research seminar 13 nov 03
TRANSCRIPT
RESEARCH SEMINAR13 Nov 03; 5:30-
6:30pmUCLan
1) Nitric oxide congeners modify the gastrotoxicity of
NSAIDs
2) but retain the ability to alter thymic function:
Nitrergic system of the thymus and autoimmune susceptibility
Dr James DowningLiverpool John Moores
University,School of Pharmacy & Chemistry
1
“Nitrergic” systems:
Expression ofNitric Oxide Synthase
(NOS).
1
Three Organ Systemsuse NOS to make NO
1
NERVES
3
VASCULATURE
2
IMMUNE SYS
(Inflammation;
incl Gut epithelium)
2
AbbreviationsN.O = Nitric Oxide.N.O.S = Nitric Oxide Synthase.
Enzyme activityConvert L-Arginine to L-Citrulline and N.O.
Isoforms First located (named) Regulation
NOS1 Neural/Brain (n/bNOS) Constitutive (Ca-
dept).
NOS2 'mac'/inducible (iNOS) Inducible (Ca-indept).
NOS3 endothelial (eNOS) Constitutive
(Ca-dept).
AssaysElectrochemical meter.E.S.R (using a "spin trap" eg. MGD).Histology: NADPH-diaphorase / Immunocytochemistry.Citrulline production (from 3H-Arginine).Colourimetric Greiss assay (NOx).
NITRERGIC Enzymes and Assays
3
“Nitrergic” system of THYMUS:
Expression of inducible Nitric Oxide Synthase,
iNOS (NOS2).
• Maintains "Deletional" Immune Tolerance of Self
• BioAssay for toxicological risk of autoimmunity.
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5
Cortex“outer”
Lobule
Cross-section (120um) through thymic lobule
Nitrergic cells - by diaphorase activityin paraformaldehyde-fixed tissue
Medulla“inner”
Immunological Tolerance• T-cells are produced within the thymus.
• There is an "education" or "selection" process to ensure : - T-cells can recognise antigen (have TCR) and - T-cells are not "auto-aggressive".
• SIMPLIFICATION: - T-cellularity (relative size of the thymic cortex)
= Index of "Production". - Abundance of "Nitrergic" cells (expressing iNOS)
= Index of "Quality Control" (Deletional Tolerance).
• HYPOTHESIS: - A quantitative "balance" between "production" of T-cells and their "Quality control" within the thymus. - May be disturbed by genetic or pharmacological factors.
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Thymic "Education" of T-LymphocytesCortex: Phase 1 - Positive selection
Progenitor T-cell
TCR binds MHCPositive selection
not bind MHCDeath by neglect
Medulla: Phase 2 - Negative selection
TCR hi avidityDeleted
TCR mod avidity
Allowed
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COLOCALIZATION OF NADPHd AND TUNEL (APOPTOSIS)
Thymic sections from naïve rat stained for NADPH diaphorase (Tetrazolium - blue) followed by the TUNEL reaction to visualise apoptotic cells (brown). Arrows
indicate apoptotic cells adjacent to NADPHd+ cells.
(400x, scale bar = 20 um).
murderous "Quality Control"of T-cell Production
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1) Nitric oxide congeners modify the gastrotoxicity of
NSAIDs
2) but retain the ability to alter thymic function:
Nitrergic system of the thymus and autoimmune susceptibility
Dr James DowningLiverpool John Moores
University,School of Pharmacy & Chemistry
1
OUTLINE OF SEMINAR
1) The problem with NSAIDs: Inhibition of COX; gastrotox'
2) Proposed new pro-drugs: NO-glycerol-NSAID
3) Study 1 to test the gastro-protective effects of NO-NSAIDs ; possible involvement of an effect on local synthesis of NO.
4) Study 2 to test for potential immunological side effects of these anti-inflammatory / immunosuppressant drugs.
5) Consistent with an increased risk of autoimmune disorder following damage to thymic mech' for immune tolerance:
- Thymic damage (to NOS) from use/withdrawal from CsA.
- Lewis rats: AI-susceptibility may be due to deficient NOS
- Dpp4 inhibitors trailed for MS; Dpp4 mutant Fischer rats can show disordered iNOS.
2
1) The problem with NSAIDs.
