Poster Epithelix -SOT2012 Epithelix -SOT2012...Microsoft PowerPoint - Poster Epithelix -SOT2012...
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POTENTIAL OF 3D HUMAN AIRWAY EPITHELIA RECONSTITUTED IN VITRO POTENTIAL OF 3D HUMAN AIRWAY EPITHELIA RECONSTITUTED IN VITRO (MUCILAIR™) FOR IDENTIFYING RESPIRATORY SENSITIZERS (MUCILAIR™) FOR IDENTIFYING RESPIRATORY SENSITIZERS Samuel Constant Samuel Constant (1) (1) , Ludovic Wiszniewski , Ludovic Wiszniewski (1) (1) , Erwin L. Roggen , Erwin L. Roggen (2) (2) and Song Huang and Song Huang (1) (1) (1) (1) Epithelix Epithelix Sàrl Sàrl, 14 chemin des aulx, CH , 14 chemin des aulx, CH-1228 Plan 1228 Plan-les les-Ouates, Ouates, Switzerland Switzerland, email: [email protected] , email: [email protected] (2) (2) Novozymes Novozymes A/S, A/S, Krogshoejvej Krogshoejvej 36, 2880 36, 2880 Bagsvaerd Bagsvaerd Denmark Denmark Epithelix Epithelix in vitro in vitro Solutions for Solutions for Respiratory Respiratory Diseases Diseases and and Chemical Chemical Testing Testing Respiratory sensitizers and irritants are considered as substances of higher risk. However, until now there is no validated animal model/method, nor in vitro cell model for identifying the respiratory chemical sensitizers. The aim of this study is to develop an in vitro cellular assay for identification of respiratory chemical sensitizers based on a novel in vitro human 3D airway epithelium model (MucilAir™). To evaluate the potential of MucilAir™ as a tool for identifying the respiratory chemical sensitizers, 9 chemical compounds belonging to 3 classes (irritants, dermal sensitizers and respiratory sensitizers) were tested. The cytotoxic effects of these chemicals were assessed by several endpoints: TEER measurement, Resazurin test, cilia beating monitoring, morphological observation, etc... The cytokines, such as IL-8, IL-6, Rantes, Gro-α, etc… were used as biomarkers for discriminating these molecules. Interestingly, the respiratory irritants and respiratory sensitizers greatly increased the amount of cytokines released The advantages of MucilAir™ It is composed of primary human respiratory cells. It mimics the morphology and functions of the native human airway epithelium. It has a unique shelf-life of 12 months. Epithelia from different pathologies are available (asthma, COPD, CF, allergic rhinitis). It is ready and easy to use. into the culture media, whereas the dermal sensitizers showed no obvious effect. The stimulating effects the respiratory sensitizers and irritants are observable at concentrations several folds below the toxic doses. Moreover, some long term effects of the respiratory sensitizers on cytokine release, 4 weeks after the exposure, have been observed. Further studies on a large number of donors (36), using two reference compounds (TMI and DNCB), highlighted the importance of the genetic predisposition of the donors: only the allergic subgroup reacted differentially to the respiratory chemical sensitizers and the dermal sensitizers. Our results are in line with the epidemiological and genetic studies of the asthma diseases. Testing Strategy Long Term Effect of Respiratory Sensitizers Potential biomarkers for the discrimination of Respiratory Sensitizers Summary Air Liquid Information fromApical Side TEER measurement Resazurin test Morphological modifications Cilia beating frequency monitoring Information fromBasal Side Secretion of soluble factors (cytokines/chemokines/ metalloproteinases) LDH release Apical Exposure (saline solution or solid dilution in Dextran) 0 100 200 300 400 500 600 700 800 900 RANTES (pg/ml) MDI [μg/0.33mm2] Effect of MDI on RANTES secretion 