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Systemic Lupus Erythematous
What is Biomarker?
Proteomics
Biomarker Research Design
Lupus Nephritis
Liter...
3
• It is often abbreviated to SLE or lupus, It is a systemic
autoimmune disease (or autoimmune connective tissue
disease) t...
SLE Manifestations

5
6
• Not known when Lupus first appeared
– Hippocrates noted similar diseases in Ancient Greece
• Facial rash that resembles ...
• SLE most often harms the heart, joints, skin, lungs, blood
vessels, liver, kidneys, and nervous system.
• The course of ...
• SLE is one of several diseases known as "the great imitators"
because it often mimics or is mistaken for other illnesses...
There is no one specific cause of SLE. There are, however, a number of
environmental triggers and a number of genetic susc...
• During an immune reaction to a foreign stimulus, such as
bacteria, virus, or allergen, immune cells that would
normally ...
12
• A biomarker, is a measurement, including but not limited to a
genetic, biological, biochemical, molecular or imaging eve...
14
Two strategies for biomarker discovery and validation: a “targeted” approach, based on
hypothesis-driven evaluation of spe...
The platform for biomarker discovery in serum and tissue samples at our institution combines
multiple synergistic protein ...
PI

Mr (kDa)

3

4

5

6

7

8 9

10

9: Hsp70 subfamily B suppressor 1-like protein

116
97

97

11: Serum albumin
66

45...
2D-DIGE analysis of 2 different serum samples. One serum sample is labeled with Cy3 (green
color in this example), whilst ...
Mass Software

Mass Machine
19
20
21
22
23
• Isolate and identify proteins contained in the LN
Urinary protein signature (PS) of children with
SLE
• Assess the usefu...
Lupus Nephritis(LN) is among the main
determinants of poor prognosis in Systemic lupus
erythematosus (SLE).
25
• Systemic Lupus:
– most common and affects major organs
• Discoid Lupus:
– affects only the skin
– not fatal, but can cau...
• Lupus nephritis
– one of the most serious manifestations of SLE
– typically arises within 5 years of diagnosis
• commonl...
• There is a need for high quality accurate biomarker to
judge Lupus activity and renal damage with SLE.
• By using extens...
Investigation of specific protein
• Comparison of PS-protein plasma concentration
in the three groups of SLE patients (no ...
• To develop the LN protein signature (PS) as a novel
biomarkers, these biomarkers were detected on at least
two different...
 They measured plasma and urinary transferrin (Tf), plasma ceruloplasmin
(Cp), plasma α-1-acid-glycoprotein (AGP, also: o...
Sample
Collection

Fractionation

1D-SDS-PAGE

Mass
Spectrometry

In gel Digestion

2DElectrophoresis

Statistical
Analysi...
33
Values are means and SE. Significant differences are based on Tukey post-hoc testing.
The histograms show urinary concentr...
Values are means and SE. Significant differences are based on Tukey post-hoc testing.
The histograms show urinary concentr...
Values are means and SE. Significant differences are based on Tukey post-hoc
testing. The histograms show urinary concentr...
Values are geometric means of uncorrected urinary levels of Tf (A), Cp (B), AGP (C) and L-PGDS
(D) at months -6, -3 and 0,...
• They found high and increasing urinary levels of Tf
associated with active LN and impending worsening of LN
flares.
• Tf...
• Cp plays a critical physiological role in controlling the rate of iron
efflux from cells with mobilizable iron (Hellman ...
α1-acid-glycoprotein)
• AGP is a predictive biomarker for diabetic renal disease (Gomes et
al 2004) , and we provide initi...
• Lipocalins play a role in many biological processes, among them
immune responses and prostaglandin synthesis.
• L-PGDS, ...
• There is a need for high-quality accurate biomarkers to judge LN
activity and renal damage with SLE.
• By using a proteo...
• Tf, and AGP are part of LN protein signature (Varghese et al 2007)
• At present there is no universally accepted gold st...


Tf, Cp, AGP and L-PGDS are promising LN biomarkers.

