Identification and Quantification of protein and nucleic acid based biomarkers in diagnosis and prognosis of autoimmune joint diseases.

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Research Proposal

Title : Identification and Quantification of protein and nucleic acid based biomarkers in diagnosis and prognosis of autoimmune joint diseases.

Summary

Presence of some oxidoreductases secreted under inflammatory conditions either in the circulation or in the releasate from inflammatory macrophages and peripheral blood mononuclear cells and association of their aberrant expression with onset and progression of inflammatory response under oxidative stress has opened up a new field for molecular diagnosis of these pathological states. Osteoarthritis, rheumathoid arthritis and systemic lupus erythematosus are the major causes of muscoskeletal impairment world wide. Advance approaches for identification of some oxidoreductases such as xanthine oxidoreductase (XOR), thioredoxin (Trx) and peroxiredoxin (Prx) are required to overcome impairments caused by autoimmune joint diseases. Free radicals are very reactive chemical species damage both chondrocytes, and extracellular matrix (ECM) components of articular cartilage. Free radicals shift the redox balance in articular cartilage in direction of oxidants and serve as potential biomarkers in diagnosis of various inflammatory responses. Protein and nucleic acid based biomarkers are potential markers to identify the factors affecting muscle and joint injuries. In this study, the levels of various oxidoredutive enzymes in patient samples would be identified, quantified and would try to correlate the levels of specific protein signatures, released in circulation and inflammatory macrophages, with the severity of disease and correlating the effect of oxidative stress and inflammation on the expression level of these signatures and ultimately to joint diseases. Various enzyme assays for oxidoreductases identification and estimation would be used. Quantitative RT-PCR and microarray based approachs involving a single step reaction for all signatures analysis wold be used for quantification of expression levels of these enzymes. Protein signatures would also be identified as estimated by 2- Dimensional gel electrophoresis 2D-PAGE followed by mass spectrometry. Real-time PCR and microarray analyses, mass spectrometry and enzyme assays of some oxidoreductases and other proteins would possibly reveal significantly elevated levels of these biomarkers in patients in comparison with healthy individuals. Markedly increased level of specific signatures could devlope a set of new biomarker for early detection of joint diseases caused due to inflammatory responses.

Introduction and Background

Autoimmune joint disesese are the leading cause of joint impairment in the World with higher rate in women than men. Oxidative stress is a major factor causing traumatic injury or autoimmune diseases. Oxidative stress is basically the imbalance between the free radicals produced in side the body and the antioxidant produced in the body. Articular cartilage is damaged directly or indirectly by ROS (Reactive oxigen species) and RNS (Reactive nitrogen species) through up- regulation of the mediators produced during ECM (extracellular matrix) degradation. Aggrecan, a major component of ECM, is often degraded by Reactive oxygen/ nitrogen species, e.g. ONOO-, and this degradation futher promotes cartilage destruction (Billinghurst et al., 1997). Free radicals causes excess oxidation of the redox balance in articular cartilage. (Hajdigogos et al., 2003).

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease primarily affecting synovial membranes of joints. RA is a multistep process in which cellular and humoral responses are mediated by lymphocytes (T and B cells) and non hematopoietic cells like fibroblasts, connective tissue cells, and bones. At site of inflammation, T cells and macrophages are activated following a large increase in oxygen expenditure, whose outcome is amplified release of ROS (Afonso et al., 2007).

Similarly, systemic lupus erythematosus (SLE) is an example of autoimmune, multifactorial disease categorized by the existence of auto-antibodies against various nuclear antigens such as DNA and histones, as well as protein antigens and protein–nucleic acid complexes. (Khan, Siddiqui, 2006). Autoantigens are produced by free radicals, and these antigens put in to disease progress. Oxidative stress and inflammation are interconnected in SLE. Levels of interferon-gamma (INF-g), and interleukin- 12 (IL- 12) are certainly linked with malondialdehyde (MDA) levels in lupus disease (Shah et al., 2010).

Articular cartilage is a distinctive tissue having chondrocytes, which sustain the cartilage matrix through their persistent synthesis and deprivation. The environment is avascular, hyperosmotic and acidic. Cartilage cells constitute synoviocytes, and chondrocytes. Chondrocytes maintain anerobic environment for metabolism where as synoviocytes deliver nutrients to the avascular cartilage through synovial fluid. The oxygen stress in the area is usually low due to avascular environment, but in pathological conditions, like inflammation, oxygen pressure is subjected to imbalance. These fluctuations of oxygen levels services chondrocytes to generate reactive species. The major reactive species produced by chondrocytes are O2- radical, and NO that produce other derived radicals, including ONOO-and H2O2 (Hiran et al., 1997; Stefanovic- Racic et al., 1997). Free radical levels produced by chondrocytes are important for the maintenance of ion homeostasis and fluctuation in their levels may interference with protein phosphorylation. (Gibson et al., 2008).

