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Breaking of the wall : role of allergy and histamine release
Immunology Unit Institut Pasteur de Madagascar
Immunology Unit Institut Pasteur de Madagascar
Ronan Jambou MD, PhD
Vascular
Immunology
Unit
Expected decrease of Rural Malaria transmissionExpected decrease of Rural Malaria transmission
Swaziland WHO Report 2006Swaziland WHO Report 2006Kilifi _ Kenya Malaria J 2007Kilifi _ Kenya Malaria J 2007
Kilifi _ East Africa Mwangi et al JID 2005Kilifi _ East Africa Mwangi et al JID 2005
Changes in transmission modulate Malaria profil Changes in transmission modulate Malaria profil
53%32Saharevo, Madagascar: ≤1 b.i.a./human/year
50%25Pikine, Sénégal: ≤1 b.i.a./human/year
41%62Ndiop, Sénégal: 10-30 b.i.a./human/year
23%43Dielmo, Sénégal: 100-300 b.i.a./human/year
10%25Danané, Côte d'Ivoire: ≥ 300 b.i.a./human/year
% withinadluts
Total Nb of attacks 60 years old
53%32Saharevo, Madagascar: ≤1 b.i.a./human/year
50%25Pikine, Sénégal: ≤1 b.i.a./human/year
41%62Ndiop, Sénégal: 10-30 b.i.a./human/year
23%43Dielmo, Sénégal: 100-300 b.i.a./human/year
10%25Danané, Côte d'Ivoire: ≥ 300 b.i.a./human/year
% withinadluts
Total Nb of attacks 60 years old
Ann
ual N
bof
mal
aria
atta
cks
0
1
2
3
4
5
6
0 5 10 15 20 25 30 35 40 45 50
Age (years)
60A
nnua
l Nb
of m
alar
ia a
ttack
s0
1
2
3
4
5
6
0 5 10 15 20 25 30 35 40 45 50
Age (years)
60
West Africa / Madagascar (meta-analysis)West Africa / Madagascar (meta-analysis)
No clear change in mortalityNo clear change in mortality
Schofield & Grau 2005
Hunt & Grau 2003
sequestration inflammation
(oedema)pathology
Major role in: Major role in: Major role in: ECECEC
Vascular permeability
� Diabetes� Dengue fever� Age degenerescence� ARDS� Myocardial infarction� Tumour angiogenesis� Brain injury
� Diabetes� Dengue fever� Age degenerescence� ARDS� Myocardial infarction� Tumour angiogenesis� Brain injury
VVO Junction remodelling
pinocytosis
pathogenspathogenspathogens stimulistimulistimuli
Histamine
Histamine induces vascular congestion
Guido Majno (1961)
vein
Histamine
HRF/TCTPbasophiles
cytokine network (IL4, IL2..) eicosanoid pathway
endothelial cells responseto inflammation
platelets / neutrophils
DC response to TH2 typeproduction of IgE
vascular permeability
Cerebral malaria ??
( HR1, HR2, HR3, HR4, HR5 )
IgE AntigenAllergy / helminths
Histamine can modulate endothelial and immune cells func tions
vWFPlatelet + IRBC
adhesion
PfTCTP is expressed by late trophozoites
PF
TC
TP
T
rans
crip
tRQ
2 8 14 20 3226 38 44 50 hours
0,51
1,52
2,53
3,54
4,5
05
10152025303540
P0 P6 P12 P18 P30P24 P36 P42 P48
- synchronisation + sampling every 6h over 54h = « sampli ng time »
- use of thin smear and timing genes (P David) to define th e « parasite time » (gene8h= MAL8P1.4, gene 12h=PFI1735c- normalisation of mRNA on average of (N1= PFC0255c and N 2=PFA0570), then attime with less mRNA (T44 = 1 for PfTCTP)
Parasite cycle time
Sampling time
gene T8h
gene T12h
Tim
ing
gene
s Pf TCTP
- 38% identity, 53% similarity with hTCTP
- mimics hTCTP in vitro on histamine release
- 0.1 to 1µg/ml in serum from patients with malaria
- totally conserved in 350 P falciparum field isolates => target human cells ??