• NSAIDs inhibit COX: • Inhibiting cyclooxygenase COX-1 maybe "all bad" - necessary to control gastric mucus and bicarbonate secretion. • Inhibiting COX-2 originally thought to be the source of inflammatory damage; Reconsideration "not all bad" may also be required for reparative processes of wound healing.
• Systemic levels of NSAIDs may be responsible for gastro toxicity through non-selective inhibition of COX1 and COX2.
2) Proposed new pro-drugs: NO-glycerol-NSAID
• May slow the release of NSAIDs - reducing local and systemic levels.• Provides local NO to increase enteric vascular perfusion and inhibit immune responses.
3
NO-indomethacin
N
O
Cl
CH3
OO
CH3O
OH
O NO2
• NSAIDs modified with a NO donor group attached by a linker molecule.
• NO-NSAID metabolised by esterase to release NO.
• NO has ‘gastroprotective’ effects.
NO-NSAIDs4
OH
O
O
OO
ONO2
O
Ibuprofen
NO-Ibuprofen
5
STUDY 1:Reducing
gastro toxicity(side effects)of NSAIDs
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Acute effects of ibuprofen and its nitric oxide releasing prodrug on ulceration,
erosion and nitric oxide synthase expression in rat gastric mucosa
C. Wilson, J. Southall, M. Ingram*,C. Rostron, J. Duffy, J. Downing
School of Pharmacy & Chemistry, Liverpool John Moores University, Liverpool L3 3AF,
*School of Pharmacy and Biomolecular Sciences, University of Brighton, East Sussex BN2 4GJ
Corresponding author: [email protected]
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GASTROTOXICITY OF NSAIDs - NO CONGENORS
• Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen are effective in the treatment of pain and inflammation but are associated with gastrotoxicity.
• Efficacies of NSAIDs may relate to their ability to inhibit prostaglandin synthesis by alternative cyclooxygenase enzymes, which may compromise mucosal resistance to erosive effects of stomach acid.
• Modifications of NSAIDs to incorporate releasable nitric oxide (NO) are being explored (Wallace et al 1997).
• We examined ibuprofen and its NO-prodrug (Ingram et al 2001) for gastrotoxic effects and association with endogenous levels of nitric oxide synthase (NOS).
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TREATMENTS
• Single oral treatments of 4hr duration were made with either ibuprofen or ibuprofen-NO (ea. 1.33x10-4mol/kg) and compared with vehicle (aqueous Tween 80) using 16-hour fasted male Wistar rats (~250g; n=5 per treatment).
AIMS
• Determine the effects of acute oral administration of ibuprofen and its nitric oxide (NO) congener on signs of gastric damage (mucosal ulceration and thinning).
• Establish the effects of treatments on nitric oxide synthase (NOS) activity by Nitro Blue Tetrazolium-based diaphorase histochemistry.
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Thickness
Length
*
• No OF ULCERS (*) by two alternative methods:
(a) Visual examination of stomachs to produce weighted scores (Duffy et al J. Pharmacy & Pharmacology 2001, 53: 1505-1514).
(b) Microscopic counts of ulcers/cm; 195 sections observed from 15 rats.
• NOS EXPRESSION: % perimeter length stained by diaphorase.
• MUCOSAL THICKNESS: 3775 random transects; 195 sections; 15 rats.
MEASUREMENTS:Ulceration, Thickness & NOS Expression
Fig 1
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Gastric Erosion:Effect of Treatment (vehicle, ibuprofen or NO-ibuprofen) on
the Average Mucosal Thickness ( Mean +/- SEM)
0
10
20
30
40
50
60
70
80
90
Vehicle Ibuprofen NO-Ibuprofen
Treatment
Av
era
ge
Mu
cosa
l T
hic
kn
ess
(Mic
ron
s)
• Analysis of variance (ANOVA) P value < 0.001.• Tukeys follow up test shows all the results to be
significantly different from each other.• NO-ibuprofen caused significantly less erosion than
ibuprofen.
RESULTS: Erosion of mucosal thickness
Fig 2
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Gastric Ulceration:Effect of treatment (vehicle, ibuprofen or NO-Ibuprofen)
on the average number of ulcers per cm of Gastric Mucosa (Mean +/- SEM)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Vehicle Ibuprofen NO-IbuprofenTreatment
Mea
n N
um
ber
of
Ulc
ers
per
Cen
tim
etre
RESULTS: Ulceration by microscopic sampling
• Analysis of variance (ANOVA) P value = 0.639.• Tukeys test shows that no treatments are
statistically different from one another.• Number of ulcers per centimetre were not
statistically significant between treatments.
