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OBJECTIVE: In vivo optical imaging can delineate at the macroscopic level processes that are occurring at the cellular and molecular levels. E-selectin, a leukocyte adhesion molecule expressed on endothelium, is induced by tumor necrosis factor α (TNFα) and other cytokines involved in the pathogenesis of rheumatoid arthritis (RA). Collagen-induced arthritis (CIA) in mice is widely used to study the disease mechanisms and identify new treatments for RA. The purpose of this study was to demonstrate E-selectin-targeted fluorescence imaging in vivo in a mouse model of paw edema generated by local injection of TNFα as well as in mice with CIA. METHODS: Animals with either CIA or TNFα-induced paw edema were injected with anti-E-selectin or control antibodies labeled with a DyLight 750-nm near-infrared (NIR) probe. In vivo imaging studies were undertaken using an NIR optical imaging system, and images were coregistered with plain radiographic images. RESULTS: The mean fluorescence intensity measured over the time-course of TNFα-induced edema demonstrated a 1.97-fold increase (P<0.001) in signal in inflamed paws at 8 hours following injection of anti-E-selectin antibody, as compared to that in the isotype control. In the CIA model, a 2.34-fold increase in E-selectin-targeted signal was demonstrated (P<0.01). Furthermore, significant E-selectin-targeted signal was observed in the paws of animals immunized with collagen that did not display overt signs of arthritis. CONCLUSION: E-selectin-targeted fluorescence in vivo imaging is a quantifiable method of detecting endothelial activation in arthritis and can potentially be applied to the quantification of disease and the investigation of the effects of new therapies. Importantly, this approach may also be useful for the detection of subclinical disease in RA.
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Artrite Experimental/metabolismo , Selectina E/metabolismo , Endotélio Vascular/metabolismo , Análise de Variância , Animais , Artrite Experimental/imunologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Selectina E/imunologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Imuno-Histoquímica , Masculino , Camundongos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In vivo molecular optical imaging has significant potential to delineate and measure, at the macroscopic level, in vivo biological processes that are occurring at the cellular and molecular level. Optical imaging has already been developed for in vitro and ex vivo applications in molecular and cellular biology (e.g. fluorescence confocal microscopy), but is still at an early stage of development as a whole-animal in vivo imaging technique. Both sensitivity and spatial resolution remain incompletely defined. Rapid advances in hardware technology and highly innovative reporter probes and dyes will be expected to deliver significant insight into perturbations of molecular pathways that occur in disease, ultimately with the potential of translating into future molecular imaging techniques for patients with arthritis. This review will focus on currently available technologies for live in vivo animal optical imaging, including fluorescence reflectance imaging, potential novel tomographic techniques, bioluminescence reporter technology and potential novel labelling techniques, highlighting in particular the potential application of in vivo fluorescence imaging in arthritis.
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Artrite Experimental/diagnóstico , Diagnóstico por Imagem/métodos , Imageamento Tridimensional/métodos , Tomografia Óptica/métodos , Animais , Modelos AnimaisRESUMO
OBJECTIVE: To develop enzyme-activatable Förster resonance energy transfer (FRET) substrate probes to detect matrix metalloproteinase 12 (MMP-12) and MMP-13 activities in vivo in mouse models of inflammatory arthritis. METHODS: Peptidic FRET probes activated by MMP-12 and MMP-13 were reverse designed from inhibitors selected from a phosphinic peptide inhibitor library. Selectivity of the probes was demonstrated in vitro using MMP-1, MMP-2, MMP-3, MMP-12, and MMP-13. In vivo activation of the probes was tested in the zymosan-induced mouse model of inflammation, and probe specificity was evaluated by the MMP inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to monitor these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. RESULTS: The MMP-12 and MMP-13 activity probes (MMP12ap and MMP13ap, respectively) discriminated between the activities of the 2 enzymes. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85, P < 0.0001). MMP13ap activation increased gradually after disease onset and correlated with disease severity over a longer period of 15 days (r = 0.58, P < 0.0001). CONCLUSION: We generated two selective FRET probes that can be used to monitor MMP-12 and MMP-13 activities in live animals. MMP12ap follows the initial stage of inflammation in CIA, while MMP13ap follows the progression of the disease. The specificity of these probes is useful in monitoring the efficacy of MMP inhibitors.
