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2. Systemic Lupus Erythematous What is Biomarker? Proteomics Biomarker Research Design Lupus Nephritis Literature Discussion Conclusion 2 3. 3 4. It is often abbreviated to SLE or lupus, It is a systemic autoimmune disease (or autoimmune connective tissue disease) that can affect any part of the body. As occurs in other autoimmune diseases, the immune system attacks the body's cells and tissue, resulting in inflammation and tissue damage. It is a Type III hypersensitivity reaction in which antibody-immune complexes precipitate and cause a further immune response. 4 5. SLE Manifestations5 6. 6 7. Not known when Lupus first appeared Hippocrates noted similar diseases in Ancient Greece Facial rash that resembles the markings of a wolf 1851 French-man named Pierre Cazenave first clinical records More than 1.4 million Americans are affected by SLE7 8. SLE most often harms the heart, joints, skin, lungs, blood vessels, liver, kidneys, and nervous system. The course of the disease is unpredictable, with periods of illness (called flares) alternating with remissions. The disease occurs nine times more often in women than in men, especially in women in child-bearing years ages 15 to 35, and is also more common in those of non-European descent There is no cure for SLE. It is treated with immunosuppression, mainly with cyclophosphamide, corticosteroids and other immunosuppressants. SLE can be fatal. The leading cause of death is from cardiovascular disease due to accelerated atherosclerosis 8 9. SLE is one of several diseases known as "the great imitators" because it often mimics or is mistaken for other illnesses. SLE is a classical item in differential diagnosis because SLE symptoms vary widely and come and go unpredictably. Diagnosis can thus be elusive, with some people suffering unexplained symptoms of untreated SLE for years. Common initial and chronic complaints include fever, malaise, joint pains, myalgia, fatigue, and temporary loss of cognitive abilities. Because they are so often seen with other diseases, these signs and symptoms are not part of the diagnostic criteria for SLE. When occurring in conjunction with other signs and symptoms however, they are considered suggestive. 9 10. There is no one specific cause of SLE. There are, however, a number of environmental triggers and a number of genetic susceptibilities @ One manifestation of SLE is abnormalities in apoptosis, a type of programmed cell death in which aging or damaged cells are neatly disposed of as a part of normal growth or functioning. @ In SLE, the body's immune system produces antibodies against itself, particularly against proteins in the cell nucleus. @ SLE is triggered by environmental factors that are unknown. @ In order to preserve homeostasis, the immune system must balance between being sensitive enough to protect against infection, and becoming sensitized to attack the body's own proteins (autoimmunity).10 11. During an immune reaction to a foreign stimulus, such as bacteria, virus, or allergen, immune cells that would normally be deactivated due to their affinity for self tissues can be abnormally activated by signaling sequences of antigen-presenting cells. Thus triggers may include viruses, bacteria, allergens (both IgE and hypersensitivity), and can be aggravated by environmental stimulants such as ultraviolet light and certain drug reactions11 12. 12 13. A biomarker, is a measurement, including but not limited to a genetic, biological, biochemical, molecular or imaging event whose alternations correlate with disease pathogenesis and/or manifestations and can be evaluated qualitatively and/or quantitatively in laboratories. Several criteria are required for a laboratory measure to serve as a reliable biomarker, including: 1. It must be biologically and patho physiologically relevant 2. It must be simple for routine practice 3. It must accurately and sensitively respond to change in disease activity 13 14. 14 15. Two strategies for biomarker discovery and validation: a targeted approach, based on hypothesis-driven evaluation of specific biomarker candidates, and a de novo discovery approach using different proteomic technologies followed by validation of potential Biomarker Candidate...2008 by American Physiological SocietyMatt P et al. Physiol. Genomics 2008;33:12-1715 16. The platform for biomarker discovery in serum and tissue samples at our institution combines multiple synergistic protein fractionation and separation methods including one- and twodimensional gel electrophoresis (1-DE, 2-DE), differential in-gel electrophoresis...2008 by American Physiological SocietyMatt P et al. Physiol. Genomics 2008;33:12-1716 17. PIMr (kDa)345678 9109: Hsp70 subfamily B suppressor 1-like protein116 979711: Serum albumin 6645 3114: Epidermal growth factor receptor kinase substrate 8like protein 214 71D- SDS-PAGE GEL20 Putative serine/threonine-protein phosphatase 4 regulatory subunit 1-like2D- Gel Electrophoresis 17 18. 