Spinal release of tumour necrosis factor activates c-Jun N-terminal kinase and mediates inflammation-induced hypersensitivity.

BackgroundMounting evidence points to individual contributions of tumour necrosis factor-alpha (TNF) and the c-Jun N-terminal kinase (JNK) pathway to the induction and maintenance of various pain states. Here we explore the role of spinal TNF and JNK in carrageenan-induced hypersensitivity. As links between TNF and JNK have been demonstrated in vitro, we investigated if TNF regulates spinal JNK activity in vivo.MethodsTNF levels in lumbar cerebrospinal fluid (CSF) were measured by enzyme-linked immunosorbent assay, spinal TNF gene expression by real-time polymerase chain reaction and TNF protein expression, JNK and c-Jun phosphorylation by western blotting. The role of spinal TNF and JNK in inflammation-induced mechanical and thermal hypersensitivity was assessed by injecting the TNF inhibitor etanercept and the JNK inhibitors SP600125 and JIP-1 intrathecally (i.t.). TNF-mediated regulation of JNK activity was examined by assessing the effect of i.t. etanercept on inflammation-induced spinal JNK activity.ResultsTNF levels were increased in CSF and spinal cord following carrageenan-induced inflammation. While JNK phosphorylation followed the same temporal pattern as TNF, c-jun was only activated at later time points. Intrathecal injection of TNF and JNK inhibitors attenuated carrageenan-induced mechanical and thermal hypersensitivity. TNF stimulation induced JNK phosphorylation in cultured spinal astrocytes and blocking the spinal actions of TNF in vivo by i.t. injection of etanercept reduced inflammation-induced spinal JNK activity.ConclusionsHere we show that spinal JNK activity is dependent on TNF and that both TNF and the JNK signalling pathways modulate pain-like behaviour induced by peripheral inflammation

Termination of psychotherapy: The journey of ten psychoanalytic and psychodynamic therapists

Objectives: Literature on termination originates mainly from clinical and theoretical accounts as well as practitioners' autobiographical reports. There is, however, a paucity of psychological research on termination, and studies of therapists' experiences are particularly rare. The purpose of this study is to examine the process of termination of therapy based on therapists' narratives of experiences of endings with patients. Design: Grounded Theory methodology has been applied in this study in order to conceptualise the process of termination from the therapist’s perspective. Methods: Ten psychoanalytic and psychodynamic therapists were interviewed for this study. Results: Grounded Theory analysis of the data revealed five central categories: therapist as a person, therapist's awareness of termination, development of therapeutic relationship, working through termination, and the aftermath (post-termination phase). Conclusions: The results offer a Grounded Theory model of the therapist's journey through termination of therapy with patients. Subcategories and their relationships will be explored. Implications for clinical practice, limitations and suggestions for further research will be discussed

Targeted delivery of anti-inflammatory therapy to rheumatoid tissue by fusion proteins containing an IL-4-linked synovial targeting peptide

We provide first-time evidence that the synovial endothelium-targeting peptide (SyETP) CKSTHDRLC successfully delivers conjugated IL-4 to human rheumatoid synovium transplanted into SCID mice. SyETP, previously isolated by in vivo phage display and shown to preferentially localize to synovial xenografts, was linked by recombinant technology to hIL-4 via an MMP-cleavable sequence. Both IL-4 and the MMP-cleavable sequence were shown to be functional. IL-4-SyETP augmented production of IL-1ra by synoviocytes stimulated with IL-1[beta] in a dose-dependent manner. In vivo imaging confirmed increased retention of SyETP-linked-IL-4 in synovial grafts which was enhanced by increasing number of copies (one to three) in the constructs. Strikingly, SyETP delivered bioactive IL-4 in vivo as demonstrated by increased pSTAT6 in synovial grafts. Thus, this study provides proof of concept for peptide-tissue-specific targeted immunotherapy in rheumatoid arthritis. This technology is potentially applicable to other biological therapies providing enhanced potency to inflammatory sites and reducing systemic toxicity

