The TWEAK receptor Fn14 is a therapeutic target in melanoma: immunotoxins targeting Fn14 receptor for malignant melanoma treatment.

Fibroblast growth factor-inducible protein 14 (Fn14), the cell surface receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is overexpressed in various human solid tumor types and can be a negative prognostic indicator. We detected Fn14 expression in ∼60% of the melanoma cell lines we tested, including both B-Raf WT and B-Raf(V600E) lines. Tumor tissue microarray analysis indicated that Fn14 expression was low in normal skin, but elevated in 173/190 (92%) of primary melanoma specimens and in 86/150 (58%) of melanoma metastases tested. We generated both a chemical conjugate composed of the recombinant gelonin (rGel) toxin and the anti-Fn14 antibody ITEM-4 (designated ITEM4-rGel) and a humanized, dimeric single-chain antibody of ITEM-4 fused to rGel (designated hSGZ). Both ITEM4-rGel and hSGZ were highly cytotoxic to a panel of different melanoma cell lines. Mechanistic studies showed that both immunotoxins induced melanoma cell necrosis. In addition, these immunotoxins could upregulate the cellular expression of Fn14 and trigger cell-signaling events similar to the Fn14 ligand TWEAK. Finally, treatment of mice bearing human melanoma MDA-MB-435 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared with controls. We conclude that Fn14 is a therapeutic target in melanoma and the hSGZ construct appears to warrant further development as a therapeutic agent against Fn14-positive melanoma

Applications and life cycle assessment of shape memory polyethylene terephthalate in concrete for crack closure

YesShape memory polymer (SMP) products have been developed for application as crack closure de-vices in concrete. They have been made from PET in the form of both fibres and hollow tubes. Here, manufacturing methods using die-drawing and mandrel-drawing to induce shape memory are reported. The fibre-based devices are incorporated into concrete and, upon triggering, exert shrinkage restraint forces that close cracks in the concrete. The evolution of shrinkage restraint force in the fibres as manufactured was measured as a function of temperature, showing stresses in excess of 35 MPa. Tendons consisting of fibre bundles are incorporated into concreate beams subjected to controlled cracking. When activated, the tendons reduce the crack widths by 80%. The same fibres are used to produce another class of device known as knotted fibres, which have knotted ends that act as anchor points when they incorporated directly into concrete. Upon acti-vation within the cracked concrete, these devices are shown to completely close cracks. The tubes are used to enclose and restrain prestressed Kevlar fibres. When the tubes are triggered, they shrink and release the prestress force in the Kevlar, which is transferred to the surrounding con-crete in the form of a compressive force, thereby closing cracks. The Kevlar fibres also provide substantial reinforcement after activation. The devices are shown to be able to partially and fully close cracks that have been opened to 0.3 mm and achieve post-activation flexural strengths com-parable to those of conventional reinforced and prestressed structural elements. Finally, a pre-liminary life cycle assessment study was used to assess the carbon footprint a nominal unit of concrete made with SMPs fibres compared to conventional concrete.This research was funded by the UKRI-EPSRC Resilient Materials 4 Life (RM4L), grant number EP/P02081X/1 and the support of ARUP via the UKIMEA research funding

TWEAK Affects Keratinocyte G2/M Growth Arrest and Induces Apoptosis through the Translocation of the AIF Protein to the Nucleus