24h 48h 72h 1 week 3 weeks 0 100 200 300 400 500 600 700 800 RANTES (pg/ml) DNCB [μg/0.33mm2] Effect of DNCB on RANTES secretion 24h 48h 72h 1 week 3 weeks 0 100 200 300 400 500 600 700 800 RANTES (pg/ml) Salicilic Acid [μg/0.33mm2] Effect of Salicilic Acid on RANTES secretion 24h 48h 72h 1 week 3 weeks Respiratory Sensitizers Dermal Sensitizers Irritants and weak Pre/Pro-Hapten 1- Washing of the apical surface 2- Measurement of end-points Exposure Recovery Phase 24h 48h; 72h; 1, 3 and 6 weeks Measurement of end-points Multiple End-point Analysis Protocol 1: It may be possible to discriminate the respiratory sensitizers from dermal sensitizers or respiratory irritants using a right panel of biomarkers (cytokines) and recovery time, based on MucilAir™. 2: Dextran Carrier Method is an easy and powerful method for delivering non soluble or non water compatible insoluble material on apical surface ALI cultures. 3: Standard Operating procedures are accessible. Conclusions Acknowledgements 0 100 200 300 400 500 600 700 800 900 RANTES (pg/ml) TMA [μg/0.33mm2] Effect of TMA on RANTESsecretion 24h 48h 72h 1 week 3 weeks 0 100 200 300 400 500 600 700 800 RANTES (pg/ml) Cinnamic Aldehyde [mM] Effect of Cinnamic Aldehyde on RANTES secretion 24h 48h 72h 1 week 3 weeks 0 100 200 300 400 500 600 700 800 RANTES (pg/ml) Phenol [mM] Effect of Phenol on RANTES secretion 24h 48h 72h 1 week 3 weeks 0 100 200 300 400 500 600 700 800 900 RANTES (pg/ml) HCPt [μg/0.33mm2] Effect of HCPt on RANTES secretion 24h 48h 72h 1 week 3 weeks 0 100 200 300 400 500 600 700 800 RANTES (pg/ml) TMTD [μg/0.33mm2] Effect of TMTD on RANTES secretion 24h 48h 72h 1 week 3 weeks 0 100 200 300 400 500 600 700 800 RANTES (pg/ml) Cinnamic Alcohol [mM] Effect of Cinnamic Alcohol on RANTES secretion 24h 48h 72h 1 week 3 weeks The figures show some examples of the results obtained on RANTES secretion at non cytotoxic concentrations. Interestingly, two days after removal of the compounds (e.g. at 72h-time point), RANTES was highly up-regulated when exposed to respiratory chemical sensitizers. This up-regulation was not observed for respiratory irritants and for dermal sensitizers. Discrimination potential Recommended Cytokine and Recovery Time Respiratory sensitizers vs Dermal Sensitizers RANTES (72 h) IL-6 (24h, 48h, 72h; 1, 3 and 6 weeks) MCP-1 (3 and 6 weeks) Respiratory sensitizers vs Respiratory Irritants RANTES (72 h) Gro-α (24 h) 0 25 50 75 100 125 D1 W1 W3 IL-8 [ng/ml] Effect of 144µg/cm² TMA and 3µg/cm² DNCB on IL-8 secretion (Asthmatic donors, N=3) Dextran TMA DNCB Basal 15.9 ±1.8 ng/ml Asthma 0 25 50 75 100 125 D1 W1 W3 IL-8 [ng/ml] Effect of 144µg/cm² TMA and 3µg/cm² DNCB on IL-8 secretion (Allergic donors, N=5) Dextran TMA DNCB Basal 14.3 ±2.3 ng/ml Allergic 0 25 50 75 100 125 D1 W1 W3 IL-8 [ng/ml] Effect of 144µg/cm² TMA and 3µg/cm² DNCB on IL-8 secretion (Asthmatic/Allergic donors, N=2) Dextran TMA DNCB Basal 15.2 ±2.0 ng/ml As/All 0 25 50 75 100 125 D1 W1 W3 IL-8 [ng/ml] Effect of 144µg/cm² TMA and 3µg/cm² DNCB on IL-8 secretion (Normal donors, N=22) Dextran TMA DNCB Basal 20.7 ± 3.3 ng/ml Normal Genetic Predisposition Statistically, after 24 hours exposure to TMA, the amount of IL-8 released at D1 was significantly increased for all pathologies. It is also the case for DNCB, except for allergic group, where DNCB seems to have no significant stimulatory effect on IL-8 release. After 6 and 20 days recovery (week 1 and 3), Il-8 secretion returned to the normal level for both sensitizers and for all pathologies. More Information
Transcript of Poster Epithelix -SOT2012 Epithelix -SOT2012...Microsoft PowerPoint - Poster Epithelix -SOT2012...
