Engineering of TIMP-3 as a LAP-fusion protein for targeting to sites of inflammation

Tissue inhibitor of metalloproteinase (TIMP)‐3 is a natural inhibitor of a range of enzymes that degrade connective tissue and are involved in the pathogenesis of conditions such as arthritis and cancer. We describe here the engineering of TIMP‐3 using a novel drug‐delivery system known as the ‘LAP technology’. This involves creating therapeutic proteins in fusion with the latency‐associated peptide (LAP) from the cytokine TGF‐? to generate proteins that are biologically inactive until cleavage of the LAP to release the therapy. LAP‐TIMP‐3 was successfully expressed in mammalian cells and the presence of the LAP resulted in a 14‐fold increase in the quantity of recombinant TIMP‐3 produced. LAP‐TIMP‐3 was latent until release from the LAP by treatment with matrix metalloproteinase when it could inhibit proteases of the adamalysins and adamalysins with thrombospondin motifs families, but not matrix metalloproteinases, indicating that this version of TIMP‐3 is a more specific inhibitor than the native protein. There was sufficient protease activity in synovial fluid from human joints with osteoarthritis to release TIMP‐3 from the LAP fusion. These results demonstrate the potential for development of TIMP‐3 as a novel therapy for conditions where upregulation of catabolic enzymes are part of the pathology

Precipitation of soluble uric acid is necessary for in vitro activation of the NLRP3 inflammasome in primary human monocytes

Objective. To investigate the effects of soluble uric acid (UA) on expression and activation of the NOD-like receptor (NLR) pyrin domain containing protein 3 (NLRP3) inflammasome in human monocytes to elucidate the role of hyperuricemia in the pathogenesis of gout. Methods. Primary human monocytes and the THP-1 human monocyte cell line were used to determine the effects of short- and long-term exposure to UA on activation of the NLRP3 inflammasome and subsequent interleukin-1β (IL-1β) secretion by enzyme linked immunosorbent assay (ELISA) and cell-based assays. Expression of key NLRP3 components in monocytes from patients with a history of gout were analysed by quantitative PCR. Results. Precipitation of UA was required for the activation of the NLRP3 inflammasome and subsequent release of IL-1β in human monocytes. Neither monosodium urate (MSU) crystals nor soluble UA had any effect on activation of the transcription factor, NF-κB. Prolonged exposure of monocytes to soluble UA did not alter these responses. However, both MSU crystals and soluble UA did result in a 2-fold increase in reactive oxygen species (ROS). Gout patients (n=15) had significantly elevated serum UA concentrations compared to healthy individuals (n=16), yet secretion of IL-1β and expression of NLRP3 inflammasome components in monocytes isolated from these patients were not different from healthy controls. Conclusion. Despite recent reports indicating that soluble UA can prime and activate the NLRP3 inflammasome in human peripheral blood mononuclear cells (PBMCs), precipitation of soluble UA into MSU crystals is essential for in vitro NLRP3 signalling in primary human monocytes

Simvastatin inhibits TLR8 signaling in primary human monocytes and spontaneous TNF production from rheumatoid synovial membrane cultures

Simvastatin has been shown to have anti-inflammatory effects that are independent of its serum cholesterol lowering action, but the mechanisms by which these anti-inflammatory effects are mediated have not been elucidated. To explore the mechanism involved, the effect of simvastatin on Toll-like receptor (TLR) signalling in primary human monocytes was investigated. A short pre-treatment with simvastatin dose-dependently inhibited the production of tumor necrosis factor-α (TNF) in response to TLR8 (but not TLRs 2, 4, or 5) activation. Statins are known inhibitors of the cholesterol biosynthetic pathway, but intriguingly TLR8 inhibition could not be reversed by addition of mevalonate or geranylgeranyl pyrophosphate; downstream products of cholesterol biosynthesis. TLR8 signalling was examined in HEK 293 cells stably expressing TLR8, where simvastatin inhibited IKKα/β phosphorylation and subsequent NF-κB activation without affecting the pathway to AP-1. Since simvastatin has been reported to have anti-inflammatory effects in RA patients and TLR8 signalling contributes to TNF production in human RA synovial tissue in culture, simvastatin was tested in these cultures. Simvastatin significantly inhibited the spontaneous release of TNF in this model which was not reversed by mevalonate. Together, these results demonstrate a hitherto unrecognized mechanism of simvastatin inhibition of TLR8 signalling that may in part explain its beneficial anti-inflammatory effects