The soluble TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) binds to the fibroblast growth factor-inducible 14 receptor (FN14, TNFRSF12A) on the cell membrane and induces multiple biological responses, such as proliferation, migration, differentiation, angiogenesis and apoptosis. Previous reports show that TWEAK, which does not contain a death domain in its cytoplasmic tail, induces the apoptosis of tumor cell lines through the induction of TNFα secretion. TWEAK induces apoptosis in human keratinocytes. Our experiments clearly demonstrate that TWEAK does not induce the secretion of TNFα or TRAIL proteins. The use of specific inhibitors and the absence of procaspase-3 cleavage suggest that the apoptosis of keratinocytes follows a caspase- and cathepsin B-independent pathway. Further investigation showed that TWEAK induces a decrease in the mitochondrial membrane potential of keratinocytes. Confocal microscopy showed that TWEAK induces the cleavage and the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus, thus initiating caspase-independent apoptosis. Moreover, TWEAK induces FOXO3 and GADD45 expression, cdc2 phosphorylation and cdc2 and cyclinB1 degradation, resulting in the arrest of cell growth at the G2/M phase. Finally, we report that TWEAK and FN14 are normally expressed in the basal layer of the physiological epidermis and are greatly enhanced in benign (psoriasis) and malignant (squamous cell carcinoma) skin pathologies that are characterized by an inflammatory component. TWEAK might play an essential role in skin homeostasis and pathology

TWEAK Appears as a Modulator of Endometrial IL-18 Related Cytotoxic Activity of Uterine Natural Killers

BACKGROUND: TWEAK (Tumor necrosis factor like WEAK inducer of apoptosis) is highly expressed by different immune cells and triggers multiple cellular responses, including control of angiogenesis. Our objective was to investigate its role in the human endometrium during the implantation window, using an ex-vivo endometrial microhistoculture model. Indeed, previous results suggested that basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Endometrial biopsies were performed 7 to 9 days after the ovulation surge of women in monitored natural cycles. Biopsies were cut in micro-pieces and cultured on collagen sponge with appropriate medium. Morphology, functionality and cell death were analysed at different time of the culture. We used this ex vivo model to study mRNA expressions of NKp46 (a uNK cytotoxic receptor) and TGF-beta1 (protein which regulates uNK cytokine production) after adjunction of excess of recombinant IL-18 and either recombinant TWEAK or its antibody. NKp46 protein expression was also detailed by immunohistochemistry in selected patients with high basic mRNA level of IL-18 and either low or high mRNA level of TWEAK. The NKp46 immunostaining was stronger in patients with an IL-18 over-expression and a low TWEAK expression, when compared with patients with both IL-18 and TWEAK high expressions. We did not observe any difference for TWEAK expression when recombinant protein IL-18 or its antibody was added, or conversely, for IL-18 expression when TWEAK or its antibody was added in the culture medium. In a pro-inflammatory environment (obtained by an excess of IL-18), inhibition of TWEAK was able to increase significantly NKp46 and TGF-beta1 mRNA expressions. CONCLUSIONS/SIGNIFICANCE: TWEAK doesn't act on IL-18 expression but seems to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed. Thus, TWEAK appears as a crucial physiological modulator to prevent endometrial uNK cytotoxicity in human

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

Extent: 10p.INTRODUCTION: TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro. METHODS: TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry. RESULTS: TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts. CONCLUSIONS: The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.Anak A. S. S. K. Dharmapatni, Malcolm D. Smith, Tania N. Crotti, Christopher A. Holding, Cristina Vincent, Helen M. Weedon, Andrew C. W. Zannettino, Timothy S. Zheng, David M. Findlay, Gerald J. Atkins and David R. Hayne

Morphine Induces Expression of Platelet-Derived Growth Factor in Human Brain Microvascular Endothelial Cells: Implication for Vascular Permeability

