Pathways to Discovery Blog

Cutting edge Readouts for Rheumatoid Arthritis Model (part 2)

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Continuing the discussion of imaging technologies, this week we will cover biofluorescence and bioluminescence as readouts for RA models.

Traditional extrinsic fluorescent dyes fluoresce within the range of 300-500nm. Unfortunately, at these wavelengths, biological samples autofluoresce. Recent advancement in technology and development of dyes that fluoresce close to the near infrared (NIR) range (700-900nm) has produced dyes better suited for in vivo studies. A charged-coupled detector (CCD) is used to detect the photons emitted from these dyes after appropriate excitation. Initially these studies were limited due to interference from thermal energy. However, cooling of the chip markedly increased the quality of the image (1). Imaging using these techniques, coined fluorescence molecular tomography (FMT) or optical fluorescence tomography (OFT) in some studies, has been used to follow therapeutic agents (2), assess cell surface molecule expression (3) and to determine disease state by quantifying enzymatic activity (4, 5). Enzymatic activity is assessed using activity based probes (APBs). ABPs consist of a dye and quencher attached to opposite ends of a peptide linker. When the peptide is cleaved by a protease the signal is released. Commercially available APBs Prosense750 (which detects Cathepsin activity) and ProSense680 (which detects MMP activity) were used by Peterson et al (5) in their study assessing the performance of different therapeutic agents. There are also non-specific dyes which accumulate due to increased vascular leakage (6).

Bioluminescence

Like fluorescence imaging, bioluminescence imaging offers a non-toxic, non-invasive means of following specific events longitudinally in live animals. However, unlike fluorescence imaging, bioluminescence imaging requires no exogenous excitation. The most commonly used bioluminescence assay involves the luciferase gene. The luciferase gene, found naturally in glow worms and fireflies, oxidises Luciferin. This reaction results in the release of a photon, which can be quantified and localised with imaging systems. Luciferase-expressing cells can be transferred into hosts or Tg mice expressing luciferase under specific promoters of interest can be used in bioluminescence studies. After injection of the substrate luciferin the cells of interest can be imaged. Nakajima et al (7) used luciferase bioluminescence to show that adoptively transferred CII-specific T cells home to the joints of CIA arthritic mice. In other studies mice expressing luciferase under the Nf-kB (8) or human IL-1b (9) reporter were used to investigate arthritis models and the animal’s response to therapy. One group used bioluminescence in the CIA model to directly visualise their novel therapy (a cytolytic adenovirus) (10). Luminol is also a bioluminescent agent. When exposed to an appropriate oxidising species, luminol emits a blue luminescence. MPO activity at sites of inflammation can generate the necessary oxidative species to catalyse this reaction. Investigators have exploited this using a model of LPS-induced arthritis where luminol bioluminescence was shown to co-localise with sites of inflammation (11).

View pre-clinical models of Rheumatoid Arthritis

References

1. Spibey CA, Jackson P, Herick K.  Electrophoresis. 2001 22(5):829-36.
2. Paiframan R, Airey M, Moore A, Vulger A, Nesbitt A. J Immunol Methods. 31;348(1-2):36-41.
3. Hansch A, Frey O, Sauner D, Hilger I, Haas M, Malich A, Bräuer R, Kaiser WA.  Arthritis Rheum. 2004 50(3):961-7.
4. Wunder A, Tung CH, Muller-Ladner U, Weissleder R, Mahmood U.  Arthritis Rheum. 2004 50(8):2459-65.
5. Peterson JD, Labranche T, Vasquez KO, Kossodo S, Melton M, Rader R, Listello JT, Abrams MA, Misko TP.  Arthritis Res Ther. 2010 12(3):R105.
6. Hansch A, Frey O, Hilger I, Sauner D, Haas M, Schmidt D, Kurrat C, Gajda M, Malich A, Brauer R, Kaiser WA.  Invest Radiol. 2004 39(10):626-32.
7. Nakajima A, Seroogy CM, Sandora MR, Tarner IH, Costa GL, Taylor-Edwards C, Bachmann MH, Contag CH, Fathman CG.  J Clin Invest. 2001 107(10):1293-301.
8. Carlsen H, Moskaug JO, Fromm SH, Blomhoff R. J Immunol 2002 168(3):1441.
9. Li L, Fei Z, Ren J, Sun R, Liu Z, Sheng Z, Wang L, Sun X, Yu J, Wang Z, Fei J. BMC Immunol. 2008 9:49.
10. Chen SY, Shiau AL, Shieh GS, Su CH, Lee CH, Lee HL, Wang CR, Wu CL. Arthritis Rheum. 2009 60(11):3290-302.
11. Gross S, Gammon ST, Moss BL, Rauch D, Harding J, Heinecke JW, Ratner L, D. Nat Med. 2009 15(4):455-61.