Fig 3
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Ulceration and Irritation Visible to the EyeData produced by visible examination of the Rat Stomachs
0123456789
10
Vehicle Ibuprofen NO-Ibuprofen
Treatment
Irrit
ati
on
Sca
led
Sco
re
RESULTS: Ulceration by visual score
• Results based on a scaled score, Vehicle = 1.• Compare data with the number of ulcers per
centimetre sampled microscopically.• Suggests a trend towards NO-ibuprofen causing
less ulceration.
Fig 4
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NOS EXPRESSION (BY DIAPHORASE)
• Three isoforms of nitric oxide synthase (NOS) can be identified in paraformaldehyde-fixed tissue by NADPH-diaphorase histochemistry.
• High levels of NO produced by the inducible NOS may contribute to tissue damage at the site of inflammation.
• Lower levels of NO expected from constitutive (neural and endothelial) isoforms may support gastro-protective functions, such as the inhibition of platelet aggregation and increased vascular perfusion (Nathan 1997).
• NSAIDs may modify endogenous NO synthesis and NO-conjugated prodrugs may substitute for the effects of endogenous NO.
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DEGREE OF STAINING BY DIAPHORASE (NOS EXPRESSION):Mean percent length of the gastric mucosa positivley stained by NADPH
Diaphorase (a marker of nitric oxide synthase)
0
5
10
15
20
25
30
35
Vehicle Ibuprofen NO-Ibuprofen
Treatment
Av
e %
le
ng
th s
tain
edRESULTS: NOS expression by diaphorase labelling
• Analysis of variance (ANOVA) P value = 0.001.• Tukeys test shows difference between ibuprofen
and vehicle to be insignificant.• NOS expression is significantly increased by
NO-ibuprofen, but not ibuprofen.
Fig 5
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CONCLUSIONS• Effects of NO-ibuprofen reported here:
- Reduced mucosal erosion significantly.- Appeared to reduce the number of ulcers / cm (shown by scaled score but not statistically resolved by microscopic sampling).- Increased the expression of diaphorase (NOS).
• Complexing of NO with ibuprofen reduces gastro-toxicity when compared to ibuprofen and this effect is associated with higher levels of expression of mucosal diaphorase, likely to be NOS, indicating a possible mechanism for gastroprotection.
Acknowledgements Thanks to Francis Essuman.
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STUDY 2:Altered thymic function:Risk of additional side effects
to autoimmune susceptibility
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Acute reduction of nitric oxide synthase (NOS) in rat thymus without change in
T-cellularity following oral non-steroidalanti-inflammatory drug (NSAID) treatments
R. Clark, M. Whitty, M. Ingram*,C. Rostron, J. Duffy, J. Downing
School of Pharmacy & Chemistry, Liverpool John Moores University, Liverpool L3 3AF,
*School of Pharmacy and Biomolecular Sciences, University of Brighton, East Sussex BN2 4GJ
Corresponding author: [email protected]
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INTRODUCTION
• Conjugation of NSAIDs with esterase-releasable nitric oxide (NO) is being explored for its potential to reduce gastrotoxicity (Ingram et al 2001).
• Additional side effects are reported to include disruption of T-cell development within the thymus (Xu et al 2001) and the appearance of over active, possibly autoreactive, T-cells in the periphery (Yamamura et al 1996).
• Pharmacological inhibition of a thymic mechanism using nitric oxide synthase (NOS) and possibly underlying central immune tolerance has also been reported (Kosaka et al 1990).
• The effect of acute oral dosing with NSAIDs and their NO-congeners was therefore investigated for possible disturbance of thymic organisation.
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METHODS
• Single 4hr duration oral treatments with either indomethacin, indomethacin-NO (ea. 2x10-5 mol/kg), ibuprofen or ibuprofen-NO (ea. 1.33x10-4 mol/kg) were compared with vehicle (aqueous Tween 80) using 16-hour fasted male Wistar rats (~250g; n=4).
• Paraformaldehyde-fixed thymi were stored frozen (-20oC) before parallel processing for enzyme histochemistry.