TLR expression profiles are a function of disease status in rheumatoid arthritis and experimental arthritis

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation

Pattern recognition receptors as potential therapeutic targets in inflammatory rheumatic disease

The pattern recognition receptors of the innate immune system are part of the first line of defence against pathogens. However, they also have the ability to respond to danger signals that are frequently elevated during tissue damage and at sites of inflammation. Inadvertent activation of pattern recognition receptors has been proposed to contribute to the pathogenesis of many conditions including inflammatory rheumatic diseases. Prolonged inflammation most often results in pain and damage to tissues. In particular, the Toll-like receptors and nucleotide-binding oligomerisation domain-like receptors that form inflammasomes have been postulated as key contributors to the inflammation observed in rheumatoid arthritis, osteoarthritis, gout and systemic lupus erythematosus. As such, there is increasing interest in targeting these receptors for therapeutic treatment in the clinic. Here the role of pattern recognition receptors in the pathogenesis of these diseases is discussed, with an update on the development of interventions to modulate the activity of these potential therapeutic targets

Dreams and adjustment following marital separation : implications for the function of dreaming

Arguably the most popular current theories of dreaming are the functional theories, including the emotional adaptation or problem-solving theory. These theories revolve around the idea that dreams may serve an independent adaptive function, helping us to adjust to, cope with, or resolve emotionally difficult life circumstances, problems and concerns. Contrary to these theories, other researchers have argued that dreams may have no function of their own, but are an epiphenomenon of REM sleep. The cognitive theories of dreaming suggest that dream content is continuous with waking concerns and preoccupations, and that dreaming about waking concerns is not adaptive but reflective, in a similar way that waking thought or daydreaming is reflective, of what is uppermost in the mind of the dreamer. A relatively small body of research (e.g., Barrett, 1993; Cartwright, 1991; Kramer, 1993) relating to individuals who have experienced major stressful life events, is often cited as support for the theory that dreams serve the specific function of helping us to adjust or adapt to current events. Until recently, this body of work has gone largely unexamined and unreplicated, though some have questioned the findings and their implications for the function of dreaming. The research presented in this thesis examined whether dream content reflects a process of adjustment in people who had recently experienced a marital separation, by investigating the relationship between their dream content in relation to measures of adjustment over time. In Study 1, 97 recently separated participants and 93 married controls were tested on personality and coping factors, asked to answer questions about their dream content, and then monitored over 12 months for change in their adjustment. In Study 2, a subset of 42 separated participants kept dream logs for a period of four weeks. Their dream reports were subjected to a qualitative analysis of thematic content, including threat and threat mastery, and analyses were conducted to explore the relationship between threat content, mastery and adjustment. In Study 3, a subset of eight Study 2 participants participated in a case study analysis which investigated contextual information about their individual situations in relation to their dream content and adjustment, in order to explore, in a more detailed way, the relationship between dream themes, adjustment, and waking concerns. Study 4 was designed to compare the findings of the previous studies with a separate sample, using three different methodologies for the collection of dream content data. This study was carried out to replicate the previous studies with the addition of a laboratory-based data collection technique. In Study 4, 18 separated participants spent one night in the sleep laboratory, monitored with a Nightcap, which allowed dream data to be collected from them via questionnaires, dream logs, and REM awakenings. Across all of the studies, and regardless of the method used to measure dream recall and content, there was a significant concurrent relationship between better adjustment and fewer dreams relating to participants’ marital situations. Those with the most distress were the same ones who were dreaming excessively about their separation. These findings suggest that dreams are continuous with waking preoccupation, and do not function to aid adjustment. As such, they did not support the functional adaptation theories of dreaming. The findings were more consistent with the cognitive theories of dreaming, including the theory that dreams have meaning, but no independent function of their own. A significant relationship was, however, found between ego strength, coping style and adjustment, highlighting the greater influence of internal personal resources in adjusting to difficult life circumstances. While these findings do not discount the suggestion that individuals derive significant personal meaning from their dreams, nor the possibility that dreams may reflect something of the function of REM sleep, they do suggest that “adaptationist” assumptions of functional theories of dreaming may be unfounded

Modulation of toll-like receptor function has therapeutic potential in autoimmune disease