Somatostatin Regulates the Extracellular Regulated Kinase Cascade of Human Rheumatoid Arthritis Synoviocytes

Somatostatin (SRIF, somatotropin release inhibitory factor) is a ubiquitously expressed neuropeptide, interacting with cells via five SRIF receptor subtypes (sst.-sst.), belonging to the guanine nucleotide binding protein-coupled receptor (GPCR) superfamily. SRIF receptors have been well documented for their ability to inhibit cell proliferation and secretion in a variety of animal tissues. Over the past l S years, pre-clinical and clinical studies implicate that SRIF and SRIF analog therapy improve symptoms in patients suffering from rheumatoid arthritis (RA). In this study, we investigate SRIF\u27s effects on intracellular signaling in the synovium, the cellular layer that lines synovial joints, using in vitro cultures of human synovial fibroblasts. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of synovial isolated mRNA established the presence of sst, in these cells. The sst, receptor links somatostatin action to control of synovial intracellular signaling. We have examined SRIF effects on the synoviocyte extracellular regulated kinase pathway (ERK). Using phospho-specific antisera, we demonstrate that SRIF and SRIF analogs decrease phospho-ERK.1/2 levels, suggesting basal ERK.1/2 is upregulated in RA synoviocytes. SRIF also suppressed basal levels of activated Raf and MEKl/2, two upstream regulatory kinases ofERKl/2. Furthermore, SRIF suppressed 1NFa­ stimulated activation ofERK.112 in RA synoviocytes. We also observed that SRIF increases a sodium vanadate-sensitive intracellular protein phosphatase activity in RA synoviocytes, indicating a possible intracellular mechanism by which SRIF controls the phosphorylation status of the ERK.112 kinases. Fluorescent confocal scanning microscopy reveals that TNF-o. stimulated the localization of phospho-ERK.112 in the nuclei of RA synoviocytes, which was inhibited upon co-treatment with SRIF. Taken together our results demonstrate that SRIF regulates intracellular signaling in rheumatoid synoviocytes

Somatostatin Regulates the Extracellular Regulated Kinase Cascade of Human Rheumatoid Arthritis Synoviocytes

Somatostatin (SRIF, somatotropin release inhibitory factor) is a ubiquitously expressed neuropeptide, interacting with cells via five SRIF receptor subtypes (sst.-sst.), belonging to the guanine nucleotide binding protein-coupled receptor (GPCR) superfamily. SRIF receptors have been well documented for their ability to inhibit cell proliferation and secretion in a variety of animal tissues. Over the past l S years, pre-clinical and clinical studies implicate that SRIF and SRIF analog therapy improve symptoms in patients suffering from rheumatoid arthritis (RA). In this study, we investigate SRIF\u27s effects on intracellular signaling in the synovium, the cellular layer that lines synovial joints, using in vitro cultures of human synovial fibroblasts. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of synovial isolated mRNA established the presence of sst, in these cells. The sst, receptor links somatostatin action to control of synovial intracellular signaling. We have examined SRIF effects on the synoviocyte extracellular regulated kinase pathway (ERK). Using phospho-specific antisera, we demonstrate that SRIF and SRIF analogs decrease phospho-ERK.1/2 levels, suggesting basal ERK.1/2 is upregulated in RA synoviocytes. SRIF also suppressed basal levels of activated Raf and MEKl/2, two upstream regulatory kinases ofERKl/2. Furthermore, SRIF suppressed 1NFa­ stimulated activation ofERK.112 in RA synoviocytes. We also observed that SRIF increases a sodium vanadate-sensitive intracellular protein phosphatase activity in RA synoviocytes, indicating a possible intracellular mechanism by which SRIF controls the phosphorylation status of the ERK.112 kinases. Fluorescent confocal scanning microscopy reveals that TNF-o. stimulated the localization of phospho-ERK.112 in the nuclei of RA synoviocytes, which was inhibited upon co-treatment with SRIF. Taken together our results demonstrate that SRIF regulates intracellular signaling in rheumatoid synoviocytes

