determine if disc cells can proteolytically process Link N, human disc cells were exposed to native Link N over a 48 h period and mass spectrometric analysis revealed that a peptide spanning residues 1-8 was generated in the presence of AF cells but not NP cells. Link N 1-8 significantly induced proteoglycan production in the presence of IL-1b NP and AF cells, confirming that the biological effect is maintained in the first 8 amino acids of the peptide and indicating that the effect is sustained in an inflammatory environment. Thus Link-N 1-8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration. ß

Introduction Intervertebral disk (IVD) degeneration has been strongly associated with and named a major cause of back pain. At present, little is known about the molecular mechanisms involved in the degeneration of IVD and how these may differ from normal turnover of the tissue. As a result of this, a biomarker for disk degeneration has not yet been identified and we propose chondroadherin (CHAD) fragmentation as a potential option. CHAD, a protein of the leucine rich repeat (LRR) family, is one of the proteins predominantly expressed in the extracellular matrix of cartilaginous tissue, including that of the IVD. This restricted distribution is unusual among LRR proteins, which commonly show a wide tissue distribution. CHAD is primarily found close to the cells, where it can interact with collagen fibrils of the ECM and molecules at the cell surface, providing a mechanism for regulating cell metabolism and ECM structure. These interactions may also aid in promoting matrix homeostasis, and variation in CHAD abundance or structure might therefore lead to pathological changes in the tissue over time. The aims of this study were to determine whether CHAD fragmentation occurs and is unique to disk degeneration, and to characterize the cleavage site within CHAD at which fragmentation occurs. Materials and Methods Healthy and degenerate lumbar IVDs were obtained through organ donations via Transplant Quebec. IVDs from patients with degenerative disk disease and from patients with scoliosis were obtained at the time of surgery. Punches of 4 mm were taken and disk tissue then extracted using 15 volumes of extraction buffer (4 M GuCl, 10 mM EDTA, COMPLETE, 50 mM NaAc, pH 5.8) on a wet weight per volume basis. Extracted proteins were ethanol precipitated, and CHAD fragmentation was studied using SDS-PAGE and western blotting in combination with specific antibodies. To characterize the CHAD cleavage site, a degenerate disk extract was subjected to CsCl density gradient centrifugation to remove proteoglycans. CHAD was purified from the protein fraction by chromatography through carboxymethyl 52. The CHAD-containing samples were then fractionated on an SDS-PAGE gel and stained with Coomassie Blue. Gel portions containing the CHAD fragment were excised, then lyophilized, reduced and alkylated, and subjected to trypsin digestion. Peptides were then identified by liquid chromatography mass spectrometry. Antineoepitope antibodies specifically recognizing the fragmented CHAD were generated by immunizing rabbits with synthetic peptides conjugated to KLH. The peptides represented the terminal amino acid sequences at the site of CHAD fragmentation. Results Proteolytic fragmentation of CHAD was observed in IVDs from patients with DDD and in some individuals with adolescent idiopathic scoliosis (AIS). Its presence appeared to be related to the degree of degeneration in both cases (Fig. 1). This phenomenon was found to be specific to disk degeneration, as CHAD fragmentation was not observed in healthy adolescent and adult disks from...