Conférence de la 8ème édition du Breaking of the wall : role of allergy and histamine release. Cours international « Atelier Paludisme » - JAMBOU Ronan

1. Breaking of the wall : role of allergy and
histamine release
Ronan Jambou MD, PhD
Immunology Unit
Institut Pasteur de Madagascar
Vascular
Immunology
Unit

2. Expected decrease of Rural Malaria transmission
Kilifi _ Kenya Malaria J 2007
Kilifi _ Kenya Malaria J 2007 Swaziland WHO Report 2006
Swaziland WHO Report 2006

3. Changes in transmission modulate Malaria profil
Total Nb of attacks 60 % within
years old adluts
6
Danané, Côte d'Ivoire: ≥ 300 b.i.a./human/year 25 10%
Dielmo, Sénégal: 100-300 b.i.a./human/year 43 23%
5
Ndiop, Sénégal: 10-30 b.i.a./human/year 62 41%
A n n u a l N b o f m a l a r i a a tt a c k s
4 Pikine, Sénégal: ≤1 b.i.a./human/year 25 50%
Saharevo, Madagascar: ≤1 b.i.a./human/year 32 53%
3
2
1
0
0 5 10 15 20 25 30 35 40 45 50 60
Age (years)
Kilifi _ East Africa Mwangi et al JID 2005
Kilifi _ East Africa Mwangi et al JID 2005 West Africa // Madagascar (meta-analysis)
West Africa Madagascar (meta-analysis)
No clear change in mortality

4. Hunt & Grau 2003
Schofield & Grau 2005
sequestration inflammation
pathology
(oedema)

5. Vascular permeability
Histamine
pathogens
pathogens stimuli
stimuli
Major role in:
EC
Diabetes
Diabetes
Dengue fever
Dengue fever
Age degenerescence
Age degenerescence
ARDS
ARDS
Myocardial infarction
Myocardial infarction
Tumour angiogenesis
Tumour angiogenesis Junction
VVO pinocytosis
Brain injury
Brain injury remodelling

6. Histamine induces vascular congestion
Guido Majno (1961)
vein

7. Histamine can modulate endothelial and immune cells functions
Allergy / helminths IgE Antigen
platelets / neutrophils basophiles HRF/TCTP
Histamine
( HR1, HR2, HR3, HR4, HR5 )
cytokine network (IL4, IL2..) vascular permeability DC response to TH2 type
eicosanoid pathway production of IgE
endothelial cells response Platelet + IRBC
to inflammation
vWF adhesion
Cerebral malaria ??

8. PfTCTP is expressed by late trophozoites
Timing genes 40
35 gene T8h Pf TCTP
30
gene T12h
25
- 38% identity, 53%
20
15 similarity with hTCTP
10
5 - mimics hTCTP in vitro
0
P0 P6 P12 P18 P24 P30 P36 P42 P48 on histamine release
Sampling time - 0.1 to 1µg/ml in serum
from patients with
malaria
4,5
Transcript RQ
4
3,5
- totally conserved in 350
PFTCTP
3 P falciparum field
2,5
2
isolates => target human
1,5 cells ??
1
0,5
2 8 14 20 26 32 38 44 50 hours
Parasite cycle time Plasmo-DB
- synchronisation + sampling every 6h over 54h = « sampling time »
- use of thin smear and timing genes (P David) to define the « parasite time » (gene
8h= MAL8P1.4, gene 12h=PFI1735c
- normalisation of mRNA on average of (N1= PFC0255c and N2=PFA0570), then at
time with less mRNA (T44 = 1 for PfTCTP)

9. Allergic response in seasonal transmission area
« The background »

10. Area of Dakar
Gouly Couly
Seasonal 17°
W 16° 15° 14° 13° 12°
transmission 16°
N
15°
4 M inhabitants Dakar
= 1/3 Senegal SENEGAL
14°
3.5% territory 13°
An arabiensis

11. Longitudinal study: Gouly couly
Enrolment End
Jun 04 Jun 06
july 04 Nov. 04 Jun 05
IgE Baso, IgE Baso, IgE
dry season Rainy season dry season Rainy season dry season
Drug resistance study
Follow up