Oxygen radicals can directly alter collagen, that resists the swelling pressure of aggrecan- hyaluronate aggregates by providing tensile strength and forming a network,. Free radicals prime collagen to proteolytic enzymes. (Monboisse & Borel, 1992). Interestingly, free radicals may destruct collagen synthesis indirectly. NO inhibits collagen synthesis via interleukin-1 (IL-1) (Cao et al., 1997).

Synoviocytes are the second kind of articular cells. These cells consume larger amount of oxygen when compared to chondrocytes (Schneider et al., 2005). An indirect evidence of oxidative stress in synoviocytes is the incidence of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase in these cells (Mattey et al., 1993). Oxidative stress makes synoviocytes to undergo a cell death of an apoptotic nature (Galleron et al., 1999). The ROS scavenger system of synoviocytes protects chondrocytes from toxic effects of free radicals.

In autoimmune joint diseases systemic inflammation exists long before it exerts local effects on synovial membrane. At one point, systemic inflammation is translocated into synovium where it initiates the inflammatory response often leading to oxidative burst. Oxidative burst in rheumatoid joints is a result of the activation of innate immune system cells. Activated phagocytic cells such as neutrophils, and macrophages both produce free radicals in the joint area. Activated phagocytes produce reactive oxidants by enzymes: the NADPH- oxidase, and the nitric oxide synthase (NOS).

Pro-inflammatory cytokines´ presence and activity undoubtedly subjects to rheumatoid synovitis governing a variety of pathological processes including cell activation, cell proliferation, tissue resorption and chemotaxis (Schett et al., 2000). Experimental evidence confirms cytokine- induced oxidative stress in rheumatoid synovium. Thioredoxin, a cellular catalyst induced by oxidative stress, is found in high amounts in RA synovial cells and tissue. Thioredoxin acts as a co- factor for tumor necrosis factor- alpha (TNF-induced synthesis of interleukins (IL-6 and IL-8) in synovial fibroblastlike cells (Yoshida et al., 1999).

ROS are documented as mediators of synovial inflammation. Excessive production of ROS at the site of inflammation contributes to the inflammatory process in general, by induction of the expression of adhesion molecules, pro-inflammatory cytokines, and chemoattractants. (Maurice et al., 1997).

A short lifetime of free radicals in body fluids restricts their direct estimation instead effects of oxygen on lipids, proteins, and nucleic acids molecules are used. There are many chemical modifiers of protein, and lipid structures, whose activity is accelerated by oxidative stress. N-carboxymethyllysine (CML) represents a chemically modified amino acid and originates in vivo from carbohydrate as well as from lipid derived precursors.

Oxidative damage to proteins is also reflected by increased levels of advanced oxidation protein products (AOPP), which form by the reaction between chlorinated oxidants (HOCl/OCl_) and proteins. AOPP are defined as dityrosine-containing cross-linked proteins. MDA is one of the end products of lipid peroxidation induced by ROS and marker of oxidative stress in lipids. 8-Hydroxy-2´-deoxyguanosine (8-OHdG) has been recognized as a biomarker of oxidative DNA damage by endogenously generated oxygen radicals. In addition, oxidative stress may be estimated by levels of enzymes like thioredoxin, a protein with reduction / oxidation active disulfide / dithiol groups in its active site. Signs of oxidative stress are apparent on cells and tissue affected by arthritis. SODs exert protective effects in animal models of inflammation. In mice, genetically deficient in SOD III, both the severity of collagen- induced arthritis and the production of pro- inflammatory cytokines are increased (Ross et al., 2004).

Biomarkers are used for timely diagnosis, growing number of novel markers have been identified to predict outcome following inflammatory responses under oxidative stress. This may facilitate to give reasonable therapy to high-risk patients (Chan and Ng, 2010).Presently, the biochemical markers used for diagnosis of autoimmune joint diseases are conventional markers. Therefore, other biochemical markers must be sought that provide important information and might give an earlier sign of an ongoing inflammatory response against oxidative stress.(Labugger et al., 2000).