Citrullination – small change with a great consequence

Citrullination is one of the possible post-translational modifications of proteins. It is based on a conversion of L-arginine residue (L-Arg) to L-citrulline residue (L-Cit). The reaction is catalyzed by peptidylarginine deiminases (PAD). The change of L-Arg imino moiety results in a loss of a positive charge. This slight modification can contribute to significant changes in physicochemical properties of proteins, which may also cause a change of their functions. Citrullination is the modification observed in physiological processes such as epidermal keratinization, regulation of gene expression and the reorganization of myelin sheaths. The changes in the efficacy of citrullination may contribute to the pathogenesis of many different diseases including: psoriasis, multiple sclerosis, rheumatoid arthritis and cancer

Expression and regulation of VCAM-1 and CD44 by cultured fibroblast-like synoviocytes.

The fibroblast-like synoviocyte (FLS) has been implicated in the destructive process associated with rheumatoid arthritis. In this thesis I demonstrate the expression and regulation of several adhesion molecules expressed on cultured FLSs obtained from inflamed synovium. Unlike fibroblasts from other areas of the body, FLSs constitutively express vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 on RA FLSs is constitutively expressed at high levels during the first 2 weeks of culture. At later time points (4 weeks in culture), VCAM-1 expression declined to low basal levels. A number of strategies were employed to determine the exogenous factors that determine the initially high levels of VCAM-1 in FLSs. Of the extracellular matrix components examined only collagen type I enhanced VCAM-1 expression but this had only limited success. The pro-inflammatory cytokines, IL-1β (10 ng/ml) and TNF-α (10 ng/ml), were also tested and found to induce only transient increases in cell surface VCAM-1 expression. However, the chronic administration of IL-4 or IL-13 in combination with TNF-α resulted in elevated levels of VCAM-1 with prolonged expression Prolonged VCAM-1 expression was found to result in part from the capacity of IL-4 and IL-13 to stabilize VCAM-1 mRNA transcripts. CD44 splice variants, isoforms of the CD44 receptor, that are implicated in the progression of a number of human tumours were also expressed by FLSs isolated from inflamed synovium. Immunohistochemistry, flow cytometry and RT-PCR analysis of CD44 splice variant expression revealed differential expression of a number of variant isoforms. Splice variant expression, at both the mRNA and cell surface protein level, was observed in a large percentage of RA FLSs, it is variable in those from OA synovium, and is absent in cells isolated from non-inflamed joints. However, RA FLSs showed a greater intensity of staining for variants v3, v5 and v7/8. VCAM-1-positive FLSs also demonstrated complex splice variant mRNA transcripts, comprising v3, v6, v7, v8, v9 and v10 in a variety of splicing combinations. These results indicate that the nature of CD44-splice variant expression is closely linked to the inflammatory state of the synovial joint. Moreover, expression of the CD44v7/8 epitope is associated with an increased cellular proliferation rate and could thus be functionally implicated in the hyperplasia observed in RA synovium

Up the nose of the beholder? Aesthetic perception in olfaction as a decision-making process

Is the sense of smell a source of aesthetic perception? Traditional philosophical aesthetics has centered on vision and audition but eliminated smell for its subjective and inherently affective character. This article dismantles the myth that olfaction is an unsophisticated sense. It makes a case for olfactory aesthetics by integrating recent insights in neuroscience with traditional expertise about flavor and fragrance assessment in perfumery and wine tasting. My analysis concerns the importance of observational refinement in aesthetic experience. I argue that the active engagement with stimulus features in perceptual processing shapes the phenomenological content, so much so that the perceptual structure of trained smelling varies significantly from naive smelling. In a second step, I interpret the processes that determine such perceptual refinement in the context of neural decision-making processes, and I end with a positive outlook on how research in neuroscience can be used to benefit philosophical aesthetics