Plasmo-DB
Allergic response in seasonal transmission area
« The background »
15°
14°
13°
16°N
Dakar
17°W 16° 15° 14° 13° 12°
SENEGAL
Area of Dakar
Seasonal transmission
4 M inhabitants = 1/3 Senegal
3.5% territory
An arabiensis
Gouly Couly
Longitudinal study: Gouly couly Longitudinal study: Gouly couly
Nov. 04Baso, IgE
Follow up
EnrolmentJun 04
Jun 05Baso, IgE
july 04IgE
EndJun 06
Rainy season Rainy seasondry season dry seasondry season
Drug resistance study
CD203Surface marker
activation of basophiles
CD63granule marker
Release
Low
high
medium
0
20
40
60
80
100
9-19 20-39 40-80
0
2
4
6
8
10
Total IgE ug/L
9-19 20-39 40-80
0
0,5
1
1,5
july 04 Nov 04 July 05
IgE
ug/
mL
0
0,5
1
1,5
2
2,5
IgE
ug/
mL
0
5
10
15
20
25
30
1 2 3
IgE
ug/
mL
% of subjects with > 0.4µg/l
0
5
10
15
20
25
30
35
40
45
9-19 20-39 40-80
% Ig
E_P
f pos
itive
nov_04Jun_05
% of subjects with IgE-Pf
No difference according to age for
high IgE level
Stability of IgE level« High is high »
No change in [IgE] according to season
Higher percent of Pf-IgE during dry
season
IgE responses : Gouly couly IgE responses : Gouly couly
Basophiles responses : Gouly couly Basophiles responses : Gouly couly
standard antigens
flmpD farinaeD pteronyssinus
0102030405060708090
9-19y 20-39y 40-80y
Per
cent
res
pond
ers
nov jun
Percent of responders maximum during rainy
season
Pf can induce response for 10% of villagers during rainy season
High response for salivary glandsHemozoin
Pf ghostSalivary glands
0
10
20
30
40
50
60
70
80
9-19y 20-39y 40-80y
nov jun nov jun nov jun
Per
cent
res
pond
ers
Mosquitoes can trigger basophiles
activation
Pf antigens
Mosquitos bites induce mast cells activationJI Demeure (2005)
Mosquitos bites induce mast cells activationJI Demeure (2005)
No difference according to age for
high IgE level
Stability of IgE level« High is high »
Higher percent of Pf-IgE during dry
season
Percent of responders maximum during rainy
season
Pf can induce response for 10% of villagers during rainy season
High response for salivary glands
Mosquitoes can trigger basophiles
activation
IgE responsesIgE responsesBasophiles responsesBasophiles responses
Anti-IgE present“consummed” ?
Players are there !
Basophyle activation during Cerebral malaria
« Is it a relevant question ??? »
Area of Dakar
Patients
Severe malaria (SM) cases were recruited at the Int ensive Care Unit (CHNU)
Mild malaria (MM) matched cases were recruited at t he Saint-Martin Dispensary
Healthy matched subjects attending the laboratory for routine examination
Mild malaria have = lower basal level of CD203c exp ression
Basal level of CD203c expression
Cerebral malaria = higher reactivity to IgE-indepen dent stimulation
IgE independant activation of basophiles
HZ hemozoin (malaria pigment)
Severe malaria=
higher reactivity to IgE-dependent
stimulation+
No over-stimulation
A
B
Basophiles response to anti-IgE in healthy subjects
Ex vivo priming with Pf- or h-TCTP increases IgE dependant response and induces over stimulation
hTCTP
Pf-TCTPAnti-IgE 1micro/ml
Basophiles response to anti-IgE in healthy subjects
Modulation by Pf-TCTP
(horizontal lines represent median for positive individuals only)
ELISA with Pf-specific monoclonal antibodies
Pf-TCTP / anti Pf-TCTP
Cerebral malaria = higher level of Pf-TCTP
PfTCTP in serum and response to anti-IgE
Stimulation with anti-IgE 0 (PBS), 0.1, 1 or 10 µg/mL
Presence of PfTCTP is associated with higher IgE depe ndant response
Anti-PfTCTP in serum and response to IgE
Presence of anti-PfTCTP is associated with protection against excessive IgE dependant response
Summary
Cerebral malaria is associated with higher basophile response to both IgE independent and dependant
response, without overstimulation
rPf-TCTP enhances basophile response during CM and induces overstimulation in healthy subjects
Presence of PfTCTP in serum enhances IgE dependant response; presence of anti-Pf-TCTP decreases this
response.