In the light of this introduction and background it could be hypothesized that the specific oxidoreductases are affected by the free radicals under oxidative stress and might release into the circulation or present in macrophages as a result of inflammatory response and could be used to detect and monitor the expression profile of autoimmune joint diseases and serve as potential biomarkers for the diagnosis of joint and other inflammatory diseases.

Objectives

  • Identification of protein based changes in patients with autoimmune joint diseases like rheumatoid arthritis and Systemic lupus erythematosus.
  • To develope assays for the measurement of oxidoreductases in biological samples and exploration of their levels in plasma and synovial fluids of the patients and healthy subjects.
  • Correlation of oxidoreductase levels with those of TNF and other standard markers of inflammation.
  • Providing evidence that these oxidoreductases including xanthine oxidoreductase (XOR) and two protein thiol-disulfide oxidoreductases: thioredoxin (Trx) and peroxiredoxin (Prx) are secreted under inflammatory conditions and can be detected either in the circulation or in the releasate from inflammatory macrophages.
  • Identification of set of new inflammatory biomarkers in chronic inflammatory diseases and for observing the patients’ status and the effectiveness of drugs in clinical trials.

Methodology

  • Sample collection, processing and storage

Blood samples and synovial fluid samples will be collected. Blood samples will be collected in EDTA tubes from patients and controls. Samples will be collected on the onset of the symptoms. These samples will be centrifuged at 3000g for 4 minutes at 4°C. Plasma will be collected in sterile tubes. Aliquots will be prepared for proteome analysis and will be stored at -80°C.

  • Proteome Analysis

Protein quantification

Total protein contents in plasma and synovial fluid would be estimated by Bradford assay (Bradford, 1976).

  • 1-D-PAGE (Sodium dodecyl Sulphate polyacrylamide gel electrophoresis) (SDS-PAGE)

For total plasma protein analysis SDS-PAGE would be done. All reagents will be prepared using a modification of the procedure described by Laemmli (1970).

  • 2-D-PAGE (2-Dimensional gel electrophoresis)
  • Mass spectrometric analysis of the spots (MALDI) analysis
  • Proteins analysis by liquid chromatography-tandem mass spectrometry.
  • Nucleic acid isolation and quantification

RNA of plasma will be isolated using Tri-Reagent. The quantity of isolated RNA will be measured and PBMCs ( peripheral blood mononuclear cells) will be isolated from heparinised blood on Ficoll gradients.

  • Idenification of gene expression signature in peripheral blood mononuclear cells by microarrays and real-ime polymerase chain reaction (qPCR)

For quantitative analysis of RNA present in patient sample and PBMCs in normal samples quantitative real-time PCR will be performed using Real-Time System and specific probes. Quantification will be based on determination of the quantification cycle (Cq).

Expected Outcomes :

Characterization of patients’ samples.

  • The biochemical and clinical history reports of patients collected from Pathology laboratory having study cohort including Age, gender, Smoking, Family History, Hypertension, Diabetes, would provide correlation of these factors and parameters with joint impairments.
  • Protein analysis would provide evidences for the protein based biomarkers. It would provide valuable insight of the changes at protein level of plasma of patients.
  • While Quantitative real time analysis of enzymes at their expression levels would provide nucleic acid based biomarkers of autoimmune joint diseases.
  • This study would provide clinical evidence that oxidoreductases that can be detected in plasma and synovial fluids are related to inflammatory response due to oxidative stress. By examining few measurable proteins, and comparing expression levels of these proteins in patients with joint diseases like rheumatoid arthiritus, we could begin to define a set of new biomarkers that are altered and provide potential targets that influence autoimmune joint diseases. Our study would demonstrate circulating as well cell specific oxidoreductases as a marker of diseases like rheumatoid arthiritus and Systemic lupus erythematosus. Furthermore the use of “qRT-PCR and 2-D PAGE followed by mass spectrometric analysis would be the most susceptible and explicit means for the assessment of expression profiling to detect changes at DNA or RNA and protein profiles, elucidating clinical diagnosis and prognosis. It would be possible that the detection of a set of new biomarkers can be included in future routine clinical examinations for the diagnosis purpose and for monitoring the patients’ status and the efficacy of drugs in clinical trials.

References

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Billinghurst, R.C. Dahlberg, L. Ionescu, M., Reiner, A. Bourne, R. Rorabeck, C. Mitchell, P. Hambor, J. Diekmann, O. Tischesche, H. Chen, J. Van Wart, H. Poole, A.R.. (1997). Enhanced cleavage of type ll collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest, Vol. 99, No. 7, pp.1534–1545.

