CDP571, a humanized monoclonal antibody to tumour necrosis factor-alpha, for steroid-dependent Crohn's disease: a randomized, double-blind, placebo-controlled trial

Background More than 50% of patients with Crohn's disease become either steroid resistant or dependent. Accordingly, development of new treatments for steroid-dependent Crohn's disease is a research priority. Aim To evaluate CDP571, a humanized antibody to tumour necrosis factor-α, for the treatment of steroid-dependent Crohn's disease. Methods Patients with steroid-dependent Crohn's disease (n = 271) were enrolled in a 36-week, double-blind, placebo-controlled trial. Steroid dependence was defined as use of prednisolone or prednisone (15–40 mg/day) or budesonide (9 mg/day) for ≥8 weeks, a previous failed attempt to decrease or discontinue steroids within 8 weeks of screening, and a Crohn's Disease Activity Index score of ≤150 points. Patients were randomized to receive intravenous CDP571 10 mg/kg or placebo 8-weekly through to week 32. Steroids were then tapered using a defined schedule. The primary efficacy endpoint was the percentage of patients with steroid sparing, defined as discontinuation of steroid therapy without a disease flare (Crohn's Disease Activity Index score ≥220 points) at week 36. Results Steroid sparing occurred in 53 of 181 (29.3%) CDP571 patients and 33 of 90 (36.7%) placebo patients (P = 0.24). Adverse events occurred at similar frequencies in both treatment groups. Conclusions CDP571 was ineffective for sparing steroids in patients with steroid-dependent Crohn's disease. CDP571 was well tolerated.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha(TNF alpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNF alpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor ( SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNF alpha trafficking and secretion. TNF alpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNF alpha surface distribution revealed these puncta to be the initial points of TNF alpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

Genetics and genomics of osteoclast differentiation: Integrating cell signaling pathways and gene networks

The regulation of osteoclast differentiation in the bone microenvironment is critical for normal bone remodeling, as well as for various human bone diseases. Over the last decade, our knowledge of how osteoclast differentiation occurs has progressed rapidly. We highlight some of the major advances in understanding how cell signaling and transcription are integrated to direct the differentiation of this cell type. These studies used genetic, molecular, and biochemical approaches. Additionally, we summarize data obtained from studies of osteoclast differentiation that used the functional genomic approach of global gene profiling applied to osteoclast differentiation. This genomic data confirms results from studies using the classical experimental approaches and also may suggest new modes by which osteoclast differentiation and function can be modulated. Two conclusions that emerge are that osteoclast differentiation depends on a combination of fairly ubiquitously expressed transcription factors rather than unique osteoclast factors, and that the overlay of cell signaling pathways on this set of transcription factors provides a powerful mechanism to fine tune the differentiation program in response to the local bone microenvironment.

Recent developments in C5/C5a inhibitors

Complement factor 5a (C5a) is formed upon complement system activation in response to infection, injury or disease. Whilst C5a is a potent mediator of immune and inflammatory processes, excessive production or inadequate regulation of C5a has been implicated in the pathogenesis of numerous immuno-inflammatory diseases, predominantly through experimental studies utilising animal models of disease. Both acute and chronic conditions may benefit from C5a inhibition, including rheumatoid arthritis, inflammatory bowel disease, asthma, psoriasis, haemorrhagic shock and neurodegenerative conditions. The potentially broad clinical application for treatments that inhibit the activity of C5a at C5a receptors and the large global market for anti-inflammatory therapeutics have made C5a and the C5a receptor attractive targets for academic and commercial drug development programmes. in the past 5 years, interest in C5a as a drug target has grown substantially, and this activity has resulted in a collection of patents and scientific papers reporting novel C5a and C5a receptor inhibitors and antagonists, and generated a secondary stream of patent applications broadly claiming the use of C5/C5a inhibitors as a method of treating various immune and inflammatory conditions. This paper will review the physiology and pathophysiology of C5a and discuss the development of C5a and C5a receptor inhibitors in light of the recent scientific and patent literature.