Despite the advent of antiretroviral therapy, complications of HIV-1 infection with concurrent drug abuse are an emerging problem. Morphine, often abused by HIV-infected patients, is known to accelerate neuroinflammation associated with HIV-1 infection. Detailed molecular mechanisms of morphine action however, remain poorly understood. Platelet-derived growth factor (PDGF) has been implicated in a number of pathological conditions, primarily due to its potent mitogenic and permeability effects. Whether morphine exposure results in enhanced vascular permeability in brain endothelial cells, likely via induction of PDGF, remains to be established. In the present study, we demonstrated morphine-mediated induction of PDGF-BB in human brain microvascular endothelial cells, an effect that was abrogated by the opioid receptor antagonist-naltrexone. Pharmacological blockade (cell signaling) and loss-of-function (Egr-1) approaches demonstrated the role of mitogen-activated protein kinases (MAPKs), PI3K/Akt and the downstream transcription factor Egr-1 respectively, in morphine-mediated induction of PDGF-BB. Functional significance of increased PDGF-BB manifested as increased breach of the endothelial barrier as evidenced by decreased expression of the tight junction protein ZO-1 in an in vitro model system. Understanding the regulation of PDGF expression may provide insights into the development of potential therapeutic targets for intervention of morphine-mediated neuroinflammation

The Expression of BAFF, APRIL and TWEAK Is Altered in Eczema Skin but Not in the Circulation of Atopic and Seborrheic Eczema Patients

The TNF family cytokines BAFF (B-cell activating factor of the TNF family) and APRIL (a proliferation-inducing ligand) are crucial survival factors for B-cell development and activation. B-cell directed treatments have been shown to improve atopic eczema (AE), suggesting the involvement of these cytokines in the pathogenesis of AE. We therefore analyzed the expression of these TNF cytokines in AE, seborrheic eczema (SE) and healthy controls (HC). The serum/plasma concentration of BAFF, APRIL and a close TNF member TWEAK (TNF-like weak inducer of apoptosis) was measured by ELISA. The expression of these cytokines and their receptors in skin was analyzed by quantitative RT-PCR and immunofluorescence. Unlike other inflammatory diseases including autoimmune diseases and asthma, the circulating levels of BAFF, APRIL and TWEAK were not elevated in AE or SE patients compared with HCs and did not correlate with the disease severity or systemic IgE levels in AE patients. Interestingly, we found that the expression of these cytokines and their receptors was altered in positive atopy patch test reactions in AE patients (APT-AE) and in lesional skin of AE and SE patients. The expression of APRIL was decreased and the expression of BAFF was increased in eczema skin of AE and SE, which could contribute to a reduced negative regulatory input on B-cells. This was found to be more pronounced in APT-AE, the initiating acute stage of AE, which may result in dysregulation of over-activated B-cells. Furthermore, the expression levels of TWEAK and its receptor positively correlated to each other in SE lesions, but inversely correlated in AE lesions. These results shed light on potential pathogenic roles of these TNF factors in AE and SE, and pinpoint a potential of tailored treatments towards these factors in AE and SE

The cytokine tumor necrosis factor-like weak inducer of apoptosis and its receptor fibroblast growth factor-inducible 14 have a neuroprotective effect in the central nervous system

<p>Abstract</p> <p>Background</p> <p>Cerebral cortical neurons have a high vulnerability to the harmful effects of hypoxia. However, the brain has the ability to detect and accommodate to hypoxic conditions. This phenomenon, known as preconditioning, is a natural adaptive process highly preserved among species whereby exposure to sub-lethal hypoxia promotes the acquisition of tolerance to a subsequent lethal hypoxic injury. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) are found in neurons and their expression is induced by exposure to sub-lethal hypoxia. Accordingly, in this work we tested the hypothesis that the interaction between TWEAK and Fn14 induces tolerance to lethal hypoxic and ischemic conditions.</p> <p>Methods</p> <p>Here we used <it>in vitro </it>and <it>in vivo </it>models of hypoxic and ischemic preconditioning, an animal model of transient middle cerebral artery occlusion and mice and neurons genetically deficient in TWEAK, Fn14, or tumor necrosis factor alpha (TNF-α) to investigate whether treatment with recombinant TWEAK or an increase in the expression of endogenous TWEAK renders neurons tolerant to lethal hypoxia. We used enzyme-linked immunosorbent assay to study the effect of TWEAK on the expression of neuronal TNF-α, Western blot analysis to investigate whether the effect of TWEAK was mediated by activation of mitogen-activated protein kinases and immunohistochemical techniques and quantitative real-time polymerase chain reaction analysis to study the effect of TWEAK on apoptotic cell death.</p> <p>Results</p> <p>We found that either treatment with recombinant TWEAK or an increase in the expression of TWEAK and Fn14 induce hypoxic and ischemic tolerance <it>in vivo </it>and <it>in vitro</it>. This protective effect is mediated by neuronal TNF-α and activation of the extracellular signal-regulated kinases 1 and 2 pathway via phosphorylation and inactivation of the B-cell lymphoma 2-associated death promoter protein.</p> <p>Conclusions</p> <p>Our work indicate that the interaction between TWEAK and Fn14 triggers the activation of a cell signaling pathway that results in the induction of tolerance to lethal hypoxia and ischemia. These data indicate that TWEAK may be a potential therapeutic strategy to protect the brain from the devastating effects of an ischemic injury.</p

