Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans
Hepatic Inflammation and Cellular Therapy - Vital...
Transcript of Hepatic Inflammation and Cellular Therapy - Vital...
Hepatic Inflammation and Cellular Therapy
Lee K. Landeen, Jessica Van Allen, Patricia W. Bedard
Vital Therapies, Inc., San Diego, CA, USA
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Alcoholic Hepatitis: Role of Inflammation
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Leaky Gut
Inflammatory Mediators (PAMPs, DAMPs)
Immune
Dysfunction
Cytokine
Storm
Secondary Organ Failure
Activation
Infections
Anti-inflammation Therapies for Alcoholic Hepatitis
• Anti-tumor necrosis factor alpha (TNF) (infliximab)
– Double-blind randomized controlled trial (prednisolone infliximab)
– Naveau S, et al. 2004 Hepatology
– Did not demonstrate clinical benefit (higher infection rates, death rates, lower ex vivo stimulation capacity of neutrophils in treatment group)
• Thalidomide
– Pilot study to reduce TNF
– N0265006466, DH Adams (Queen Elizabeth Hospital, Birmingham, UK), 2006-2007
– Study abandoned
• Steroids (prednisolone, pentaxifylline)
– STOPAH
– Thursz MR, 2015 N Eng J Med
– Did not demonstrate medium- to long-term clinical benefit
• IL-1ra (anakinra)
– NCT01809132, anakinra + pentoxifylline + zinc sulfate vs. methylprednisolone
– On-going
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Porcine Hepatocytes Human Hepatoblastoma-derived C3A
Cell Line
Allogeneic Human Hepatocytes Human Hepatocellular Carcinoma-
derived HepaRG Cell Line
The Case for Cell-based Liver Therapies/Treatments
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• Cells are capable of producing multiple factors
• Cells can respond dynamically over time
• Cells can potentially respond to individual patients
HepatAssist
(Circe Biomedical
>Arbios Systems)
SRBAL
(Mayo
Clinic)
MELS
(Charite Virchow
Clinic)
ELAD
(Vitagen>Hepatix>
Vital Therapies)
(BALANCE,
Netherlands)
• Previous emphasis was on factors that ELAD C3A cells could remove:
– Bilirubin, ammonia, toxins, etc.
Mechanism of Action: Soluble Factors
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• Current focus is on what ELAD C3A cells can contribute:
− Anti-inflammation, regeneration, coagulation,
transport, anti-oxidation, etc.
VTL C3A Cells Secrete Many Potentially Useful Proteins
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• 148 proteins identified to date via immunoassay; 62 proteins via LC/MS
• 39 proteins secreted above 1 mg/day/cartridge
• 15 proteins secreted 10x above normal serum levels
Inflammation:
Alpha-1-Antitrypsin
Complement C3
Ferritin
Gelsolin
Haptoglobin
Intercellular Adhesion
Molecule 1
Interleukin-1 Receptor
Antagonist
Interleukin-8
Tumor Necrosis Factor Alpha
Regeneration:
Amphiregulin
Growth/differentiation factor 15
Heat-Shock protein 70
Heparin-Binding EGF-like Growth
Factor
Hepatocyte Growth Factor
Platelet-Derived Growth Factor-BB
Tissue Inhibitor of
Metalloproteinases 1
Tissue Inhibitor of
Metalloproteinases 2
Tissue Inhibitor of
Metalloproteinases 3
Transforming Growth Factor Alpha
Transport:
Albumin
Alpha-Fetoprotein
Apolipoprotein A-I
Apoliproprotein A-II
Apolipoprotein A-IV
Apolipoprotein B
Apoliproprotein C-I
Apolipoprotein C-II
Apoliproprotein C-III
Apolipoprotein E
Apoliproprotein H
Fatty Acid-Binding Protein,
liver SerotransferrinAngiogenesis:
Angiopoietin-2
Placental Growth Factor
Vascular Endothelial Growth
Factor
Vascular Endothelial Growth
Factor-C
Hematopoiesis:
Erythropoietin
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Oxidative Stress:Peroxiredoxin-4
Fatty Acid-Binding Protein,
liver
*Proteins may have multiple roles depending on concentration and targeted cell type
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Death
Alcohol
Consumption
Liver
Failure
Stabilization
Recovery/
Regeneration
Immune Dysfunction
Cholestasis
Leaky Gut
Toxic Injury
Apoptosis/Necrosis
Alcoholic
Hepatitis (Severe
Inflammation/
Dysfunction)
Coagulopathy
Respiratory
Failure
Kidney Failure
Immune/Sepsis
Re
du
ce
d in
flam
ma
tion
Me
tab
olic
Su
pp
ort
Imm
un
e F
un
ctio
n
Re
ge
ne
ratio
n
Re
du
ce
d A
po
pto
sis
Imp
rov
ed
Bile
Flo
w
ELAD Treatment
C3A Cell Proteins
Multi-organ failure:
Working Model of ELAD Therapeutic Effect
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ELAD C3A Cell Cartridge
Inflammatory Factors:
IL-1, IL-6, TNF, LPS
?