12. CD203 CD63
Surface marker granule marker
activation of basophiles Release

13. IgE responses : Gouly couly
100
10 1,5 Low
80
IgE ug/mL
8
1
60
6
40 0,5
4
2 20 0
0 july 04 Nov 04 July 05
0
9-19 20-39 40-80 9-19 20-39 40-80
2,5 medium
Total IgE ug/L % of subjects with > 0.4µg/l 2
IgE ug/mL
1,5
No difference 1
nov_04 according to age for
45 Jun_05 0,5
40
high IgE level
0
35
% IgE_Pf positive
30 Stability of IgE level
25
« High is high » 30
high
20
25
IgE ug/mL
15
20
10 No change in [IgE]
5 15
0
according to season 10
9-19 20-39 40-80 5
Higher percent of 0
% of subjects with IgE-Pf 1 2 3
Pf-IgE during dry
season

14. Basophiles responses : Gouly couly
standard antigens
nov jun
90
Percent responders
80 flmp
70 D farinae
60 D pteronyssinus
50 Percent of responders
40 maximum during rainy
30
20
season
10
0 Pf can induce response
9-19y 20-39y 40-80y for 10% of villagers
during rainy season
80 nov jun nov jun nov jun
High response for
70
Pf antigens salivary glands
Percent responders
Hemozoin
60
Pf ghost
50 Salivary glands
40
30
Mosquitoes can
20
trigger basophiles
10
activation
0
9-19y 20-39y 40-80y

15. Mosquitos bites induce mast cells activation
JI Demeure (2005)

16. Basophiles responses IgE responses
Percent of responders No difference
maximum during rainy according to age for
season high IgE level
Pf can induce response Stability of IgE level
for 10% of villagers « High is high »
during rainy season
Higher percent of
High response for Pf-IgE during dry
salivary glands season
Mosquitoes can Anti-IgE present
trigger basophiles “consummed” ?
activation
Players are there !

17. Basophyle activation during Cerebral malaria
« Is it a relevant question ??? »

18. Area of Dakar
Patients
Severe malaria (SM) cases were recruited at the Intensive Care Unit (CHNU)
Mild malaria (MM) matched cases were recruited at the Saint-Martin Dispensary
Healthy matched subjects attending the laboratory for routine examination

19. Basal level of CD203c expression
Mild malaria have = lower basal level of CD203c expression

20. IgE independant activation of basophiles
HZ hemozoin (malaria pigment)
Cerebral malaria = higher reactivity to IgE-independent stimulation

21. A
Severe malaria
=
higher reactivity to
IgE-dependent
stimulation
B +
No over-stimulation

22. Basophiles response to anti-IgE in healthy subjects

23. Ex vivo priming with Pf- or h-TCTP increases IgE dependant
response and induces over stimulation
Anti-IgE 1micro/ml
Pf-TCTP
hTCTP

24. Basophiles response to anti-IgE in healthy subjects
Modulation by Pf-TCTP

25. Pf-TCTP / anti Pf-TCTP
Cerebral malaria = higher level of Pf-TCTP
ELISA with Pf-specific
monoclonal antibodies
(horizontal lines represent median for positive individuals only)

26. 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 dependant response

27. Anti-PfTCTP in serum and response to IgE
Presence of anti-PfTCTP is associated with protection against
excessive IgE dependant response

28. 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 CM
Relevant with higher histamine detection

29. Histamine and brain endothelial cell interaction
« to treat or not to treat »

30. Opening the junction
Histamine
(Vandenbrouke et al 2008)

31. IRBC
P < 10-6
440
NS
420
400
P < 10-5
380
360
Surface (pixel)
340
320
300
280
260
240
220
200
180
NRBC Histamine IRBC
Histamine 100µM

32. Monitoring of cells monolayer impedance to measure junction permeability
ECIS: Electric Cell-substrate Impedance Sensing
HBEC-D3

33. IRBC, NRBC
Histamine, TNF
washing
Spreading: 5 days 1.5 h 24 h
HBEC
Attachment and spreading of the cells TEER
20000
Resistance (ohms)
15000
10000
5000
0
0 20 40 60 80 100
Time (hours)