Allergic response is enhanced during CMRelevant with higher histamine detection
Histamine and brain endothelial cell interaction« to treat or not to treat »
Opening the junction Opening the junction
(Vandenbrouke et al 2008)
Histamine
HistamineNRBC IRBC180
200
220
240
260
280
300
320
340
360
380
400
420
440
Sur
face
(pi
xel)
P < 10-5
P < 10-6
NS
Histamine 100µM
IRBC
ECIS: Electric Cell-substrate Impedance Sensing
Monitoring of cells monolayer impedance to measure jun ction permMonitoring of cells monolayer impedance to measure jun ction perm eabilityeability
HBEC-D3
HBEC1.5 h
washingHistamine, TNF
Spreading: 5 days 24 h
IRBC, NRBC
TEERAttachment and spreading of the cells
0
5000
10000
15000
20000
0 20 40 60 80 100
Time (hours)
Res
ista
nce
(ohm
s)
Histamine or platelet supernatant induce opening of junctions
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
29 29.5 30 30.5 31 31.5 32
stimulationHistamine
Control medium
hours
Resistanceohm
LPS
PLT SN
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
23 28 33 38 43
stimulationMedium change
Histamine
Control medium
hours
Resistanceohm
LPS
PLT SN
H1 receptor mediates histamine effect on HBECsra
tio a
t T0
-0,35
-0,3
-0,25
-0,2
-0,15
-0,1
-0,05
0
0,05
0,1
1 2 3 4 5 6 7 8 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25
his100-anti_H1
histamine100
TNF
hist100-antiH2
control
Anti H1 inhibits histamine effect on HBEC-D3 monola yer
Histamine 100 µM
Histamine induce calcium mobilization thru HR1Histamine induce calcium mobilization thru HR1
Histamine 1 µM
-100
0
100
200
300
400
500
0 5 10 15 20 25
Time (min)
Ca
(nM
)
Histamine 10 µM
Histamine
Time (min)
0.6
1.6
2.6
3.6
0 20 40 60Time (min)
Nor
mal
ised
Rat
io
Diphenylhydramine (20µM)
Histamine
Cimetidine (40µM)
0.6
1.6
2.6
3.6
4.6
0 20 40 60 80
Nor
mal
ised
Rat
io Histamine
(Pre-incubation 40 min)
H1
H2
Histamine increases VCAM but not ICAM expression
0
2
4
6
8
10
12
14
16
18
controlHistamine100Histamine100
+ antiH1H2
Histamine_10 Histamine_1
Per
cent
of p
ositi
ve c
ells
*
Histamine 100µM, 10µM, 1µMCimetidine 20µM, diphenylhydramine 20µM,
*Flow cytometry analysis after trypsinization of the HBEC-D3
Anti H1/H2 inhibits histamine effect on VCAM1 expres sion
The murine model of CM
Score Observation
0 No discernible clinical signs
1 Hunched posture, slightly
ruffled fur
2 Very ruffled fur, incipient
motor impairment
3 Very ruffled fur, severe
motor impairment such
as ataxia, hemiplegia
and paraplegia,
convulsions, fitting and
the Wooley white sign
(CBA only).
4 Very little movement, cold
to the touch
neurological
phase
cerebral
malaria
0 7 14
Blood collected
Platelet-free plasma
hyperparasitaemia
severe
anaemia
21days
39.
128.
4237.
246.
5.
DPHCiticoline ControlDay post-PbA
infection
ControlCiticholineDiphenyhydramine (anti-H1)
0 7 140
25
50
75
100
Day post-PbA infection
Per
cent
sur
viva
l
Anti_H1 increases survival of P berghei infected mice
Beghdadi et al 2008
Conclusion Conclusion
Allergy : 20% of population with high IgE level = st ableSeasonal variation of basophiles activation => Impact of mosquitoes bites on histamine release
Anti-histamine = can improve the treatment of CM ?