 Their initial validation suggests superior measurement
propertie...
45
46
The AUCROC was calculated to assess the concurrent validity of the PSproteins and the traditional renal biomarkers to diag...
 In Mass Spectrometry the Proteins are analyzes on a normal binding Protein chip to
confirm the aimed mass spectrum.
 Pe...
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Biomarker Discovery and Validation

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Biomarkers Discovery and Validation

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Biomarker Discovery and Validation

  1. 1. 1
  2. 2. • • • • • • • • Systemic Lupus Erythematous What is Biomarker? Proteomics Biomarker Research Design Lupus Nephritis Literature Discussion Conclusion 2
  3. 3. 3
  4. 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. 5. SLE Manifestations 5
  6. 6. 6
  7. 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 SLE 7
  8. 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. 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. 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. 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 reactions 11
  12. 12. 12
  13. 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. 14
  15. 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 Society Matt P et al. Physiol. Genomics 2008;33:12-17 15
  16. 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 Society Matt P et al. Physiol. Genomics 2008;33:12-17 16
  17. 17. PI Mr (kDa) 3 4 5 6 7 8 9 10 9: Hsp70 subfamily B suppressor 1-like protein 116 97 97 11: Serum albumin 66 45 31 14: Epidermal growth factor receptor kinase substrate 8like protein 2 14 7 1D- SDS-PAGE GEL 20 Putative serine/threonine-protein phosphatase 4 regulatory subunit 1-like 2D- Gel Electrophoresis 17
  18. 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 Society 18
  19. 19. Mass Software Mass Machine 19
  20. 20. 20
  21. 21. 21
  22. 22. 22
  23. 23. 23
  24. 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. 25. Lupus Nephritis(LN) is among the main determinants of poor prognosis in Systemic lupus erythematosus (SLE). 25
  26. 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 away 26
  27. 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. 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) → α 1-acid-glycoprotein (AGP) → lipocalin- type prostaglandin-D-synthetase (L-PGDS) → Albumin and Albumin related fragments 28
  29. 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. 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. 31.  They measured plasma and urinary transferrin (Tf), plasma ceruloplasmin (Cp), plasma α-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. 32. Sample Collection Fractionation 1D-SDS-PAGE Mass Spectrometry In gel Digestion 2DElectrophoresis Statistical Analysis Conformational Assay Validation 32
  33. 33. 33
  34. 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<0.004; **= P<0.002; ¶= P<0.00001. 34
  35. 35. 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. PS-protein excretion standardized by urine creatinine (mg/mL urine) is shown. Significant differences between groups are indicated as follows: *= P<0.0005; **= P<0.0001; = P<0.05; ¶ = P<0.001. 35
  36. 36. 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. PS-protein excretion standardized by nonselective proteinuria is depicted with significant differences between groups indicated as follows: * = P<0.05; ** = P<0.005; = P<0.02; ¶ = P<0.009. 36
  37. 37. Values are geometric means of uncorrected urinary levels of Tf (A), Cp (B), AGP (C) and L-PGDS (D) at months -6, -3 and 0, respectively. Month 0 is the time point when the clinical diagnosis of the course of LN is made and months -3 corresponds to the time point of 3 months prior to the clinical diagnosis of the LN flare. ‘Improved LN’ describes the course of LN with decreasing renal SLEDAI scores; ‘worse LN’ describes the course of LN associated with an increase of the renal SLEDAI scores; ‘stable active LN’ describes patients with stable renal SLEDAI scores > 0; and ‘inactive LN’ describes the course of continuously inactive LN (renal SLEDAI = 0). Significant differences in the levels between two consecutive visits are indicated in the plots as follows. = P< 0.009; ¶ = P<0.0001; * = P<0.001. The above defined LN courses are depicted as follows: Improved LN, squares; Worsened LN, circles; Stable active LN, triangles; Inactive LN, diamonds. 37
  38. 38. • They found high and increasing urinary levels of Tf associated with active LN and impending worsening of LN flares. • Tf is co-regulated by interferon-α, involved in iron delivery, and the innate immune system. • Plasma Tf levels were correlated to global SLE disease activity in the past (Yilmaz.A et al 2005). • Thus, this study confirms these earlier findings in SLE, and new evidences provided that urinary Tf excretion may represent a predictive biomarker for LN. 38
  39. 39. • Cp plays a critical physiological role in controlling the rate of iron efflux from cells with mobilizable iron (Hellman et al 2003) . • Like Tf, plasma Cp has been recommended as a marker of global SLE disease activity (Yilmaz et al 2005, Hrycek.A et al 2007) . • Conversely, the results support that urinary Cp concentrations only differ with LN activity rather than extra renal disease activity. • Possibly, because urinary Cp levels vary widely in SLE, they were unable to detect a meaningful relationship to the course of LN. 39
