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Clinical BiomarkerDefined Biomarkers in Every Disease in Human Blood
Clinical Biomarker
F.rezaee@erasmusmc.nlF.rezaee@ACTA.nlF.rezaee@UMCG.nlFarhad.Rezaee@clinicabiomarkers.comwww.clinicabiomarkers.com
Contact with Dr. F. Rezaee (associated professor in Erasmus Medical Center)
www.clinicabiomarkers.com
Biomarkers
1- Clinical Biomarkers (Disease state)2- Cellular Biomarkers (Cell function)
What is a Clinical Biomarker?
An indicator for the presence of a disease state in Human
Clinical Biomarkers
1- Diagnostic Clinical Biomarkers (Indicator or predictive of existed Diseases)2- Prognostic Clinical Biomarkers (provide knowledge about the cause or development of a disease or even recovery of a disease)
Identification of Clinical Biomarkers in Human Body
1- Human Tissues (e.g. Liver, Spleen, Pancreas, etc.)2- Human Primary Cells (e.g. Adipocytes, Macrophages,
Platelets,etc.)3- Human Body Fluids (e.g. Cerebrospinal fluid (CSF),
Blood (Plasma, Serum), Saliva (extracellular fluid, etc.)
Identification of Clinical Biomarkers in Human Plasma versus other Human Materials
Simple 1- Hard Intervention2- High Risk3- Pain4- Blood Contamination5- Unknown Source 6- No clear Biomarker?
1- Defined Biomarkers2- Source independet3- No Intervention 4- No Risk5- No Pain
Advantages DisadvantagesDisadvantagesAdvantages
1- Very Complex2- ≈ 15000 Proteins3- 40 Proteins (≈95% of plasma protein Concentrations)
Other Human Materials Human Plasma
PlasmaBuffy Coat
Red Cells
Syst
ems B
iolo
gy
Transcriptomics
Proteomics
Phospholipidomics
Imageomics
Metabolomics
Glycomics
Kinomics
NanoBioMedicine
Conventionaltechnology
Human Plasma of patients
Human Plasma of Controls
Standard Systems Biology: Identification of Biomarkers for different diseases in
Human Plasma
S ystem s B io lo g yD e f in it io n
In a scientifically context, Systems Biology can be referred as “the use of a combination of all techniques and knowledge (or develop novel
ones) to find answer to any biological and medical questions
Standard Systems Biology: Identification of Biomarkers for different diseases in
Human Plasma?
1- Biological Knowledge
2- Molecular Knowledge
3- Cellular Knowledge
Advantages Disadvantages
1- Not suitable to find clinical biomarkers (proteins) for diseases in blood at present
2- Not suitable for the detection of very low abundant proteins at present
This indicates that an innovative and original strategy is essential to detect the disease at a very early stage in the patients for the
therapy
Systems Biology: Identification of Biomarkers for different diseases in Human Plasma
Syst
ems B
iolo
gy
Transcriptomics
Proteomics
Phospholipidomics
Imageomics
Metabolomics
Glycomics
Kinomics
NanoBioMedicine
Conventionaltechnology
I have developed a breakthrough method, which make it possible to detect and identify the defined clinical Biomarkers for diseases in human plasma (100x more coverage)
The transfer of basic science knowledge to the clinic to prevent and recover the diseases and/or extend healthy aging via generation of
defined clinical biomarkers for different diseases for the development of new therapeutics.
Transfer Basic Science to the Clinic
T h e im p ac t o f C lin ic al B io m arkers o n 1-Pu b lic H ealth , 2- E c o n o m y, 3-
S o c ie ty
1- T he d iagno sis o f the d isease at early stage by physic ian2- T he therapy start o n tim e3- T he therapy will be effic ient and m o re effec tive4- T he m o rb id ity and m o rtality are extrem ely reduc ed5- T he health insuranc e c o sts go do wn6- T o prevent the d isease, find new drug target and extend healthy ag ing .
