Michael Seed

The impact of endogenous annexin A1 on glucocorticoid control of inflammatory arthritis

Objectives To establish the role and effect of glucocorticoids and the endogenous annexin A1 (AnxA1) pathway in inflammatory arthritis. Methods Ankle joint mRNA and protein expression of AnxA1 and its receptors were analysed in naive and arthritic mice by real-time PCR and immunohistochemistry. Inflammatory arthritis was induced with the K/BxN arthritogenic serum in AnxA1+/+ and AnxA1−/− mice; in some experiments, animals were treated with dexamethasone (Dex) or with human recombinant AnxA1 or a protease-resistant mutant (termed SuperAnxA1). Readouts were arthritic score, disease incidence, paw oedema and histopathology, together with pro-inflammatory gene expression. Results All elements of the AnxA1 pathway could be detected in naive joints, with augmentation during ongoing disease, due to the infiltration of immune cells. No difference in arthritis intensity of profile could be observed between AnxA1+/+ and AnxA1−/− mice. Treatment of mice with Dex (10 µg intraperitoneally daily from day 2) afforded potent antiarthritic effects highly attenuated in the knockouts: macroscopic changes were mirrored by histopathological findings and pro-inflammatory gene (eg, Nos2) expression. Presence of proteinase 3 mRNA in the arthritic joints led the authors to test AnxA1 and the mutant SuperAnxA1 (1 µg intraperitoneally daily in both cases from day 2), with the latter one being able to accelerate the resolving phase of the disease. Conclusion AnxA1 is an endogenous determinant for the therapeutic efficacy of Dex in inflammatory arthritis. Such an effect can be partially mimicked by application of SuperAnxA1 which may represent the starting point for novel antiarthritic therapeutic strategies.

The Melanocortin Agonist AP214 Exerts Anti-Inflammatory and Proresolving Properties

Synthetic and natural melanocortin (MC) peptides afford inhibitory properties in inflammation and tissue injury, but characterization of receptor involvement is still elusive. We used the agonist AP214 to test MC-dependent anti-inflammatory effects. In zymosan peritonitis, treatment of mice with AP214 (400 to 800 μg/kg) inhibited cell infiltration, an effect retained in MC receptor type 1, or MC(1), mutant mice but lost in MC(3) null mice. In vitro, cytokine release from zymosan-stimulated macrophages was affected by AP214, with approximately 80%, 30%, and 40% reduction in IL-1β, tumor necrosis factor-α, and IL-6, respectively. Inhibition of IL-1β release was retained in MC(1) mutant cells but was lost in MC(3) null cells. Furthermore, AP214 augmented uptake of zymosan particles and human apoptotic neutrophils by wild-type macrophages: this proresolving property was lost in MC(3) null macrophages. AP214 displayed its pro-efferocytotic effect also in vivo. Finally, in a model of inflammatory arthritis, AP214 evoked significant reductions in the clinical score. These results indicate that AP214 elicits anti-inflammatory responses, with a preferential effect on IL-1β release. Furthermore, we describe for the first time a positive modulation of an MC agonist on the process of efferocytosis. In all cases, endogenous MC(3) is the receptor that mediates these novel properties of AP214. These findings might clarify the tissue-protective properties of AP214 in clinical settings and may open further development for novel MC agonists.