POTENTIAL OF 3D HUMAN AIRWAY EPITHELIA RECONSTITUTED IN VITRO POTENTIAL OF 3D HUMAN AIRWAY EPITHELIA RECONSTITUTED IN VITRO
(MUCILAIR™) FOR IDENTIFYING RESPIRATORY SENSITIZERS(MUCILAIR™) FOR IDENTIFYING RESPIRATORY SENSITIZERS
Samuel ConstantSamuel Constant(1)(1), Ludovic Wiszniewski, Ludovic Wiszniewski(1)(1), Erwin L. Roggen, Erwin L. Roggen(2)(2) and Song Huangand Song Huang(1)(1)
(1)(1) Epithelix Epithelix SàrlSàrl, 14 chemin des aulx, CH, 14 chemin des aulx, CH--1228 Plan1228 Plan--lesles--Ouates, Ouates, SwitzerlandSwitzerland, email: [email protected], email: [email protected](2) (2) NovozymesNovozymes A/S, A/S, KrogshoejvejKrogshoejvej 36, 2880 36, 2880 BagsvaerdBagsvaerd DenmarkDenmark
EpithelixEpithelixin vitroin vitro Solutions for Solutions for RespiratoryRespiratory
DiseasesDiseases and and ChemicalChemical TestingTesting
Respiratory sensitizers and irritants are considered as substances of higher risk. However, until now there is no validated animal
model/method, nor in vitro cell model for identifying the respiratory chemical sensitizers. The aim of this study is to develop an in
vitro cellular assay for identification of respiratory chemical sensitizers based on a novel in vitro human 3D airway epithelium
model (MucilAir™).
To evaluate the potential of MucilAir™ as a tool for identifying the respiratory chemical sensitizers, 9 chemical compounds
belonging to 3 classes (irritants, dermal sensitizers and respiratory sensitizers) were tested. The cytotoxic effects of these
chemicals were assessed by several endpoints: TEER measurement, Resazurin test, cilia beating monitoring, morphological
observation, etc... The cytokines, such as IL-8, IL-6, Rantes, Gro-α, etc… were used as biomarkers for discriminating these
molecules. Interestingly, the respiratory irritants and respiratory sensitizers greatly increased the amount of cytokines released
The advantages of MucilAir™
� It is composed of primary human respiratory cells.
� It mimics the morphology and functions of the native human airway
epithelium.
� It has a unique shelf-life of 12 months.
� Epithelia from different pathologies are available (asthma, COPD, CF,
allergic rhinitis).
� It is ready and easy to use.
molecules. Interestingly, the respiratory irritants and respiratory sensitizers greatly increased the amount of cytokines released
into the culture media, whereas the dermal sensitizers showed no obvious effect. The stimulating effects the respiratory
sensitizers and irritants are observable at concentrations several folds below the toxic doses. Moreover, some long term effects of
the respiratory sensitizers on cytokine release, 4 weeks after the exposure, have been observed.
Further studies on a large number of donors (36), using two reference compounds (TMI and DNCB), highlighted the importance
of the genetic predisposition of the donors: only the allergic subgroup reacted differentially to the respiratory chemical sensitizers
and the dermal sensitizers. Our results are in line with the epidemiological and genetic studies of the asthma diseases.
Testing Strategy
Long Term Effect of Respiratory Sensitizers
Potential biomarkers for the discrimination of Respiratory Sensitizers
Summary
Air
Liquid
Information from Apical Side
� TEER measurement
� Resazurin test
�Morphological modifications
� Cilia beating frequency monitoring
Information from Basal Side
� Secretion of soluble factors
(cytokines/chemokines/
metalloproteinases)
� LDH release
Apical Exposure
(saline solution or solid dilution in Dextran)
0
100
200
300
400
500
600
700
800
900
RA
NT
ES
(p
g/
ml)
MDI [μg/0.33mm2]
Effect of MDI on RANTES secretion
24h
48h
72h
1 week
3 weeks
0
100
200
300
400
500
600
700
800
RA
NT
ES
(p
g/
ml)
DNCB [μg/0.33mm2]
Effect of DNCB on RANTES secretion
24h
48h
72h
1 week
3 weeks
0
100
200
300
400
500
600
700
800
RA
NT
ES
(p
g/
ml)
Salicilic Acid [μg/0.33mm2]
Effect of Salicilic Acid on RANTES secretion
24h
48h
72h
1 week
3 weeks
Respiratory Sensitizers Dermal Sensitizers Irritants and weak Pre/Pro-Hapten
1- Washing of the apical surface
2- Measurement of end-points
ExposureRecovery
Phase
24h 48h; 72h;
1, 3 and 6 weeks
Measurement of end-points
Multiple End-point Analysis
Protocol
1: It may be possible to discriminate the respiratory sensitizers from dermal sensitizers or respiratory
irritants using a right panel of biomarkers (cytokines) and recovery time, based on MucilAir™.