Pu b lic H ealth
T h e im p ac t o f C lin ic al B io m arkers o n 1-Pu b lic H ealth , 2- E c o n o m y, 3-
S o c ie ty
1- C enter o f d iagno stic2- Sell the Bio m arkers3- C ap ital stream s to c o untry and C ity (m o no po ly)4- H ealth c are budget go do wn 5- Pro sperity6- Investm ent o n o ther issues
Ec o n o m y
The impact of Clinical Biomarkers on 1-Public Health, 2- Economy, 3- Society
Society
1- Prosperity2- Economy growth3- Healthy aging4- Excellent education
Markets of clinical Biomarkers
The Compound Annual Growth Rate (CAGR)
1- Biomarkers Market worth $45.55 Billion by 2020(Markets and Markets) and will reach over $78.2 Billion by
2024 (PharmExec.com)
2- For many decades Clinical Biomarkers will be major focus of medical science and pharmaceutical Companies
http://www.pharmexec.com/global-biomarkers-market-reach-over-782-billion-2024
http://www.marketsandmarkets.com/Market-Reports/biomarkers-advanced-technologies-and-global-Market-43.html
7.5 billion inhabitants 9% T2D affected.
USA: 30.3 million T2DEU: 66 million T2DWorldwide: 700 million T2D The global cost of diabetes $825 billion per year Diagnosis diabetes in the U.S.A. $245 billion per year Diagnosis diabetes in EU $120 billion per year
Global Market
Glo b al Marke t
1 trillion dollars T2D diagnosis per year, €1500 per patient per year
Novel Biomarker for T2D found by BIO-CLINICA BIOMARKERS will lead to a very high profitable investment
BIO-CLINICA BIOMARKERS Company (CEO; Dr. F. Rezaee)
UK Researchers Identify 750 Biomarkers for Potential Early Cancer Screening
Is correct or not correct to use the word Biomarker ?
NO
Approximately 100 biomarkers for Cardiovascular Diseases (CVDs) NO
Approximately 40 biomarkers for Diabetes (T2D) NO
WHY THE ANSWER IS NO?
Based on defined clinical Biomarkers, it is not allowed to preselect for discovery of clinical Biomarkers. It is not allowed to remove any fraction from whole proteome samples. Clinical Biomarkers Discovery has to be executed in whole intact System.
The best situation 1-4 Clinical Biomarkers for one specific disease, Multifactorial diseases are exceptions from this potential law.
EXAMPLES
EXAMPLES
Systems Biology of Aged-Induced Diseases
FISH EYE DISEASE (FED)
LCAT mutation (T123→I),
Two heterozygotes with mutation (V309→M )
One Compound heterozygote patient with the both mutations (T123→I and V309→M)
FLD (familial LCAT deficiency)
FED (Fish Eye Disease)
Lecithin cholesterol acyltransferase (LCAT)
DIGE (Diffrences in gel electrophoresis)
800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000M/z0
100
%
Farhad8AMC0668 Center 11 (Cen,2, 80.00, Ht) TOF (800:4000) LD+
831.22
1283.61
832.19
1012.59
896.46
1031.53
1226.58
1724.03
1284.621453.77
1380.77
1454.77
1707.00
1468.86
1469.85
1725.03
1726.03
1815.93
1817.933594.01
3034.542645.341879.18 2963.73 3184.74 3592.063344.153721.13 3881.61
APO A-I
1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 2200 2250m/z0
100
%
far0030 25 (0.896) Cn (Cen,2, 80.00, Ht); Sb (20,40.00 ); Sm (SG, 2x6.00); Cm (1:28) TOF LD+ 4021497.69
1313.60
1292.96
1276.98
1173.461244.47
1314.60
1335.58
1336.58
1351.55
1352.56
1373.49
1411.521416.60
1498.69
1499.70
1753.76
1520.67
1730.701663.661642.681535.63
1536.63
1557.601558.61
1665.67
1755.77
1775.72
1776.742248.891792.67 2233.89
1819.672250.85
FISH EYE DISEASE
Adipose tissue and inflammation, adipocytes and Insulin regulation adipocytes and plasma lipoproteins
Adipose tissue and inflammation, adipocytes and Insulin regulation adipocytes and plasma lipoproteins
F.Rezaee@med.umcg.nlFarhad Rezaee
In this seminar:
Aadipose tissue
Eye catcher
Obesity is a problem of epidemic-pandemic proportions and is related to poor health
Body needs fat
Abdominal Obesity is a major cause of Abdominal Aortic Aneurysm (AAA)
Adipose Tissue Structure
Adipose tissue (1)
1- Storage of energy in the form of triglycerides (TG) for lean times
2- The largest endocrine organ
What is the main cause of obesity?