Cutting Edge Readouts for Rheumatoid Arthritis Models (part 1)

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Rheumatoid arthritis is a chronic and progressive inflammatory condition estimated to affect between 0.5% and 1% of the world’s population, with more women being affected than men. RA is a systemic disease manifesting mainly as a disabling destruction of the synovial joints of the hands and feet.  In addition to the disability and decreased quality of life caused by RA, patients are at increased risk of developing cardiovascular disease. Joint destruction is induced by dysregulated immune activation of both the innate and adaptive immune responses resulting in alterations in the synovium, cartilage and bone.  The normal joint has a thin synovial lining (intimal lining layer), 1-3 cells thick. Beneath this is a sub-lining layer of connective tissue scattered with immune cells, blood vessels and nerve cells.  Together these layers form the synovium, which produces the synovial fluid that serves to lubricate the joint. Disease initiation results in profound changes in the structure and composition of the synovium and synovial fluid; with the infiltration of inflammatory cells, synovial cell hyperplasia, increased angiogenesis, fibroblast proliferation and extracellular matrix production. This increase in synovial cell proliferation can result in the lining increasing up to five times its original size and can result in pannus formation. The culmination of these events is bone and cartilage erosion and loss of joint function.

Extensive research spanning five decades has failed to elucidate the precise aetiology of RA. However, it is clear that the disease is complex, heterogenous and can probably be initiated by several mechanisms. The strongest association is with HLA II, although both genetic and environmental factors have been implicated in disease. Several animal models have been developed to study the mechanisms of disease and to screen potentially therapeutic agents. There are several commonly used induced models including Collagen-Induced Arthritis (CIA), Collagen-Antibody Induced Arthritis (CAIA), and Zymosan-induced arthritis. As well as several spontaneously arthritic mouse models: TNFa over-expressing transgenic (Tg) mice, K/BxN mice, SKG mice, Human DR4-CD4 mice, IL-1Ra^-/- mice. However, it is recent advances in imaging technology that has allowed these models to provide significantly better information about disease and potential therapies. Here, we discuss state of the art imaging modalities paying particular attention to the advantages and disadvantages of using these new technologies in RA models.

Magnetic Resonance Imaging (MRI)

MRI employs powerful magnets and radiowaves to create excellent 3D images with superb spatial resolution. Furthermore, information about metabolic processes, physiology and tissue status can be obtained with MRI scanning. The magnetic field created by the scanner causes the body’s hydrogen atoms to line up in a specific orientation. Radiowaves are then sent towards these atoms and a computer records the signals that return. Bone erosion, synovitis, tendonopathy, and bone oedema can all be detected using this technique. In contrast to CT, MRI has improved soft tissue contrast and does not expose animal to low dose radiation. In addition, MRI does not always require contrast enhancing agents, minimising side effects on subjects. However, contrast enhancing agents such as gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) and ultra-small super paramagnetic iron oxide (USPIO) particles can be used to maximise the information retrieved by MRI. Gd-DTPA can generate information about vascular flow and permeability as well as information about intra-articular extracellular space, whereas, USPIO particles can generate information about articular content. Several studies that used this technique have shown that MRI technology can follow disease progression using synovial inflammation and draining lymph node volume as biomarkers. Importantly, these biomarkers respond to therapy and thus can be used to screen new potential therapies^1-4. IV injection of USPIO particles leads to their accumulation within macrophages of the endoreticular system. These macrophages can be tracked and are recruited to the joint during disease⁵. MR technology has also been used to follow T cell fate in vivo. In these studies T cells are loaded ex vivo and reintroduced into the mouse which is then scanned to detect where the T cell localise ^{6, 7}. MR scanning can detect disease before irreversible damage occurs. This in conjunction with the ability to image the same animal repeatedly results in MR scanning being an extremely powerful technique allowing longitudinal studies in the same animal where early disease can be followed and the response to therapy assessed.

Download whitepaper: Collagen antibody-induced arthritis. A short, more synchronized alternative to the CIA model.