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Are ELAD C3A Cells Capable of an Acute-Phase Response?
VTL C3A Experimental Model
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IL-1β,
IL-6,
&/or
TNFα
or
LPS
VTL C3A Cells
?
Supernatants
Collected
24, 48, 54h
Protein
Secretion
(immunoassay)
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VTL C3A Cells Exhibit an Acute-Phase Response
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C o n t r o l I L - 1 x I L - 6
0
2
4
6
8
Alb
um
in (
g/
mL
)
p = 0 . 0 4 7
Fib
rin
og
en
(n
g/m
L)
C3AIL-1β
+
IL-6
24
hr
Decreased
Albumin
Increased
Fibrinogen
VTL C3A Cells Produce Anti-Inflammatory Proteins
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AATIL-1Ra
C3AIL-1β
+
IL-6
24
hr
Increased
IL-1Ra
No Change
AAT
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C o n t r o l I L - 1 + I L - 6
0
1 0
2 0
3 0
IL-1
Ra
(n
g/m
L)
p = 0 . 0 0 7
C o n t r o l I L - 6 + I L - 1
0
2 0 0
4 0 0
6 0 0
AA
T (
ng
/mL
)
p = 0 . 5 1 4 0
VTL C3A Cells Produce Anti-Inflammatory Proteins
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0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0
0
5 0
1 0 0
1 5 0
L P S (E U / m L )
IL-1
Ra
(p
g/
mL
)
0 .0 0 1 0 .0 1 0 .1 1 1 0 1 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
L P S (E U / m L )
AA
T (
ng
/m
L)
C3ALPS
24
hr
Increased
IL-1Ra
Increased
AAT
Will Healthy Neutrophils Respond to ELAD-Treated AH Plasma?
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Increasing phagocytic capacity with Days on ELAD Treatment suggests restored function
FIT
C
Phagocytosis
Normal T0 T1 T2 T3Plasma
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Normal D0 24h EOT 30d
• Healthy peripheral blood neutrophils
• Exposed to plasma from Normal individuals or ELAD-Treated subjects
– D0=before treatment
– 24 h after treatment initiated
– EOT=end of treatment
– 30 d after treatment initiated
• Measure fluorescent signal of neutrophils actively phagocytizing FITC-labeled E. coli
R Jalan, RP Mookerjee, NA Davies (University College London)
Will Macrophages Respond to ELAD C3A Factors?