An overload of energy (TG) in the adipose tissue and in particular adipocyte-associated LDs is the
main cause of obesity
What is main function of adipose tissue?
Obesity is defined as BMI (kg/m2) determined by the increasing mass of adipose tissue (BMI above 30 is obese)
Adipose tissue (2)
An inflammatory state of adipose tissue contributes to the development of insulin resistance (IR) and type 2 diabetes (T2D)
Obesity promotes an inflammatory state
What is the link between obesity and pathophysiological diseases?
Obesity
Inflammation
Cancer
Infection
Cachexia
Insulin ResistanceCoronary Heart Disease
Hypertension
Stroke
Atherosclerosis
Adipose tissue (3)Metabolic disorder Obesity or Cachexia pathophysiological states
Regional fat metabolismTwo main fat types:
LIPID DROPLETS
ADIPOCYTES
Plasma lipoproteins
Lipoprotein structure VLDL HDL
Disturbances in plasma lipoprotein homeostasis result in dyslipidemia, e.g. hypertriglyceridemia, hypercholesterolemia and etc., which in turn have serious pathophysiological consequences like CVDs
Lipoproteins are classified by density and size, which are inversely related
VLDL/CM
LDLIDL
Lp(a)
HDL2-RichHDL3-Rich
VHDL
PLFF
Plasma Lipoproteins
Den
sity
(g/m
l)
Size (nm)
HDL Lipid Droplet
Similarities between LP and LDLP LD
Challenges
Inflammation
Insulin RegulationLipoproteins
?
Cardivascular diseases
Adipose tissue is considered as an immune organ
Objective
Why do I make this statement?
Conventional wisdom: immune functionality is the result of adipose tissue macrophages (ATM).
My research challenges this notion: Adipose tissue adipocytes (ATA) exhibit immune cell function
No body measured such function in human primary adipocytes or other human AT-associtaed cells alone
1-Classical hypothesis: necrotic AT-adipocytes (ATA) in obese state activate AT-macrophages (ATM) that then lead to a sustained chronic inflammation in AT (Only in AT).
2-The link between human adipocytes and the source of inflammation in AT has not been systematically studied.
3-AT is considered as an immune organ
3-Our hypothesis: human primary adipocytes are able to prime inflammation in AT.
Classical hypothesis versus my research challenges
Measure to know?
Challenges
1-the adipose tissue adipocytes (ATA) exhibit immune cell function
2-Adipose tissue can be potentially considered as a new organ to compensate hormone/enzyme disorders
3-Mitochondria is inactive in obese adipocytes and LDs are “the only boss” in adipocytes
4-A homeostatic balance between lipoproteins may guarantee a regulated function of lipoproteins, -and not the quantity of a specific lipoprotein
5-Human primary adipocytes produce and secrete Insulin
Macrophages Characteristic production1-Cytokines
2-Chemokines
3-MHC-II molecules
4-CDs
5-Adhesion Molecules
We must show that adipocytes are also able to produce similar products!
RESULTS 1
Differentiation of human preadipocytes to adipocytes
Pre-adipocytes Adipocytes
AdipogenesisTo establish our research model!