References:

1. Dardzinski BJ et al., Magn Reson Imaging. 2001 (9):1209-16.
2. Proulx ST, et al., Arthritis Rheum. 2007 56(12):4024-37.
3. Guo R, et al., Arthritis Rheum. 2009 60(9):2666-76.
4. Lee SI et al., J Radiol. 2009 10(6):651.
5. Beckmann N et al., Magn Reson Med. 2003 49(6):1047-55.
6. Dodd SJ et al.,  Biophys J. 1999 76(1 Pt 1):103-9.
7. Josephson L et al., Bioconjug Chem. 2002 13(3):554-60.

Targeting fibroblast-like synovioctyes: preclinical screening assays.

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Rheumatoid arthritis (RA) is a chronic autoimmune joint disease characterized by inflammation of the synovium and destruction of cartilage and bone. During synovial inflammation, inflammatory cells (macrophages, mast cells, dentritic cells and lymphocytes) are recruited while resident cells (fibroblast synoviocytes, chondrocytes, osteoclasts, and osteoblasts) are altered to support the inflammatory process.  Together, these events create a pathological tissue response.
 
The synovium consists of two layers, the sublining and intimal lining.  In RA, the sublining becomes infiltrated with mononuclear cells, B lymphocytes produce autoantibodies, blood vessels proliferate, lymphoid aggregates form and the intimal lining shows increased cellularity.  Macrophages in the synovium produce pro-inflammatory cytokines, chemokines and growth factors which in turn activate fibroblast-like synoviocytes (FLS) to produce their own array of mediators (e.g. proteolytic enzymes, chemokines and cytokines).  This produces a paracrine/autocrine network that leads to synovitis, the recruitment of new cells and the destruction of the extracellular matrix.  Fibroblast-like synoviocytes have emerged as key pro-inflammatory cells promoting the disease, largely due to their ability to produce massive amounts of degradative enzymes.
 
The availability of biological therapies has improved clinical outcomes by decreasing inflammation and joint destruction, however only about half of the patients exhibit substantial efficacy. Targeting FLS may further improve clinical outcomes without suppressing systemic immunity.  In vitro FLS assays can be used to evaluate effective therapies for arthritis. Using FLS obtained from normal, RA and OA patients, we can evaluate a compound's effect on the production of pro-inflammatory mediators in a preclinical in vitro model. 

MD Biosciences preclinical services has established an in vitro cytokine-stimulated synoviocyte screening assay. Example data shown below is from normal, OA-postiive and RA-positive tissue (see more data on the assay page). Contact a scientist to establish a protocol relevant to your compound.

Effiacy of anti-CD20 therapy in Rhuematoid Arthritis

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We read an interesting article published this week in Journal of Immunology (v184 Bottaro & co.) on the efficacy of anti-CD20 therapy in RA. The article highlights the continuing uncertaintity over the mode of action of B-cell directed therapy in Rheumatoid Arthritis (RA) [review of the differing theories is presented in Clin Exp Immunol. 2009 Aug;157(2):191-7].

Therapies such as rituximab (anti-CD20) may be involved in one or more of the following:

• remove plasma cell precursors thereby decreasing auto-reactive monoclonal antibodies
• deplete the B cells that act as antigen presenting cells to auto-reactive T cells
• remove a cytokine producing B-cell population
• disrupt peripheral lymphoid tissue
• or a combination of the above.

The authors previously used MRI to measure the synovial volume in the TNF-Tg mice and demonstrated a relationship to popliteal lymph node (PLN) volume. They observed that synovial volume is relatively constant while PLN volume increases however when the PLN "collapses" then synovial volume increases dramatically. This lead to the question of what happens when the PLN collapses that it appears to induce the pathological synovial changes.

In this paper the authors show, by immunohistochemistry, huge changes in the PLN architecture after collapse, characterised by influx of B cells into the paracortical sinuses and T cell area. The authors characterise these B cells as a unique population of previously undefined B cells which are also present in the KBxN mouse model of RA. The authors then to go on to show that this population of B cells are depleted by anti-CD20 therapy which is also surprisingly efficacious in the TNF-Tg model despite its previous appearance of being T and B cell independent model. The overall message from the paper is definition of a unique B cell population that may be the target of anti-CD20 therapy.

Pre-clinical efficacy models of Rheumatoid Arthritis:

Collagen-induced arthritis
Collagen-antibody induced arthritis
Adjuvant-induced arthritis

Download the Whitepaper:
Collagen-antibody induced arthritis: A short, synchronized and rapid alternative to the Collagen-induced arthritis model.