The roles of exercise-induced immune system disturbances in the pathology of heat stroke - The dual pathway model of heat stroke

Heat stroke is a life-threatening condition that can be fatal if not appropriately managed. Although heat stroke has been recognised as a medical condition for centuries, a universally accepted definition of heat stroke is lacking and the pathology of heat stroke is not fully understood. Information derived from autopsy reports and the clinical presentation of patients with heat stroke indicates that hyperthermia, septicaemia, central nervous system impairment and cardiovascular failure play important roles in the pathology of heat stroke. The current models of heat stroke advocate that heat stroke is triggered by hyperthermia but is driven by endotoxaemia. Endotoxaemia triggers the systemic inflammatory response, which can lead to systemic coagulation and haemorrhage, necrosis, cell death and multi-organ failure. However, the current heat stroke models cannot fully explain the discrepancies in high core temperature (Tc) as a trigger of heat stroke within and between individuals. Research on the concept of critical Tc: as a limitation to endurance exercise implies that a high Tc may function as a signal to trigger the protective mechanisms against heat stroke. Athletes undergoing a period of intense training are subjected to a variety of immune and gastrointestinal (GI) disturbances. The immune disturbances include the suppression of immune cells and their functions, suppression of cell-mediated immunity, translocation of lipopolysaccharide (LPS), suppression of anti-LPS antibodies, increased macrophage activity due to muscle tissue damage, and increased concentration of circulating inflammatory and pyrogenic cytokines. Common symptoms of exercise-induced GI disturbances include diarrhoea, vomiting, gastrointestinal bleeding, and cramps, which may increase gut-related LPS translocation. This article discusses the current evidence that supports the argument that these exercise-induced immune and GI disturbances may contribute to the development of endotoxaemia and heat stroke. When endotoxaemia can be tolerated or prevented, continuing exercise and heat exposure will elevate Tc to a higher level (> 42 degrees C), where heat stroke may occur through the direct thermal effects of heat on organ tissues and cells. We also discuss the evidence suggesting that heat stroke may occur through endotoxaemia (heat sepsis), the primary pathway of heat stroke, or hyperthermia, the secondary pathway of heat stroke. The existence of these two pathways of heat stroke and the contribution of exercise-induced immune and GI disturbances in the primary pathway of heat stroke are illustrated in the dual pathway model of heat stroke. This model of heat stroke suggests that prolonged intense exercise suppresses anti-LPS mechanisms, and promotes inflammatory and pyrogenic activities in the pathway of heat stroke.

Systematic inflammatory responses to maximal versus submaximal lengthening contractions of the elbow flexors

We compared changes in markers of muscle damage and systemic inflammation after submaximal and maximal lengthening muscle contractions of the elbow flexors. Using a cross-over design, 10 healthy young men not involved in resistance training completed a submaximal trial (10 sets of 60 lengthening contractions at 10% maximum isometric strength, 1 min rest between sets), followed by a maximal trial (10 sets of three lengthening contractions at 100% maximum isometric strength, 3 min rest between sets). Lengthening contractions were performed on an isokinetic dynamometer. Opposite arms were used for the submaximal and maximal trials, and the trials were separated by a minimum of two weeks. Blood was sampled before, immediately after, 1 h, 3 h, and 1-4 d after each trial. Total leukocyte and neutrophil numbers, and the serum concentration of soluble tumor necrosis factor-alpha receptor 1 were elevated after both trials (P < 0.01), but there were no differences between the trials. Serum IL-6 concentration was elevated 3 h after the submaximal contractions (P < 0.01). The concentrations of serum tumor necrosis factor-alpha, IL-1 receptor antagonist, IL-10, granulocyte-colony stimulating factor and plasma C-reactive protein remained unchanged following both trials. Maximum isometric strength and range of motion decreased significantly (P < 0.001) after both trials, and were lower from 1-4 days after the maximal contractions compared to the submaximal contractions. Plasma myoglobin concentration and creatine kinase activity, muscle soreness and upper arm circumference all increased after both trials (P < 0.01), but were not significantly different between the trials. Therefore, there were no differences in markers of systemic inflammation, despite evidence of greater muscle damage following maximal versus submaximal lengthening contractions of the elbow flexors.