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24-well plate
1x106 cells/well
0 1 6 7
THP-1
monocytic cells
PMA
24-hrs
Polarize
24-hrs
Cytokine
Immunoassays
Collect
Supernatants
Time (Days)
Rest
5-Days
M1
Adherent
macrophages
IFN-γ
+ LPS
+/- ELAD C3A Cell
Conditioned
Medium
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Pro-Inflammatory
Phenotype
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M1 Macrophages Reduce Pro-Inflammatory IL-1 When Treated with ELAD Conditioned Medium
M 1 M 1 + C M
0
1 0
2 0
3 0
4 0
I L - 1
IL
-1
(p
g/m
L)
M 1
M 1 + C M
***
*** <0.001, Student’s T-Test, n=6 biological replicates tested in duplicate
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Pro-Inflammatory Cytokines Decrease During ELAD Treatment of AH Subjects
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TNFa
day 0
day 1
0
50
100
150
200
pg
/ml
IL-8
Day
0
Day
1
0
200
400
600
800
1000
pg
/ml
IL-6
Day
0
Day
1
0
500
1000
1500
2000
2500
pg
/ml
IL1b
Day
0
Day
1
0
10
20
30
40
pg
/ml
R Jalan, RP Mookerjee, NA Davies (University College London)
TNF IL-8
IL-6 IL-1
Day 0 Day 1 Day 0 Day 1
Day 0 Day 1 Day 0 Day 1
VTL C3A Cells Produce IL-8 In Response to IL-1
• But not in response to IL-6 or LPS
• IL-8 is elevated in patients with chronic alcoholic liver disease
– Swiatkowska-Stodulska 2006 Med Sci Monit
• IL-8 can rescue human hepatocytes from TNF-induced apoptosis, and induce DNA synthesis
– Osawa 2002 Infect Immun
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LPS
Is There Evidence of an Anti-Inflammatory Response in ELAD-Treated AH Subjects?
• Case Study
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• Plasma collected prior to, during, and after ELAD Treatment
• Immunoassayed for IL-1Ra
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IL-1Ra Levels Increased During ELAD Treatment of AH Subject
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0.0
0.5
1.0
1.5
2.0
2.5
0 5 10 15 20 25 30
Co
ncen
trati
on
(n
g/m
L)
Study Day
IL-1Ra
Translational Biomarkers for Survey of VTI-208 Samples
Inflammation:
• IL-1Ra - anti-inflammatory protein expressed by VTL C3A cells
• AAT - anti-inflammatory protein expressed by VTL C3A cells
• IL-1β - Inflammatory protein in AH
• IL-6 - Inflammatory protein in AH
• TNFα - Inflammatory protein in AH
• C-reactive protein - increased in AH with Systemic Inflammatory Response Syndrome (SIRS)
• Pro-calcitonin – marker of SIRS
Anti-Apoptosis:
• Cytokeratin 18 (CK18) - a marker of caspase-mediated apoptosis
• Amphiregulin - EGFR ligand most highly expressed by VTL C3A cells
• Soluble Fas - blocks Fas-ligand, expressed by VTL C3A cells
• VEGF- reduces HAEC apoptosis, expressed by VTL C3A cells
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Conclusions
• Single-drug therapies have not proved successful in treating AH
• ELAD cell-based treatment may provide improved clinical benefit by reducing inflammation, among other mechanisms of action
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Death
Alcohol
Consumption
Liver
Failure
Stabilization
Recovery/
Regeneration
Immune Dysfunction
Cholestasis
Leaky Gut
Toxic Injury
Apoptosis/Necrosis
Alcoholic Hepatitis
(Severe
Inflammation/
Dysfunction)
Coagulopathy
Respiratory Failure
Kidney Failure
Immune/SepsisR
ed
uced
infla
mm
atio
n
Meta
bo
lic S
up
po
rt
Imm
un
e F
un
ctio
n
Reg
en
era
tion
Red
uced
Ap
op
tosis
Imp
roved
Bile
Flo
w
ELAD Treatment
C3A Cell Proteins
Multi-organ failure:
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Acknowledgements
• Neutrophil studies were completed by the Liver Failure Group, University College London Institute for Liver and Digestive Health, Royal Free Hospital (London, UK)
– Rajiv Jalan, MD
– Rajeshwar P Mookerjee, MBBS, PhD
– Nathan A Davies, PhD
• VTL Scientific Advisory Board
– Cliff Steer, M.D. (University of Minnesota)
– Charles Dinarello, M.D. (University Colorado Denver)
– George Michalopoulos, M.D. (University of Pittsburgh School of Medicine)
– Alan Hofmann, M.D. (emeritus) (University of California, San Diego )
– Fernando Camargo, Ph.D. (Harvard and Boston Children’s Hospital)
– Nikolaos Pyrsopoulos, M.D., M.B.A. (Rutgers New Jersey Medical School)
– Mike Millis, M.D. (University of Chicago Medicine)
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