Breedveld, F.C.& Verweij, C.L. (1997). Evidence for the role of an altered redox state in hyporesponsiveness of synovial T cells in rheumatoid arthritis. J Immunol, Vol. 158, No.3, pp. 1458- 1465.

Cao, M. ; Westerhausen-Larson, A. ; Niyibizi, C. ; Kavalkovich, H.I. ; Georgescu, H.I. ; Riyyo, C. F. ; Hebda, P.A. ; Stefanovic-Racic, M. & Evans, C.H. (1997). Nitric oxide inhibits the synthesis of type II collagen without altering Col2A1 mRNA abundance : prolyl hydroxylase as a possible target. Biochem. J, Vol. 324, No. Pt1 , pp. 305- 310.

Galleron, S.; Borderie, D.; Ponteziere, C.; Lemarechal, H.; Jambou, M.; Roch- Arveiller, M.; Ekindjan, O.G. & Calls, M.J. (1999). Reactive oxygen species induce apoptosis of synoviocytes in vitro. Alpha tocopherol provides no protection. Cell Biol Int, Vol. 23, No. 9, pp. 637- 642.

Gibson, J.S.; Milner, P.I.; White, R.; Fairfax, T.P.A. & Wilkins, R.J. (2008). Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis. Pflugers Arch - Eur J Physiol, Vol. 455, No. 4, pp.563–573. Monboisse, J.C. & Borel, J.P. (1992). Oxidative damage to collagen. EXS, Vol. 62, pp. 323- 327.

Hadjigogos, K. (2003). The role of free radicals in the pathogenesis of rheumatoid arthritis. Panminerva Med, Vol. 45, No. 1, pp. 7-13.

Khan, F. & Siddiqui, A.A. (2006). Prevalence of anti- 3- nitrotyrosine antibodies in the joint synovial fluid of patients with rheumatoid arthritis, osteoarthritis and systemic lupus erythematosus. Clin Chim Acta, Vol. 370, No. 1-2, pp. 100-107.

Labugger, R., Organ, L., Collier, C., Atar, D. and Van Eyk, J. E. (2000). Extensive troponin I and T modification detected in serum from patients with acute myocardial infarction. Circulation, 102:1221– 6.

Martina Škurlová (2012). Oxidative Stress in Human Autoimmune Joint Diseases, Oxidative Stress and Diseases, Dr. Volodymyr Lushchak (Ed.), ISBN: 978-953-51-0552-7

Mattey, D.L.; Nixon, N.; Alldersea, J.E.; Cotton, W.; Fryer, A.A.; Zhao, L.; Jones, P. & Strange, R.C. (1993). Alpha, mu and pi class glutathione S- transferases in human synovium and cultured synovial fibroblasts: effects of interleukin-1 alpha, hydrogen peroxide and inhibition of eicosanoid synthesis. Free Radic Res Commun, Vol. 19, No. 3, pp. 159- 617.

Maurice, M.M.; Nakamura, H.; van der Voort, E.A.; van Vliet, A.I.; Staal, F.J.; Tak, P.P;

Ross, A.D.; Banda, N.K.; Muggli, M.; Arend, W.P.(2004). Enhancement of collagen induced arthritis in mice genetically deficient in extracellular superoxide dismuthase. Arthritis Rheum, Vol. 50, No. , pp. 3702- 3711.

Schett, G.; Tohidast- Akrad, M.; Steiner, G. & Smolen, J. (2001). Commentary. The stressed synovium. Arthritis Res, Vol. 3, No. 2, pp. 80-86.

Schneider, N.; Mouithys- Mickalad, A.L.; Lejeune, J.- P.; Deby-Dupont, G.P.; Hoebeke, M. & Serteyn, D.A. (2005). Synoviocytes, not chondrocytes, release free radical after cycles of anoxia/ re-oxygenation. Biochimical and Biophysical Research Communications, Vol. 334, No. , pp. 669- 673.

Shah, D.; Kiran, R.; Wanchu, A. & Bhatnagar, A. (2010). Oxidative stress in systemic lupus erythematosus: relationship to Th1 cytokine and disease activity. Immunol Lett, Vol. 129, pp. 7-12.

Yoshida, S.; Katoh, T.; Tetsuka, T.; Uno, K.; Matsui, N. & Okamoto, T. (1999). Involvement of thioredoxin in rheumatoid arthritis: its costimulatory roles in the TNF-g- induced production of IL-6 and IL-8 from cultured synovial fibroblasts. Immunol, Vol. 163, No. 1, pp. 351-358.

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