ADIPOQ LEPLIPE
PLIN
PNPLA2LPL
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Preadipocytes Adipocytes
Adipocytes differentiation mRNA markers
1000x
Differentiation markersR
elat
ive
inte
nsity
( 2
log
)
Rel
ativ
e in
tens
ity (
2lo
g )
Cytokine / Chemokine Ligand and Receptor Genes
Adipocytes express cytokines and cytokine receptors
IL1BIL1R
1IL1R
2 IL3IL3R
A IL4IL4R IL5
IL5RA IL6
IL6RA
IL6RB IL7
IL7R IL2IL19 IL26
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
Preadipocytes Adipocytes
Cytokine and cytokine receptors
Adipocytes express cytokines and cytokine receptors R
elat
ive
inte
nsity
( 2
log
)
Adipocytes-associated Chemokine and Chemokine Receptor GenesR
elat
ive
inte
nsity
( 2
log
)
Chemokine and Chemokine Receptor Genes
Adhesion, MHC-I, MHC-II and CDs Genes
Genes
Confocal analysis of C-RP in adipocytes
CRP
merge
Fat organelle Nuclei
NegativeMerge
Immunoelectron microscopy of C-RP in adipocytes
Negative C-RP
Are these adipocytes-associated immune components biologically active?
We have measured 50 proteins treated with and without LPS
Adipocytes treated with LPS (1)
Proteins
Adipocytes treated with LPS (2)
Proteins
Adipocytes treated with LPS (3)
Proteins
Adipocytes-associated components are biologically active
Are adipocytes-associated components biologically functional?
We performed a migration assay according to Boyden chamber to measure adipocyte
functionality
Migration assay according to Boyden Chamber
Are adipocytes-associated components biologically functional?
YES
Summary
Preadipocytes and adipocytes synthesize immune-associated components (genes and proteins)
Since preadipocytes and adipocytes express immune-associated genes, expression of these genes suggested to be a bona fide property of this cell type
Since these genes and proteins response to LPS, They are biologically active
Since adipocytes-associated MCP-1 and IP-10 are able to activate CD4+, They are biologically functional.
Human primary adipocytes exhibit immune cell function.
Conclusion
Adipocytes are able to prime inflammation
Adipocytes Produce Insulin
the role of adipocytes behind the link between obesity and insulin regulation in human is unknown
The overload of lipid droplets with triglycerides in adipocytes is the main cause of obesity and its afflictions
such as insulin resistance
Adipocytes Produce Insulin?
RESULTS 2
Hypothesis: Fat determines the concentration of insulin
insulin concentration and human patients
Bariatric Surgery Heals Type 2 Diabetes
Assumption: the production of insulin by human adipocytes is not from pancreas
Insulin stain in pancreas of prediabetes BB rats
Insulin in control RATs
Insulin in other human primary cell types
The novel knowledge obtained by the present study could help to re-design future studies related to insulin and therapy of diabetes.
Human adipocytes constitutively synthesize and secrete insulin and that is biologically functional.
Human primary adipocytes secretes insulin but preadipocytes do not and that suggested to be due to the lipid droplets
Conclusion
Insulin expression in a variety of human cell types including the adipocytes is an evidence for a ubiquitous insulin expression
Data suggested a cross talk between AT and pancreas to tune up energy metabolic system
A homeostatic balance between lipoproteins may guarantee a regulated function of lipoproteins, and not the quantity of a
specific lipoprotein?