2D-DIGE analysis of 2 different serum samples. One serum sample is labeled with Cy3 (green color in this example), whilst the other is labeled with Cy5 (blue), and equal concentrations of both samples are labeled with Cy2 (red). All 3 labeled samples are then combined, separated on the same 2-D gel, and scanned at different emission wavelengths, which allows the differentially expressed proteins to be viewed as changed in color; see arrows for green or blue spots in enlarged gel area. Proteins that are equally expressed in both samples appear as white spots. Matt P et al. Physiol. Genomics 2008;33:12-17 2008 by American Physiological Society18 19. Mass SoftwareMass Machine 19 20. 20 21. 21 22. 22 23. 23 24. Isolate and identify proteins contained in the LN Urinary protein signature (PS) of children with SLE Assess the usefulness of the PS-Proteins for detecting activity of LN over time.24 25. Lupus Nephritis(LN) is among the main determinants of poor prognosis in Systemic lupus erythematosus (SLE). 25 26. Systemic Lupus: most common and affects major organs Discoid Lupus: affects only the skin not fatal, but can cause severe scarring Drug-induced Lupus: is systemic Lupus caused by medications when the medicine is stopped, the disease goes away26 27. Lupus nephritis one of the most serious manifestations of SLE typically arises within 5 years of diagnosis commonly within the first 6 to 36 months Silent lupus nephritis normal urinalysis no proteinuria normal serum creatinine levels However, renal biopsy reveals pathological changes 27 28. There is a need for high quality accurate biomarker to judge Lupus activity and renal damage with SLE. By using extensive proteomic approach for the discovery of novel LN biomarkers and identified a set of PSproteins. The set of PS-Proteins identified are Transferrin (Tf) Ceruloplasmin (Cp) 1-acid-glycoprotein (AGP) lipocalin- type prostaglandin-D-synthetase (L-PGDS) Albumin and Albumin related fragments 28 29. Investigation of specific protein Comparison of PS-protein plasma concentration in the three groups of SLE patients (no LN, active LN, inactive LN) and two groups of controls (active JIA, inactive JIA (Juvinile Idiopathic Arthritis) Studied the statistically important difference under a multivariate fixed effect model framework to analyze the PS-protein effect in SLE as well as LN. 29 30. To develop the LN protein signature (PS) as a novel biomarkers, these biomarkers were detected on at least two different protein chips The peak intensity should be > 100 fold increase in between groups. By using SDS-PAGE and 2D-Gel electrophoresis the significantly expressed bands are identified Then the bands are excised and digested with trypsin And recovered for Mass spectrometry. 30 31. They measured plasma and urinary transferrin (Tf), plasma ceruloplasmin (Cp), plasma -1-acid-glycoprotein (AGP, also: orosomucoid), as well as plasma and urine lipocalin-type prostaglandin-D synthetase (L-PGDS) by immunonephelometry (Dade Behring BNII Prospect, Marburg, Germany). Urinary Cp was quantified by ELISA (Human Ceruloplasmin ELISA Quantitation Kit; Genway Biotech, Inc., San Diego, CA, USA); and urinary AGP by ELISA (Human Orosomucoid ELISA Quantitation Kit; Genway Biotech, Inc., San Diego, CA, USA). Then they determine the concentration of plasma and the identified specific important proteins concentration by Tukey-Post hoc Testing technique. An AUCROC of 1.0 represents a perfect biomarker whereas a value of 0.5 is no better than expected by chance. Statistical computations were conducted using SAS version 9.1 (SAS, Cary, NC, USA) software. P-values < 0.05 were considered statistically significant.31 32. Sample CollectionFractionation1D-SDS-PAGEMass SpectrometryIn gel Digestion2DElectrophoresisStatistical AnalysisConformational AssayValidation 32 33. 33 34. Values are means and SE. Significant differences are based on Tukey post-hoc testing. The histograms show urinary concentrations of Tf (A), Cp (B), AGP (C) and L-PGDS (D) for the groups defined as Figure 1. Uncorrected PS-protein levels (per mL or dL of urine) are depicted. Significant differences between groups are indicated as follows: * = P