Effects of low dose ionizing radiation on murine chronic granulomatous tissue

PurposeSubstantial clinical evidence shows the efficacy of low-dose radiotherapy in the treatment of a wide variety of benign conditions. However, experimental investigations into these empirically clinical observations remain scarce. We investigated in vivo low-dose radiation effects on chronic granulomatous tissue by using the air pouch model in mice.Material and MethodsChronic granulomatous air pouches were induced in mice and dosed according to 4 protocols: group I: sham control; group II: 2 Gy on day 2; group III: 2 Gy on day 6; group IV: 5 daily doses of 0.5 Gy from day 2 to 6. On day 7 after granuloma induction the granuloma wet and dry weight was estimated, the vascular content was assessed by the formation of vascular casts incorporating carmine, the inducible nitric oxide synthase (iNOS)- and heme oxygenase 1 (HO-1)-expression in tissue homogenates was assessed by Western blot analysis, and the immunohistochemical localization of iNOS was carried out in cryostat sections of the granulomatous tissue.ResultsWe did not observe any significant reduction in granulomatous tissue wet weight or dry weight following the different radiation treatments, which indicates that anti-proliferative effects in response to the low radiation doses used, are probably not involved in the effects of anti-inflammatory radiotherapy. A single dose of 2 Gy on day 2, as well as fractionated treatment with 5×0.5 Gy lead to an increase in vascularity. iNOS-expression in the homogenized granulomatous tissue was decreased, being most pronounced after single-dose irradiation with 2 Gy on day 2, early on in the acute phase of inflammation. In contrast, the HO-1-expression was increased in all irradiated groups.ConclusionLow doses of radiation interfere with the NO- and the HO-1 pathway. Since NO contributes to several aspects of inflammation such as oedema formation and inflammatory pain, we put forward the hypothesis, that the inhibitory effect of low doses of ionizing radiation on the NO pathway is one radiobiological mechanism underlying the clinically observed efficacy of anti-inflammatory radiotherapy and might result in the reduction of swelling as well as relief of pain. Furthermore, the suppression of iNOS activity could be due to the increase in the stress protein HO-1 by low dose radiotherapy.ZusammenfassungZielEs gibt zahlreiche klinische Beweise dafür, daß die niedrig dosierte Strahlentherapie in der Behandlung verschiedener gutartiger Erkrankungen erfolgreich ist. Experimentelle Untersuchungen dieser klinisch-empirischen Beobachtungen liegen jedoch nahezu nicht vor. Wir untersuchten in vivo die Effekte der niedrig dosierten Bestrahlung auf chronisch granulomatöses Gewebe unter Verwendung des Air-Pouch-Modells in der Maus.Material und MethodenChronisch granulomatöses Gewebe wurde induziert und bestrahlt: Gruppe I: scheinbestrahlte Kontrolle; Gruppe II: 2 Gy am Tag 2; Gruppe III: 2 Gy am Tag 6; Gruppe IV: 5×0,5 Gy an den Tagen 2 bis 6. Am Tag 7 nach Induktion des Granuloms wurden das Feucht- und Trockengewicht des Granuloms bestimmt, dessen Vaskularität wurde mit Hilfe des Carmin-Assays beurteilt, die Expression der induzierbaren Stickoxydsynthase (iNOS) und der Häm-Oxygenase 1 (HO-1) wurden in homogenisiertem granulomatösem Gewebe (Western-Blot-Analyse) sowie die iNOS-Immunlokalisation in Cryostat-Schnitten untersucht.ErgebnisseWir beobachteten unter den verschiedenen Behandlungsregimen keine signifikante Verminderung des Feucht- sowie des Trockengewichts des Granuloms, was darauf schließen läßt, daß antiproliferative Effekte in Reaktion auf die applizierten niedrigen Strahlendosen für die Effektivität der Entzündungsbestrahlung keine Rolle spielen. Einzeitbestrahlung mit 2 Gy am Tag 2 sowie fraktionierte Bestrahlung mit 5×0,5 Gy führten zu einer Erhöhung der Vaskularität. Die Western-Blot-Analyse der iNOS-Expression demonstrierte in allen bestrahlten Gruppen im Durchschnitt eine verminderte iNOS-Konzentration, die am ausgeprägtesten nach Einzeitbestrahlung mit 2 Gy am Tag 2 war, das heißt in der akuten Entzündungsphase. Die immunhistochemisch bestimmte Anzahl an iNOS-positiv markierten Zellen innerhalb der verschiedenen Bestrahlungsgruppen zeigte im wesentlichen ein vergleichbares Muster. Im Gegensatz dazu demonstrierte die Western-Blot-Analyse der HO-1-Expression erhöhte HO-1-Konzentrationen in allen bestrahlten Gruppen.SchlußfolgerungNiedrig dosierte ionisierende Strahlung interferiert mit dem NO- sowie dem HO-System. Da NO zu verschiedenen Aspekten der Entzündung wie Ödem und Schmerz beiträgt, postulieren wir, daß der inhibitorische Effekt niedriger Strahlendosen auf das NO-System einer der strahlenbiologischen Mechanismen der klinisch beobachteten Effektivität der Entzündungsbestrahlung ist und zu einem Rückgang des Entzündungsödems sowie einer Verminderung des Entzündungsschmerzes führen könnte. Des weiteren könnte die Suppression der iNOS-Aktivität auf eine strahleninduzierte Expression des Streßproteins HO-1 zurückzuführen sein.