Importance of the field: The role of toll-like receptors (TLRs) in the immune response to exogenous pathogens is well characterized. These receptors have been suggested to be involved in the initiation and/or perpetuation of many inflammatory autoimmune diseases and have become attractive candidates for the modulation of inflammation. Areas covered in this review: This review discusses the evidence to support a potential role for TLRs in inflammatory diseases, focusing on rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus. The approaches to targeting TLR activation are outlined. What the reader will gain: An appreciation for the role of TLRs in inflammatory diseases and in particular the contribution of specific TLRs in rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus. This review focuses on recent developments in targeting TLR activity from ligand binding through to the resultant signaling. Take home message: As initiators of immune responses, TLRs have previously been targeted to increase the immune response with some success. However, targeting TLRs to attenuate immune responses for the treatment of chronic inflammatory diseases will require further evidence of the mechanisms of TLR involvement in the pathophysiology and a better understanding of the potential effects of modulating TLR physiology over a sustained period

Intracellular localization of endothelial cell annexins is differentially regulated by oxidative stress

Annexins are calcium-dependent phospholipid binding proteins that are implicated in the regulation of both intracellular and extracellular thrombostatic mechanisms in the vascular endothelium. Tight control of annexin gene expression and targeting of annexin proteins is therefore of importance in maintaining the health of the endothelium. Because annexins are abundant in vascular endothelial cells and could be either dysregulated by or contribute to anomalies in Ca2+ signaling, we investigated annexin gene expression and subcellular localization in human umbilical vein endothelial cells (HUVEC) in a model of chronic oxidative stress. HUVEC were cultured under mild hyperoxic conditions in a custom-built chamber to induce oxidative stress over a period of 12 days. Although annexin expression levels did not change significantly in response to hyperoxic stress, immunofluorescence analysis revealed striking effects on the subcellular localization of certain annexins, including the redistribution of annexins 5 and 6 from the cytosol to the nucleus. In addition, oxidative stress modulated the responses of certain annexins to stimulation with a range of pharmacological and physiological Ca2+-mobilizing agonists, in a manner that suggested that annexin localization is regulated via the complex integration of both Ca2+ and intracellular signaling pathways. These results show that differential regulation of annexin localization by oxidative stress may have a causative role in the cellular pathophysiology of vascular endothelial cell disease

Targeting Toll-like Receptors in Autoimmunity

In the past few years there has been an increasing appreciation of the importance of Toll-like receptors (TLRs), not just in immunity, but also in autoimmune diseases. TLRs were first identified as sensors of viral and bacterial pathogens that form an integral part of the innate immune response. It was later discovered that these receptors can also respond to endogenous ligands that are produced as a result of tissue damage. This lead to the hypothesis that TLRs may be key contributors to the pathogenesis of chronic inflammatory conditions. A large body of data supporting the role of TLRs in autoimmunity has emerged from animal models and more data is increasingly being generated from human studies as further tools to examine these receptors have become available. Developing strategies to manipulate TLR function is of great interest in autoimmunity, as well as other diseases that include allergy and cancer. This review explores the evidence that points to a role for TLRs in autoimmunity and highlights some of the potential ways in which modulation of their action may yield clinical benefits

The Yapunyah project: embedding Aboriginal and Torres Strait Islander perspectives in the nursing curriculum

The Yapunyah Project is an initiative of the Faculty of Health at Queensland University of Technology. It was instigated to further improve the development of cultural competence in health graduates with respect to Aboriginal and Torres Strait Islander perspectives. The project was informed by the cultural competence in healthcare delivery models of Campinha-Bacote (1998a) and Cross, Bazron, Dennis and Isaacs (1989) and by the cultural safety reforms to nursing curricula in New Zealand. The Yapunyah Project involved extensive consultation and collaboration with Indigenous staff and health experts in the local Aboriginal and Torres Strait Islander community. A core curriculum, and associated graduate transcultural competencies, were informed by these discussions and earlier reforms in health curricula by the Committee of Deans of Australian Medical Schools and the Royal Australian College of General Practitioners. Although the overall project involved four separate schools within the faculty, this paper details the experience of embedding Indigenous perspectives within the undergraduate nursing curriculum. The experience has been a challenging and positive one, and the reforms have been supported by a sustainable framework. This paper outlines how one university faculty is endeavouring to educationally prepare nursing students to practice with evidence-based transcultural nursing knowledge based on culture care values, beliefs, and traditional lifeways of Indigenous people of Australia. As such, the project aims to contribute to the improvement and promotion of the health and well-being of Indigenous Australians in culturally and ethnohistorically meaningful ways