• Two parameters were measured from 100 micron sections:
(i) Size of cortex relative to total surface area gave an index of T-cell production or “positive selection“, T-cellularity, by imaging auto-fluorescent cells (AFC) at the cortico-medullary junction.
(ii) Abundance of medullary nitrergic cells (counts/mm2) stained positive by NADPH-diaphorase, a marker of NOS.
20
30 μm
150 μmcortex
medulla
A) Oil Red O
D) nNOS +ve control
200 μmcortex
medulla
AFCs
B) Auto Fluorescent Cells
150 μm
cortex
medulla
debris
Nitrergic cells
C) Diaphorase
Fig. 1
Illustration of methods to observe T-cellularity and Nitrergic cells
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HYPOTHESIS - Central Immunological Tolerance
• Negative selection (deletion) of potentially autoreactiveT-cells is proposed to involve induction of NOS within thymic medulla and may reflect "quality control" of T-cells.
• Relatively low levels of nitrergic cell expression in the presence of high T-cellularity have been associated with autoimmune susceptibility of the Lewis rat compared to Fischer strain (Downing et al 1998).
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RESULTS
• Possible effects of NSAIDs on thymic organisation were compared using analysis of variance followed by Tukey's test and expressed as mean +/- SEM.
• Both ibuprofen (57.08 +/- 5.19; p<0.001) and indomethacin (62.88 +/- 4.84, p<0.001) reduced nitrergic cell abundance compared to control (94.79 +/- 6.70) in the absence of significant changes in T-cellularity.
• NO-conjugated drugs also suppressed the levels of NOS (NO-ibuprofen, 66.77 +/- 5.23, p<0.001; NO-indomethacin, 65.37 +/- 6.55, p<0.001) and also occurred in the absence of significant changes in T-cellularity.
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0
0.2
0.4
0.6
0.8
1
Control Indomethacin NO-Indomethacin
ctx
:me
du
lla a
rea
Fig. 2a: Effects of Indomethacin / NO on T-cellularity.• ANOVA P-value = 0.357 (NS).
0
20
40
60
80
100
120
control indomethacin NO-indomethacin
Me
an
ce
ll c
ou
nt
Fig. 2b: Effects of Indomethacin / NO on Nitrergic cell count. • ANOVA P-value < 0.001; No difference in treatments by Tukeys.
Me
an
ce
ll c
ou
nt
Ra
tio
ctx
:me
d a
rea
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T-Cellularity(mean +/- SEM)
0
0.25
0.5
0.75
Control Ibuprofen NO-Ibuprofen
T-C
ellu
lari
ty
Fig. 3a: Effects of Ibuprofen / NO on T-cellularity.• ANOVA P-value = 0.095 (NS).
020406080
100120
Control Ibuprofen NO-IbuprofenCell
co
un
t /
Med
ull
ary
A
rea
Fig. 3b: Effects of Ibuprofen / NO on Nitrergic cell count. • ANOVA P-value < 0.001; No difference in treatments by Tukeys.
Me
an
ce
ll c
ou
nt
Ra
tio
ctx
:me
d a
rea
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REFERENCES
Ingram, M.J. et al (2001) J. Pharmacy and Pharmacology 53: 345-350.
Xu, H. et al (2001) Cellular Immunology 214: 184-193.
Yamamura, S. et al (1996) Cellular Immunology 173: 303-311.
Downing, J.E.G. et al (1998) Immunology 95: 148-155.
Kosaka, H. et al (1990) J. Exp. Med. 172: 395.
CONCLUSIONS
• It is proposed that if the effects of acute oral treatment with either NO-conjugated or parent forms of ibuprofen or indomethacin are maintained over chronic periods they may pose a risk to effective immune tolerance and lead to the escape of autoreactive T-cells.
• A comparable autoimmune adverse drug reaction has been identified following withdrawal of the anti-inflammatory drug, cyclosporin A, which is also associated with inhibition of thymic nitric oxide synthesis (Kosaka et al 1990).
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5) OTHER EVIDENCE
Consistent with an increased risk of autoimmune disorder following damage to a thymic mechanism for immune tolerance:
- Thymic damage (to NOS) from use/withdrawal from CsA.
- Lewis rats: AI-susceptibility may be due to deficient NOS
- Dpp4 inhibitors trailed for Multiple Sclerosis (MS); Dpp4 mutant Fischer rats can show disordered iNOS.
27