International Union of Pharmacology. LXIX. Status of the calcitonin gene-related peptide subtype 2 receptor

Historically, calcitonin gene-related peptide (CGRP) receptors have been divided into two classes, CGRP(1) and CGRP(2).After the cloning of calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMPs), it became clear that the CGRP(1) receptor was a complex between CLR and RAMP1. It is now apparent that the CGRP(2) receptor phenotype is the result of CGRP acting at receptors for amylin and adrenomedullin. Accordingly, the term "CGRP(2)" receptor should no longer be used, and the "CGRP(1)" receptor should be known as the "CGRP" receptor.

Characterization of tumour necrosis factor alpha release by human granulocytes in response to procainamide challenge

The role of human granulocytes in the promotion of procainamide (PA) toxicity in vitro has been studied and one of the agents responsible for DNA strand scission and cell death in human target cells has been characterized. Crude peripheral blood mononuclear cells (cPBMNs) isolated by density centrifugation, and the lymphocyte cell lines--CCRF-HSB2 and WIL-2NS--were exposed to PA, and DNA strand breaks were quantified by fluorescent analysis of DNA unwinding. Therapeutic plasma concentrations of PA (0-50 microM) caused dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in cPBMNs incubated for 3 and 1.5 hr at 37 degrees, respectively. Using 50 microM PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with significant induction of strand breaks also being observed for 10 and 25 microM concentrations. Toxicity was much reduced in lymphocyte cell lines (maximal killing = 3.0% at 50 microM PA compared with 13.2% in cPBMNs). A similar decrease in toxicity was observed where N-acetyl procainamide (NAPA) was substituted for PA (less than 50% of strand breaks at all concentrations). Further investigations showed that the presence of a contaminating granulocyte population in the cPBMN fraction was responsible for the induction of PA toxicity. Incubation of a highly enriched granulocyte population with PA for 1 hr prior to exposure to purified peripheral blood mononuclear cells (pPBMNs) led to the complete restoration of the toxic effects. The resulting cyto- and genotoxicity were not significantly different to levels observed in cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal studies showed that the toxicity associated with the < 3000 Da fraction appeared during the first 10-15 min incubation with PA whereas the > 30,000 Da fraction did not display significant toxicity until the 40-60 min period. Further assessment of the nature of these agents indicated that the 30,000 Da fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da species appearing in granulocyte supernatants after 40 min incubation with PA. Dot blot analysis indicated that tumour necrosis factor alpha (TNF alpha) was present in the > 30,000 Da fraction. Evidence that TNF alpha was the high-molecular weight species responsible for PA-induced toxicity was obtained from neutralization assays employing an anti-TNF alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice

Background Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. Methods MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-? and tumor necrosis factor-a) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-? and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia. Results LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-?-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. Conclusion The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

Mechanisms of Cancer Cachexia

Up to 50% of cancer patients suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia, resulting in weight loss, a reduced quality of life, and a shortened survival time. Anorexia often accompanies cachexia, but appears not to be responsible for the tissue loss, particularly lean body mass. An increased resting energy expenditure is seen, possibly arising from an increased thermogenesis in skeletal muscle due to an increased expression of uncoupling protein, and increased operation of the Cori cycle. Loss of adipose tissue is due to an increased lipolysis by tumor or host products. Loss of skeletal muscle in cachexia results from a depression in protein synthesis combined with an increase in protein degradation. The increase in protein degradation may include both increased activity of the ubiquitin-proteasome pathway and lysosomes. The decrease in protein synthesis is due to a reduced level of the initiation factor 4F, decreased elongation, and decreased binding of methionyl-tRNA to the 40S ribosomal subunit through increased phosphorylation of eIF2 on the a-subunit by activation of the dsRNA-dependent protein kinase, which also increases expression of the ubiquitin-proteasome pathway through activation of NF?B. Tumor factors such as proteolysis-inducing factor and host factors such as tumor necrosis factor-a, angiotensin II, and glucocorticoids can all induce muscle atrophy. Knowledge of the mechanisms of tissue destruction in cachexia should improve methods of treatment. Copyright © 2009 the American Physiological Society