Gene Expression Profiles of the NCI-60 Human Tumor Cell Lines Define Molecular Interaction Networks Governing Cell Migration Processes

Although there is extensive information on gene expression and molecular interactions in various cell types, integrating those data in a functionally coherent manner remains challenging. This study explores the premise that genes whose expression at the mRNA level is correlated over diverse cell lines are likely to function together in a network of molecular interactions. We previously derived expression-correlated gene clusters from the database of the NCI-60 human tumor cell lines and associated each cluster with function categories of the Gene Ontology (GO) database. From a cluster rich in genes associated with GO categories related to cell migration, we extracted 15 genes that were highly cross-correlated; prominent among them were RRAS, AXL, ADAM9, FN14, and integrin-beta1. We then used those 15 genes as bait to identify other correlated genes in the NCI-60 database. A survey of current literature disclosed, not only that many of the expression-correlated genes engaged in molecular interactions related to migration, invasion, and metastasis, but that highly cross-correlated subsets of those genes engaged in specific cell migration processes. We assembled this information in molecular interaction maps (MIMs) that depict networks governing 3 cell migration processes: degradation of extracellular matrix, production of transient focal complexes at the leading edge of the cell, and retraction of the rear part of the cell. Also depicted are interactions controlling the release and effects of calcium ions, which may regulate migration in a spaciotemporal manner in the cell. The MIMs and associated text comprise a detailed and integrated summary of what is currently known or surmised about the role of the expression cross-correlated genes in molecular networks governing those processes

Over expression of Plk1 does not induce cell division in rat cardiac myocytes in vitro

BACKGROUND: Mammalian cardiac myocytes withdraw from the cell cycle during post-natal development, resulting in a non-proliferating, fully differentiated adult phenotype that is unable to repair damage to the myocardium, such as occurs following a myocardial infarction. We and others previously have shown that forced expression of certain cell cycle molecules in adult cardiac myocytes can promote cell cycle progression and division in these cells. The mitotic serine/threonine kinase, Polo-like kinase-1 (Plk1), is known to phosphorylate and activate a number of mitotic targets, including Cdc2/Cyclin B1, and to promote cell division. PRINCIPAL FINDINGS: The mammalian Plk family are all differentially regulated during the development of rat cardiac myocytes, with Plk1 showing the most dramatic decrease in both mRNA, protein and activity in the adult. We determined the potential of Plk1 to induce cell cycle progression and division in cultured rat cardiac myocytes. A persistent and progressive loss of Plk1 expression was observed during myocyte development that correlated with the withdrawal of adult rat cardiac myocytes from the cell cycle. Interestingly, when Plk1 was over-expressed in cardiac myocytes by adenovirus infection, it was not able to promote cell cycle progression, as determined by cell number and percent binucleation. CONCLUSIONS: We conclude that, in contrast to Cdc2/Cyclin B1 over-expression, the forced expression of Plk1 in adult cardiac myocytes is not sufficient to induce cell division and myocardial repair