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Artrite Experimental/enzimologia , Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Membrana Sinovial/enzimologia , Animais , Diagnóstico por Imagem , Transferência Ressonante de Energia de Fluorescência , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Membrana Sinovial/efeitos dos fármacosRESUMO
Novel molecular imaging techniques are at the forefront of both preclinical and clinical imaging strategies. They have significant potential to offer visualisation and quantification of molecular and cellular changes in health and disease. This will help to shed light on pathobiology and underlying disease processes and provide further information about the mechanisms of action of novel therapeutic strategies. This review explores currently available molecular imaging techniques that are available for preclinical studies with a focus on optical imaging techniques and discusses how current and future advances will enable translation into the clinic for patients with arthritis.
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Artrite/diagnóstico por imagem , Artrite/patologia , Interpretação de Imagem Assistida por Computador/métodos , Imagem Molecular/métodos , Humanos , Radiografia , CintilografiaRESUMO
Preclinical models for musculoskeletal disorders are critical for understanding the pathogenesis of bone and joint disorders in humans and the development of effective therapies. The assessment of these models primarily relies on morphological analysis which remains time consuming and costly, requiring large numbers of animals to be tested through different stages of the disease. The implementation of preclinical imaging represents a keystone in the refinement of animal models allowing longitudinal studies and enabling a powerful, non-invasive and clinically translatable way for monitoring disease progression in real time. Our aim is to highlight examples that demonstrate the advantages and limitations of different imaging modalities including magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging. All of which are in current use in preclinical skeletal research. MRI can provide high resolution of soft tissue structures, but imaging requires comparatively long acquisition times; hence, animals require long-term anaesthesia. CT is extensively used in bone and joint disorders providing excellent spatial resolution and good contrast for bone imaging. Despite its excellent structural assessment of mineralized structures, CT does not provide in vivo functional information of ongoing biological processes. Nuclear medicine is a very promising tool for investigating functional and molecular processes in vivo with new tracers becoming available as biomarkers. The combined use of imaging modalities also holds significant potential for the assessment of disease pathogenesis in animal models of musculoskeletal disorders, minimising the use of conventional invasive methods and animal redundancy.
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INTRODUCTION: Rheumatoid arthritis (RA) is a chronic disease associated with inflammation and destruction of bone and cartilage. Although inhibition of TNFα is widely used to treat RA, a significant number of patients do not respond to TNFα blockade, and therefore there is a compelling need to continue to identify alternative therapeutic strategies for treating chronic inflammatory diseases such as RA. The anti-epidermal growth factor (anti-EGF) receptor antibody trastuzumab has revolutionised the treatment of patients with EGF receptor-positive breast cancer. Expression of EGF ligands and receptors (known as HER) has also been documented in RA. The highly unique compound RB200 is a bispecific ligand trap that is composed of full-length extracellular domains of HER1 and HER3 EGF receptors. Because of its pan-HER specificity, RB200 inhibits responses mediated by HER1, HER2 and HER3 in vitro and in vivo. The objective of this study was to assess the effect of RB200 combined with TNF blockade in a murine collagen-induced arthritis (CIA) model of RA. METHODS: Arthritic mice were treated with RB200 alone or in combination with the TNF receptor fusion protein etanercept. We performed immunohistochemistry to assess CD31 and in vivo fluorescent imaging using anti-E-selectin antibody labelled with fluorescent dye to elucidate the effect of RB200 on the vasculature in CIA. RESULTS: RB200 significantly abrogated CIA by reducing paw swelling and clinical scores. Importantly, low-dose RB200 combined with a suboptimal dose of etanercept led to complete abrogation of arthritis. Moreover, the combination of RB200 with etanercept abrogated the intensity of the E-selectin-targeted signal to the level seen in control animals not immunised to CIA. CONCLUSIONS: The human pan-EGF receptor bispecific ligand trap RB200, when combined with low-dose etanercept, abrogates CIA, suggesting that inhibition of events downstream of EGF receptor activation, in combination with TNFα inhibitors, may hold promise as a future therapy for patients with RA.
