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ABSTRACT: STRUCTURED ABSTRACT: Study Design. We measured the expression and responses of Toll-Like Receptor 4 (TLR4) activation in the intervertebral disc (IVD) in vitro and in vivo. We hypothesize that stimulation of the IVD with the TLR4 ligand lipopolysaccharide (LPS) results in upregulation of a coordinated set of pro-inflammatory mediators and inhibition of matrix expression, both consistent with a molecular profile of degeneration.Objective. To characterize early inflammatory and morphological changes induced by TLR4 activation in the IVD.Summary of Background Data. TLR4 is a pattern recognition receptor activated in innate immunity that has been implicated in disease mechanisms of inflammatory cartilaginous degeneration. However, no study to date has examined the expression and responses of TLR4 in the IVD.Methods. IVD cells were stimulated with LPS in a dose-dependent manner, and inflammatory cytokine levels measured by quantitative RT-PCR. Histological and inflammatory changes due to in vivo injection of LPS into the rat caudal IVD were measured by ELISA and immunoblotting.Results. Baseline TLR4 expression in IVD tissue varied according to cell type. LPS stimulation resulted in significant increases in TNF-α, IL-1β, IL-6, and NO levels and significant inhibition in aggrecan and collagen-2. Intradiscal injection of LPS was found to cause moderate degenerative changes in the IVD, with increases in tissue levels of IL-1β, TNF-α, HMGB1 and MIF.Conclusions. This study provides the first evidence that IVD cells express TLR4, and are responsive to TLR4 activation by upregulating a coordinated set of inflammatory cytokines. This study suggests that intradiscal injection of LPS offers a model for triggering inflammation of the IVD, demonstrating that inflammatory insults alone may potentially trigger degenerative changes of the IVD.
Spine 07/2012; · 2.08 Impact Factor
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ABSTRACT: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of pathogenic autoantibodies, many of which are directed against nuclear antigens, in particular double-stranded (ds) DNA. Both clinical studies and animal models have shown that anti-dsDNA antibodies contribute to kidney disease, which is present in 50% of lupus patients and is a major cause of mortality. We previously demonstrated that a subset of nephrotoxic anti-dsDNA antibodies also recognizes the pentapeptide consensus sequence D/E W D/E Y S/G (DWEYS) present in the NR2A and NR2B subunits of the N-methyl-d-aspartate receptor (NMDAR). Autoantibodies with this specificity are present in ≈40% of lupus patient sera and are both nephrotoxic and neurotoxic. Elevated titers are present in cerebrospinal fluid of patients with central nervous system manifestations of SLE. Administration of the nonnaturally occurring D form of the DWEYS pentapeptide prevents these antibodies from depositing in glomeruli and from mediating neuronal excitotoxicity. To craft a more useful therapeutic, we used the structural features of the DWEYS peptide to design a unique, selective, and potent small molecule peptidomimetic, FISLE-412, which neutralizes anti-dsDNA/NMDAR lupus autoantibodies and prevents their pathogenic interaction with tissue antigens. This compound, or others derived from it, may provide a unique strategy for the development of lupus therapeutics.
Proceedings of the National Academy of Sciences 06/2011; 108(25):10255-9. · 9.68 Impact Factor
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ABSTRACT: The adaptive immune response is initiated by the presentation of peptides bound to major histocompatibility complex molecules on dendritic cells (DCs) to antigen-specific T lymphocytes at a junction termed the immunological synapse. Although much attention has been paid to cytoplasmic events on the T cell side of the synapse, little is known concerning events on the DC side. We have sought signal transduction components of the neuronal synapse that were also expressed by DCs. One such protein is spinophilin, a scaffolding protein of neuronal dendritic spines that regulates synaptic transmission. In inactive, immature DCs, spinophilin is located throughout the cytoplasm but redistributes to the plasma membrane upon stimulus-induced maturation. In DCs interacting with T cells, spinophilin is polarized dynamically to contact sites in an antigen-dependent manner. It is also required for optimal T cell activation because DCs derived from mice lacking spinophilin exhibit defects in antigen presentation both in vitro and in vivo. Thus, spinophilin may play analogous roles in information transfer at both neuronal and immunological synapses.
The Journal of Cell Biology 05/2008; 181(2):203-11. · 10.26 Impact Factor
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ABSTRACT: The maturation of dendritic cells (DCs) after exposure to microbial products or inflammatory mediators plays a critical role in initiating the immune response. We found that maturation can also occur under steady-state conditions, triggered by alterations in E-cadherin-mediated DC-DC adhesion. Selective disruption of these interactions induced the typical features of DC maturation including the upregulation of costimulatory molecules, MHC class II, and chemokine receptors. These events were triggered at least in part by activation of the beta-catenin pathway. However, unlike maturation induced by microbial products, E-cadherin-stimulated DCs failed to release immunostimulatory cytokines, exhibiting an entirely different transcriptional profile. As a result, E-cadherin-stimulated DCs elicited an entirely different T cell response in vivo, generating T cells with a regulatory as opposed to an effector phenotype. These DCs induced tolerance in vivo and may thus contribute to the elusive steady-state "tolerogenic DCs."
Immunity 11/2007; 27(4):610-24. · 21.64 Impact Factor
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ABSTRACT: During the past decade, many molecular components of clathrin-mediated endocytosis have been identified and proposed to play various hypothetical roles in the process [Nat. Rev. Neurosci. 1 (2000) 161; Nature 422 (2003) 37]. One limitation to the evaluation of these hypotheses is the efficiency and resolution of immunolocalization protocols currently in use. In order to facilitate the evaluation of these hypotheses and to understand more fully the molecular mechanisms of clathrin-mediated endocytosis, we have developed a protocol allowing enhanced and reliable subcellular immunolocalization of proteins in synaptic endocytic zones in situ. Synapses established by giant reticulospinal axons in lamprey are used as a model system for these experiments. These axons are unbranched and reach up to 80-100 microm in diameter. Synaptic active zones and surrounding endocytic zones are established on the surface of the axonal cylinder. To provide access for antibodies to the sites of synaptic vesicle recycling, axons are lightly fixed and cut along their longitudinal axis. To preserve the ultrastructure of the synaptic endocytic zone, antibodies are applied without the addition of detergents. Opened axons are incubated with primary antibodies, which are detected with secondary antibodies conjugated to gold particles. Specimens are then post-fixed and processed for electron microscopy. This approach allows preservation of the ultrastructure of the endocytic sites during immunolabeling procedures, while simultaneously achieving reliable immunogold detection of proteins on endocytic intermediates. To explore the utility of this approach, we have investigated the localization of a GTPase, dynamin, on clathrin-coated intermediates in the endocytic zone of the lamprey giant synapse. Using the present immunogold protocol, we confirm the presence of dynamin on late stage coated pits [Nature 422 (2003) 37] and also demonstrate that dynamin is recruited to the coat of endocytic intermediates from the very early stages of the clathrin coat formation. Thus, our experiments show that the current pre-embedding immunogold method is a useful experimental tool to study the molecular mechanisms of synaptic vesicle recycling.
Journal of Neuroscience Methods 06/2004; 135(1-2):169-74. · 1.98 Impact Factor
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ABSTRACT: It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.
The Journal of Cell Biology 06/2003; 161(4):737-47. · 10.26 Impact Factor
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ABSTRACT: Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster.
Proceedings of the National Academy of Sciences 11/2002; 99(22):14476-81. · 9.68 Impact Factor