Clinical trial
Cerebral malaria is associated with hyper reactivity of basophiles , without auto-regulationPfTCTP enhances / anti-TCTP decrease, this response
Rapid opening of intercellular junction thru H1R Anti-H1 can inhibit this effect
University SydneyV Combes
A Sanchez PerezF ElassaadMJ Jambou
GE. Grau
IMTSSA- Le PharoS PelleauD Parzy
Institut Pasteur de Dakar
D AldebertS Pelleau
L Marrama F Diène-Sarr
R PaulIbrahima Dia
B Diop JC Moreau
Institut Pasteur (PF5)
F Nato, P Beguin
Institut Pasteur de Madagascar
RomyTsiry
Emma
Thanks for your attention
A B C
IFNg IL10 IFN/IL10
Severe malaria = imbalanced pro/anti-inflammatory r esponse
IFNg / IL10 balance
Histamine release and HRFsHistamine release and HRFs
Human TCTP (IgE-dependent HRF)
-Gene locus 13q12-q14
- induces histamine release by basophiles of atopic p atients
- in vitro : increase reactivity of basophiles to other stimul us (IgE, ..etc)
- induce proliferation of B cells and activation of e osinophiles
Pf TCTP
- 38% identity, 53% similarity with hTCTP
- mimics hTCTP in vitro on histamine release
- 0.1 to 1µg/ml in serum from patients with malaria
- totally conserved in 350 P falciparum field isolate s => target human cells ??
TCTP
- found in all eukaryotes , with two conserved motifs (microtubules binding domain)
- control cells proliferation, division, and apoptose => overexpression in cancer
Induction of idiopathic allergy ?
Relative quantity
2
1
-5
20
6
60
ICAM-1 VCAM-1IL-1b
IL-6IL-8
TNFb TLR-2
PfTCTP down regulates expresion of TRL2 and Vcam1 in HMEC
8h of stimulation
Moderate direct effect of recombinant PfTCTP on endothelial cells- Vcam and TRL2 repression- no effect on permeability - no effect on microparticules productions
sequestration
Severe Malaria
NewbornfeverdehydrationconvulsionMetabolic disorders= impaired consciousness
ChildSevere anaemia +++High parasitaemia +++Cerebral pathology
AdultRespiratory distresscerebral malarialow parasiteamia= delay in treatment
29
15 17
53
61
54 55
85 82
42
76
3737
47
74
Monde Afrique Asie Amériquelatine/Caraïbes
Régions plusavancées
1950 2000 2030 (projections)
Source : Nations Unies, Perspectives de la population dans le monde, Edition 2003 (scénario moyen), 2004.
From rural to urban: trends of the population From rural to urban: trends of the population
Major challenge for the next 10 years
Changes of risks Changes in control strategies
Prevalence of malaria in consultations
0
0,05
0,1
0,15
0,2
0,25
0,3
septembre octobre novembre decembre
00,10,20,30,40,50,6
septembre octobre novembre decembre
00,050,1
0,150,2
0,250,3
0,350,4
sept oct nov dec
part of class of age in the malaria cases
prevalence of severe malaria.
0
200
400
600
800
1000
1200
1400
M F M F M F M F M F0-1 an 1- 4ans 5-14 ans 15- 49 ans 50ans &+
consultations by age
0-1 y1- 4 y5-14 y
15- 49 y> 50 y
From children to adults From children to adults
brain endothelial cells
TNF plays a major role in sequestrationTNF plays a major role in sequestration
Lucas et al., Eur J Immunol 27: 1719, 1997Lou, Lucas & Grau Clin Microbiol Rev 14: 810, 2001
Stoelcker et al., Infect Immun 70: 5857, 2002
monocyte bindingmonocyte bindingmonocyte bindingmonocyte bindingICAMICAMICAMICAM----1 1 1 1 upregulation upregulation upregulation upregulation
platelet bindingplatelet bindingplatelet bindingplatelet binding
memTNF
TNFR2TNFR2
sol or
memLT
PRBC bindingPRBC bindingPRBC bindingPRBC binding
Functional consequence of TNFR2 upregulation (Functional consequence of TNFR2 upregulation ( mouse modelmouse model , PbA infection), PbA infection)