  40. 40. α1-acid-glycoprotein) • AGP is a predictive biomarker for diabetic renal disease (Gomes et al 2004) , and we provide initial evidence that this is also the case for LN. • More importantly, urine concentrations of AGP (similar to Tf and L-PGDS) appear useful to anticipate LN flares, i.e. these markers may allow clinicians to preemptively adjust therapy prior to the appearance of overt worsening of LN. • Previous studies proposed plasma AGP to be a biomarker of SLE global disease activity (Lacki JK et al 1997, Meijer C et al 1993) . • The results support this (data not shown) but they also provide evidence that urinary AGP constitutes a biomarker of LN rather than extra renal disease activity. 40
  41. 41. • Lipocalins play a role in many biological processes, among them immune responses and prostaglandin synthesis. • L-PGDS, a lipocalin, is involved in nitric oxide regulation and the induction of apoptosis in the kidney. • L-PGDS has not been previously found to be a LN biomarker. Urine and plasma L-PGDS are considered sensitive indicators of chemotherapy- induced renal damage and diabetes-associated hypertension (Hirawa N 2002, Ogawa et al 2002). • They found urinary L- PGDS unrelated to the creatinine clearance in both JIA and SLE; L-PGDS also did not significantly change with cyclophosphamide exposure in SLE patients. 41
  42. 42. • There is a need for high-quality accurate biomarkers to judge LN activity and renal damage with SLE. • By using a proteomic approach for the discovery of novel LN biomarkers and identified a set of PS-proteins (i.e. Tf, Cp, AGP and L-PGDS). • In quantitative analysis, particularly urinary rather than plasma levels of the PS-proteins increased significantly with the presence of active LN. • Tf, CP, AGP and L-PGDS to be promising LN biomarkers, as their levels do not seem to change with the use of angiotensin inhibiting medications and even help discriminate patients who are at risk of a future LN flare. 42
  43. 43. • Tf, and AGP are part of LN protein signature (Varghese et al 2007) • At present there is no universally accepted gold standard for the measurement of LN activity. • The relevance of this findings is strengthened by the fact that the PS-Proteins performed similarly well to capture and anticipate the course of LN. • The sensitivity of moderately elevated protein :creatinine ratio to angiotensin blocking medications and its unproven ability for predicting LN flares . • They consider Tf, Cp, AGP, and L-PGDS to be promising LN biomarkers as their levels do not seem to change with the use of angiotensin inhibiting medications and risk of LN flare. 43
  44. 44.  Tf, Cp, AGP and L-PGDS are promising LN biomarkers.  Their initial validation suggests superior measurement properties compared to most traditional LN biomarkers and that Tf, AGP and L-PGDS are candidates of a novel set of predictive LN biomarkers.  Additional validation studies are mandatory to evaluate the usefulness of such a LN Renal Panel to predict the course of LN, the severity of kidney pathology, and the future development of renal damage with SLE. 44
  45. 45. 45
  46. 46. 46
  47. 47. The AUCROC was calculated to assess the concurrent validity of the PSproteins and the traditional renal biomarkers to diagnose the presence of active LN as measured by the SLEDAI and the BILAG, respectively (Table 3). Individual urinary PS-Proteins in performed all in the fair to good range according to current ROC interpretation standards (12), they were all better diagnostic markers of active LN than traditional renal biomarkers (all AUCROC <0.63) with the exception was the urine protein:creatinine ratio with an AUCROC at 0.91 (SLEDAI) and 0.85 (BILAG), respectively. 47
  48. 48.  In Mass Spectrometry the Proteins are analyzes on a normal binding Protein chip to confirm the aimed mass spectrum.  Peptides recovered from the in-gel digest were identified either by peptide mass fingerprints (PMF) on the SELDI-TOF platform or MALDI-TOF/MS and MS/MS fragmentation with sequencing individual peptides.  These two methods are necessary as albumin or albumin fractions are often were present.  Due to the unsuccessful removing of albumin by various methods there is a need to perform two methods to identify the specific novel proteins.  PMFs and MS/MS-fragmentation data were collected for each sample.  Both MALDI-TOF and TOF/TOF approaches were used, since the extreme abundance of albumin fragments interfered with the PMF identification for many of the bands.  The acquired peptide data from SELDI-TOF MS were searched via Mascot database search engine and the International Protein Index (IPI) human protein database.  For the MALDI-TOF MS/MS spectra, data were processed using an integrated GPSExplorer interface from Applied Bio systems coupled to a local Mascot Server with database searches against the entire NCBI database.  In either case, standard Mascot statistical criteria were used to indicate positive protein identification. 48

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