2: Dextran Carrier Method is an easy and powerful method for delivering non soluble or non water
compatible insoluble material on apical surface ALI cultures.
3: Standard Operating procedures are accessible.
Conclusions Acknowledgements
0
100
200
300
400
500
600
700
800
900
RA
NT
ES
(p
g/
ml)
TMA [μg/0.33mm2]
Effect of TMA on RANTES secretion
24h
48h
72h
1 week
3 weeks
0
100
200
300
400
500
600
700
800
RA
NT
ES
(p
g/
ml)
Cinnamic Aldehyde [mM]
Effect of Cinnamic Aldehyde on RANTES secretion
24h
48h
72h
1 week
3 weeks
0
100
200
300
400
500
600
700
800
RA
NT
ES
(p
g/
ml)
Phenol [mM]
Effect of Phenol on RANTES secretion
24h
48h
72h
1 week
3 weeks
0
100
200
300
400
500
600
700
800
900
RA
NT
ES
(p
g/
ml)
HCPt [μg/0.33mm2]
Effect of HCPt on RANTES secretion
24h
48h
72h
1 week
3 weeks
0
100
200
300
400
500
600
700
800
RA
NT
ES
(p
g/
ml)
TMTD [μg/0.33mm2]
Effect of TMTD on RANTES secretion
24h
48h
72h
1 week
3 weeks
0
100
200
300
400
500
600
700
800
RA
NT
ES
(p
g/
ml)
Cinnamic Alcohol [mM]
Effect of Cinnamic Alcohol on RANTES secretion
24h
48h
72h
1 week
3 weeks
The figures show some examples of the results obtained on RANTES secretion at non cytotoxicconcentrations. Interestingly, two days after removal of the compounds (e.g. at 72h-time point), RANTES washighly up-regulated when exposed to respiratory chemical sensitizers. This up-regulation was not observed forrespiratory irritants and for dermal sensitizers.
Discrimination potential Recommended Cytokine and Recovery Time
Respiratory sensitizers vs Dermal Sensitizers RANTES (72 h)
IL-6 (24h, 48h, 72h; 1, 3 and 6 weeks)
MCP-1 (3 and 6 weeks)
Respiratory sensitizers vs Respiratory Irritants RANTES (72 h)
Gro-α (24 h)
0
25
50
75
100
125
D1 W1 W3
IL-8
[ng
/ml]
Effect of 144µg/cm² TMA and 3µg/cm² DNCB
on IL-8 secretion (Asthmatic donors, N=3)
Dextran
TMA
DNCB
Basal 15.9 ± 1.8 ng/ml
Asthma
0
25
50
75
100
125
D1 W1 W3
IL-8
[ng
/m
l]
Effect of 144µg/cm² TMA and 3µg/cm² DNCB
on IL-8 secretion (Allergic donors, N=5)
Dextran
TMA
DNCB
Basal 14.3 ± 2.3 ng/ml
Allergic
0
25
50
75
100
125
D1 W1 W3
IL-8
[ng
/m
l]
Effect of 144µg/cm² TMA and 3µg/cm² DNCB
on IL-8 secretion (Asthmatic/Allergic donors, N=2)
Dextran
TMA
DNCB
Basal 15.2 ± 2.0 ng/ml
As/All
0
25
50
75
100
125
D1 W1 W3
IL-8
[ng
/ml]
Effect of 144µg/cm² TMA and 3µg/cm² DNCB
on IL-8 secretion (Normal donors, N=22)
Dextran
TMA
DNCB
Basal 20.7 ± 3.3 ng/ml
Normal
Genetic Predisposition
Statistically, after 24 hours exposure to TMA, the amount of IL-8 released at D1 wassignificantly increased for all pathologies. It is also the case for DNCB, except forallergic group, where DNCB seems to have no significant stimulatory effect on IL-8release. After 6 and 20 days recovery (week 1 and 3), Il-8 secretion returned to thenormal level for both sensitizers and for all pathologies.
More
Information