PLASMA LIPOPROTEINS
RESULTS 3
Phospholipidomic analysis of all defined plasma lipoproteins
VLDL/CM
LDLIDL
Lp(a)
HDL2-RichHDL3-Rich
VHDL
PLFF
Plasma Lipoproteins
Den
sity
(g/m
l)
Size (nm)
High Performance Thin Layer Chromatography
Proteomics analysis of plasmaVLDL, LDL, Lp (a), and HDL
95 proteins
51 proteins
55 proteins
Proteomic analysis of high-density lipoproteinRezaee F, Casetta B, Levels JH, Speijer D, Meijers JC (Proteomics 2006) AMC
Phospholipidomic analysis of all defined human plasma lipoproteinsDashti M, Kulik W, Hoek F, Veerman EC, Peppelenbosch MP, Rezaee F
(Nature Scientific Reports 2011) UMCG, AMC, VU, ERASMUSMC
Human plasma very low density lipoprotein carries Indian hedgehogQueiroz KC, Tio RA, Zeebregts CJ, Bijlsma MF, Zijlstra F, Badlou B, de Vries M, Ferreira CV, Spek CA, Peppelenbosch MP, Rezaee F ( J Proteome Research 2010) UMCG, AMC
Human plasma Proteome of Very Low-Density Lipoprotein and Low-Density Lipoprotein exhibits a link with coagulation and lipid metabolism
Dashty M, Motazacker MM, Levels J, de Vries M, Mahmoudi M, Peppelenbosch MP, Rezaee F (Thromb Haemost. 2014) UMCG, AMC, STANFORD, ERASMUSMC
Proteome analysis of defined human plasma Lipoprotein a (LP (a))(Manuscript will be submitted today, 2019) UMCG, ICL, VU, STANFORD,
ERASMUSMC
Papers related to proteome and lipidome of Plasma lipoprotein
Ernst Schaefer Citated VLDL/LDL paper as “Of outstanding interest”
The paper have been recommended on PUBADVANCED by A Director at ROCHE
The paper have been also recommended for Science Global Discovery
Human plasma Proteome Very Low-Density Lipoprotein and Low-Density Lipoprotein exhibits a link with coagulation and lipid metabolism
Dashty M, Motazacker MM, Levels J, de Vries M, Mahmoudi M, Peppelenbosch MP, Rezaee F (Thromb Haemost. 2014) UMCG, AMC, STANFORD, ERASMUSMC
Found very interesting paper by Head of Mass Spectrometry of OCDEM (University of Oxford)
The majority of apolipoproteins and proteins involved in lipid metabolism are conserved between the different lipoproteins.
The known phospholipids are also conserved between the different lipoproteins.
Human diseases related to proteins carried by VLDL and LDL can be divided in three major categories: 1 – dyslipidaemia, 2 –atherosclerosis and vascular disease, and 3 – coagulation disorders.
A variety of proteins present on LDL and VLDL strongly supports that protein shuttling through lipoproteins may be involved in the regulation of biological processes
SUMMARY
CONCLUSION
A homeostatic balance between lipoproteins may guarantee a regulated function of lipoproteins, and not the quantity of a
specific lipoprotein
SYSTEMS BIOLOGY
Present and future studies
Quantitative proteomics of patients with CVDs versus control
HDL; PILOT STUDY
A NOVEL METHOD FOR QUANTITATIVE PROTEOMICS
I developed a novel method, which increases the number of quantified proteins from 20 to 300 or even more in
plasma research and you do not need to run a gel (That is a revolution contribution in proteomics).
This approach is applicable to any good samples
With this approach, we are able to find easily the candidate protein(s) (diagnostic or prognostic markers) in any
patients (CVDs, T1D, T2D, Obesity, Coagulation Disorders)
This approach is applicable to all spieces (e.g. Bacteria, Viruses)
PROTEOMICS
1800 proteins
Visceral Adipocytes proteome
Cytoplasma (560)
Mitochondria (388)
Nucleus (332)
Extracelluar (198)
ER (164)
Golgi (99)
Lysosome (49)
Peroxisome (26)
Lipid droplets (7)
Distribution of proteins
0.4 %
MITOCHONDRIA
Mitochondria
Distribution of genes
NanoBioTechnology
Personalized protein coronas: a “key” factor at the nanobiointerface
MJ. Hajipour, S. Laurent, A. Aghaie, F. Rezaee* and Morteza Mahmoudi**
Composition-dependent effects of nanoparticles on blood coagulation
Kamran Bakhtiari, Joost C.M. Meijers, Sophie Laurent, Svetlana Mintova, Eng-Poh Ng, Hussein Awala, Somayyeh Mirsadeghi, Morteza Mahmoudi, Farhad Rezaee
STEM CELL BASED THERAPY
*Stem Cell Therapy: Stem Cell based Therapy including Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (IPSCs) for metabolic diseases such as T1D and CVDs.
Thank you
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