The Proinflammatory Environment in Potential Heart and Lung Donors: Prevalence and Impact of Donor Management and Hormonal Therapy

BACKGROUND: Brain stem death can elicit a potentially manipulable cardiotoxic proinflammatory cytokine response. We investigated the prevalence of this response, the impact of donor management with tri-iodothyronine (T3) and methylprednisolone (MP) administration, and the relationship of biomarkers to organ function and transplant suitability. METHODS: In a prospective randomized double-blinded factorially designed study of T3 and MP therapy, we measured serum levels of interleukin-1 and -6 (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein, and procalcitonin (PCT) levels in 79 potential heart or lung donors. Measurements were performed before and after 4 hr of algorithm-based donor management to optimize cardiorespiratory function and +/-hormone treatment. Donors were assigned to receive T3, MP, both drugs, or placebo. RESULTS: Initial IL-1 was elevated in 16% donors, IL-6 in 100%, TNF-alpha in 28%, CRP in 98%, and PCT in 87%. Overall biomarker concentrations did not change between initial and later measurements and neither T3 nor MP effected any change. Both PCT (P =0.02) and TNF-alpha (P =0.044) levels were higher in donor hearts with marginal hemodynamics at initial assessment. Higher PCT levels were related to worse cardiac index and right and left ventricular ejection fractions and a PCT level more than 2 ng x mL(-1) may attenuate any improvement in cardiac index gained by donor management. No differences were observed between initially marginal and nonmarginal donor lungs. A PCT level less than or equal to 2 ng x mL(-1) but not other biomarkers predicted transplant suitability following management. CONCLUSIONS: There is high prevalence of a proinflammatory environment in the organ donor that is not affected by tri-iodothyronine or MP therapy. High PCT and TNF-alpha levels are associated with donor heart dysfunction. (C) 2009 Lippincott Williams & Wilkins, Inc.

Regulation of IL-1β-induced NFκB by hydroxylases links key hypoxic and inflammatory signaling pathways

Hypoxia is a prominent feature of chronically inflamed tissues. Oxygen-sensing hydroxylases control transcriptional adaptation to hypoxia through the regulation of hypoxia-inducible factor (HIF) and nuclear factor ?B (NF-?B), both of which can regulate the inflammatory response. Furthermore, pharmacologic hydroxylase inhibitors reduce inflammation in multiple animal models. However, the underlying mechanism(s) linking hydroxylase activity to inflammatory signaling remains unclear. IL-1ß, a major proinflammatory cytokine that regulates NF-?B, is associated with multiple inflammatory pathologies. We demonstrate that a combination of prolyl hydroxylase 1 and factor inhibiting HIF hydroxylase isoforms regulates IL-1ß-induced NF-?B at the level of (or downstream of) the tumor necrosis factor receptor-associated factor 6 complex. Multiple proteins of the distal IL-1ß-signaling pathway are subject to hydroxylation and form complexes with either prolyl hydroxylase 1 or factor inhibiting HIF. Thus, we hypothesize that hydroxylases regulate IL-1ß signaling and subsequent inflammatory gene expression. Furthermore, hydroxylase inhibition represents a unique approach to the inhibition of IL-1ß-dependent inflammatory signaling.

Structure-activity relationship of calcitonin gene-related peptide and its receptor

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