Human single-chain variable fragment that specifically targets arthritic cartilage.

Objective To demonstrate that posttranslational modification of type II collagen (CII) by reactive oxygen species (ROS), which are known to be present in inflamed arthritic joints, can give rise to epitopes specific to damaged cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA) and to establish a proof of concept that antibodies specific to ROS-modified CII can be used to target therapeutics specifically to inflamed arthritic joints. Methods We used a semisynthetic phage display human antibody library to raise single-chain variable fragments (scFv) specific to ROS-modified CII. The specificity of anti–ROS-modified CII scFv to damaged arthritic cartilage was assessed in vitro by immunostaining articular cartilage from RA and OA patients and from normal controls. The in vivo targeting potential was tested using mice with antigen-induced arthritis, in which localization of anti–ROS-modified CII scFv in the joints was determined. The therapeutic effect of anti–ROS-modified CII scFv fused to soluble murine tumor necrosis factor receptor II–Fc fusion protein (mTNFRII-Fc) was also investigated. Results The anti–ROS-modified CII scFv bound to damaged arthritic cartilage from patients with RA and OA but not to normal preserved cartilage. When systemically administered to arthritic mice, the anti–ROS-modified CII accumulated selectively at the inflamed joints. Importantly, when fused to mTNFRII-Fc, it significantly reduced inflammation in arthritic mice, as compared with the effects of mTNFRII-Fc alone or of mTNFRII-Fc fused to an irrelevant scFv. Conclusion Our findings indicate that biologic therapeutics can be targeted specifically to arthritic joints and suggest a new approach for the development of novel treatments of arthritis.

Apoptosis Induction and Inhibition of Colon-26 Tumour Growth and Angiogenesis: Findings On COX-1 and COX-2 Inhibitors in Vitro & in Vivo and Topical Diclofenac in Hyaluronan

The chronic administration of non-steroidal anti-inflammatory drugs (NSAIDs) results in reduced colonic adenocarcinoma incidence1. When administered to existing tumours, NSAIDs induce tumour regression2. Other states susceptible to NSAIDs include basal cell carcinoma (BCC) and the precancerous lesion actinic keratosis (AK)3 using a topical delivery system, HYAL CT1101 (3% diclofenac in 2.5% hyaluronan). The advent of inducible cyclooxygenase (COX-2) raises the question as to its role in tumour growth and development. The role of COX of any isoform remains speculative. Tumour derived prostaglandin-E2 (PGE2) may result in inhibition of macrophage and NK cell function or there may be direct effects on tumour cells. COX-2 has been reported in many human colonic adenocarcinomas but not all4, 5. Studies with the dual COX-1/2 inhibitor sulindac have shown that familial intestinal polyposis is reduced in the clinic6, whilst the murine equivalent is modified by selective COX-2 inhibition or gene deletion7.

Animal models of rheumatoid arthritis: How informative are they?

Animal models of arthritis are widely used to de-convolute disease pathways and to identify novel drug targets and therapeutic approaches. However, the high attrition rates of drugs in Phase II/III rates means that a relatively small number of drugs reach the market, despite showing efficacy in pre-clinical models. There is also increasing awareness of the ethical issues surrounding the use of animal models of disease and it is timely, therefore, to review the relevance and translatability of animal models of arthritis. In this paper we review the most commonly used animal models in terms of their pathological similarities to human rheumatoid arthritis as well as their response to drug therapy. In general, the ability of animal models to predict efficacy of biologics in man has been good. However, the predictive power of animal models for small molecules has been variable, probably because of differences in the levels of target knockdown achievable in vivo.

Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

IntroductionWe previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis.MethodsViral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis.Results1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10).ConclusionsTargeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically.

I kappa B kinase alpha (IKKα) activity is required for functional maturation of dendritic cells and acquired immunity to infection

Dendritic cells (DC) are required for priming antigen‐specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto‐reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti‐inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen‐specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.

Molecular engineering of short half-life small peptides (VIP, αMSH and γ₃MSH) fused to latency-associated peptide results in improved anti-inflammatory therapeutics.

Objective To facilitate the targeting to inflammation sites of small anti-inflammatory peptides, with short half-lives, by fusion with the latency-associated peptide (LAP) of transforming growth factor β1 through a cleavable matrix metalloproteinase (MMP) linker. This design improves efficacy, overcoming the limitations to their clinical use. Methods We generated latent forms of vasoactive intestinal peptide (VIP), α-melanocyte-stimulating hormone (MSH) and γ3MSH by fusion to LAP through an MMP cleavage site using recombinant DNA technology. The biological activities of these latent therapeutics were studied in vivo using monosodium urate (MSU)-induced peritonitis and collagen-induced arthritis (CIA) models. We assessed gene therapy and purified protein therapy. Results The recruitment of the polymorphonuclear cells induced by MSU injection into mouse peritoneal cavity was reduced by 35% with γ3MSH (1 nmol), whereas administration of a much lower dose of purified latent LAP–MMP–γ3MSH (0.03 nmol) attenuated leucocyte influx by 50%. Intramuscular gene delivery of plasmids coding LAP–MMP–VIP and LAP–MMP–αMSH at disease onset reduced the development of CIA compared with LAP–MMP, which does not contain any therapeutic moiety. Histological analysis confirmed a significantly lower degree of inflammation, bone and cartilage erosion in groups treated with LAP–MMP–VIP or LAP–MMP–αMSH. Antibody titres to collagen type II and inflammatory cytokine production were also reduced in these two groups. Conclusion Incorporation of small anti-inflammatory peptides within the LAP shell and delivered as recombinant protein or through gene therapy can control inflammatory and arthritic disease. This platform delivery can be developed to control human arthritides and other autoimmune diseases.

Paracetamol-induced hypothermia is independent of cannabinoids and transient receptor potential vanilloid-1 and is not mediated by AM404

In recent years, there has been increasing interest in hypothermia induced by paracetamol for therapeutic purposes, which, in some instances, has been reported as a side effect. Understanding the mechanism by which paracetamol induces hypothermia is therefore an important question. In this study, we investigated whether the novel metabolite of paracetamol, N-(4-hydroxyphenyl)arachidonylamide (AM404), which activates the cannabinoid (CB) and transient receptor potential vanilloid-1 (TRPV1) systems, mediates the paracetamol-induced hypothermia. The hypothermic response to 300 mg/kg paracetamol in CB1 receptor (CB1R) and TRPV1 knockout mice was compared to wild-type mice. Hypothermia induced by paracetamol was also investigated in animals pretreated with the CB1R or TRPV1 antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperdinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt (AM251) or 4′-chloro-3-methoxycinnamanilide (SB366791), respectively. In CB1R or TRPV1 knockout mice, paracetamol induced hypothermia to the same extent as in wild-type mice. In addition, in C57BL/6 mice pretreated with AM251 or SB366791, paracetamol induced hypothermia to the same extent as in control mice. AM404 failed to induce hypothermia at pharmacological doses. Inhibition of fatty acid amide hydrolase (FAAH), which is involved in the metabolism of paracetamol to AM404, did not prevent the development of hypothermia with paracetamol. Paracetamol also induced hypothermia in FAAH knockout mice to the same extent as wild-type mice. We conclude that paracetamol induces hypothermia independent of cannabinoids and TRPV1 and that AM404 does not mediate this response. In addition, potential therapeutic value of combinational drug-induced hypothermia is supported by experimental evidence.