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Artrite Experimental/metabolismo , Receptores ErbB/metabolismo , Receptor ErbB-3/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Animais , Artrite Experimental/tratamento farmacológico , Bovinos , Etanercepte , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos DBA , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores do Fator de Necrose Tumoral/uso terapêuticoRESUMO
The origin of pain in osteoarthritis is poorly understood, but it is generally thought to arise from inflammation within the innervated structures of the joint, such as the synovium, capsule and bone. We investigated the role of nerve growth factor (NGF) in pain development in murine OA, and the analgesic efficacy of the soluble NGF receptor, TrkAD5. OA was induced in mice by destabilisation of the medial meniscus and pain was assessed by measuring hind-limb weight distribution. RNA was extracted from joints, and NGF and TNF expressions were quantified. The effect of tumour necrosis factor (TNF) and neutrophil blockade on NGF expression and pain were also assessed. NGF was induced in the joints during both post-operative (day 3) and OA (16weeks) pain, but not in the non-painful stage of disease (8weeks post-surgery). TrkAd5 was highly effective at suppressing pain in both phases. Induction of NGF in the post-operative phase of pain was TNF-dependent as anti-TNF reduced NGF expression in the joint and abrogated pain. However, TNF was not regulated in the late OA joints, and pain was not affected by anti-TNF therapy. Fucoidan, by suppressing cellular infiltration into the joint, was able to suppress post-operative, but not late OA pain. These results indicate that NGF is an important mediator of OA pain and that TrkAd5 represents a potent novel analgesic in this condition. They also suggest that, unlike post-operative pain, induction of pain in OA may not necessarily be driven by classical inflammatory processes.
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Analgésicos/farmacologia , Artralgia/tratamento farmacológico , Fator de Crescimento Neural/agonistas , Osteoartrite do Joelho/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Receptor trkA/uso terapêutico , Analgésicos/síntese química , Animais , Artralgia/metabolismo , Artralgia/fisiopatologia , Modelos Animais de Doenças , Etanercepte , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/inervação , Articulação do Joelho/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/fisiopatologia , Medição da Dor/métodos , Fragmentos de Peptídeos/síntese química , Peptídeos/síntese química , Polissacarídeos/farmacologia , Receptores do Fator de Necrose Tumoral/uso terapêutico , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To determine the potential clinical efficacy of combination antibiotic therapy in treating rheumatoid arthritis (RA). METHODS: Twenty-one patients with active RA despite second-line treatment were randomized to receive either combination antibiotic therapy (treatment group, n = 11) or no additional therapy (control group, n = 10). Antibiotic therapy was given for 12 months and comprised oral tetracycline 250 mg twice daily, 3 times per week, and intravenous clindamycin infused on 5 consecutive days (300, 300, 600, 600, and 900 mg) followed by weekly infusions of 900 mg for 3 weeks and then fortnightly infusions for the remainder of the 12 months. The primary outcome measure was the American College of Rheumatology 20% (ACR20) response at the end of the initial treatment period of 12 months. RESULTS: Five patients in the treatment group (45%) achieved an ACR20 response at 1 year compared to none in the control group (p = 0.04). Eight patients in the treatment group and 1 in the control group had a greater than 20% improvement in tender joint count (p = 0.008). There were also significant differences between the groups in physician and patient global assessments. Nine patients in the treatment group completed the 6 months' followup; of these, 3 sustained the ACR20 response. CONCLUSION: Combined antibiotic therapy with intravenous clindamycin and oral tetracycline may be useful in the management of active RA. A double-blind, placebo-controlled trial of therapy is justified.