34. Histamine or platelet supernatant induce opening of junctions
18000 Control medium
Resistance
ohm Histamine
stimulation
16000 LPS
PLT SN
14000
12000
10000
8000
18000 Control medium
Resistance
ohm Medium Histamine
change stimulation
16000 LPS
6000 PLT SN
14000
12000
4000 10000
8000
2000 6000
4000
2000 hours
0
hours
0
29 29.5 30 30.5 23 31 28 33 31.5 38 32 43

35. H1 receptor mediates histamine effect on HBECs
0,1
0,05
0
1 2 3 4 5 6 7 8 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25
-0,05 his100-anti_H1
ratio at T0
-0,1 histamine100
-0,15 TNF
-0,2
hist100-antiH2
-0,25 control
-0,3
-0,35
Anti H1 inhibits histamine effect on HBEC-D3 monolayer

36. Histamine induce calcium mobilization thru HR1
Normalised Ratio
Histamine 100 µM 3.6
Histamine
2.6
1.6
0.6
0 20 40 60
Time (min)
Histamine 10 µM Cimetidine (40µM)
Normalised Ratio
4.6
Histamine
3.6
2.6
H2
1.6
0.6
0 20 40 60 80
Time (min)
Diphenylhydramine
(20µM)
Histamine
Histamine 1 µM
500
400
H1
Ca (nM)
300
200
100
0
-100
0 5 10 15 20 25
(Pre-incubation 40 min)
Time (min)

37. Histamine increases VCAM but not ICAM expression
18
Histamine 100µM, 10µM, 1µM
16
Percent of positive cells*
Cimetidine 20µM,
14 diphenylhydramine 20µM,
12
10
8
6
4
2
0
Histamine100 Histamine100 Histamine_10 Histamine_1 control
+ antiH1H2 *Flow cytometry analysis after trypsinization of the HBEC-D3
Anti H1/H2 inhibits histamine effect on VCAM1 expression

38. The murine model of CM
Score Observation
0 No discernible clinical signs
neurological
phase 1 Hunched posture, slightly
hyperparasitaemia
ruffled fur
0 7 14 21
days 2 Very ruffled fur, incipient
severe motor impairment
cerebral anaemia
malaria 3 Very ruffled fur, severe
motor impairment such
as ataxia, hemiplegia
Blood collected and paraplegia,
convulsions, fitting and
Platelet-free plasma the Wooley white sign
(CBA only).
4 Very little movement, cold
to the touch

39. Anti_H1 increases survival of P berghei infected mice
100
Percent survival
Control
75 Citicholine
Diphenyhydramine (anti-H1)
50
25
0
0 7 14
Day post-PbA infection
Day post-PbA
Control Citicoline DPH
infection
5.
6. 4 2
7. 3 2 4
8. 2 1
9. 3
Beghdadi et al 2008

40. Conclusion
Allergy : 20% of population with high IgE level = stable
Seasonal variation of basophiles activation
=> Impact of mosquitoes bites on histamine release
Cerebral malaria is associated with hyper reactivity of
basophiles , without auto-regulation
PfTCTP enhances / anti-TCTP decrease, this response
Rapid opening of intercellular junction thru H1R
Anti-H1 can inhibit this effect
Anti-histamine = can improve the treatment of CM ?
Clinical trial

41. University Sydney
V Combes
A Sanchez Perez
Institut Pasteur de
Dakar F Elassaad
D Aldebert MJ Jambou
GE. Grau Institut Pasteur de
S Pelleau
Madagascar
L Marrama
Romy
F Diène-Sarr
Tsiry
R Paul Emma
Ibrahima Dia IMTSSA- Le Pharo
B Diop S Pelleau
JC Moreau D Parzy
Institut Pasteur
(PF5)
F Nato, P Beguin

42. Thanks for your attention

43. IFNg / IL10 balance
A B C
IFNg IL10 IFN/IL10
Severe malaria = imbalanced pro/anti-inflammatory response

44. Histamine release and HRFs
TCTP
- found in all eukaryotes , with two conserved motifs (microtubules binding domain)
- control cells proliferation, division, and apoptose => overexpression in cancer
Human TCTP (IgE-dependent HRF)
-Gene locus 13q12-q14
- induces histamine release by basophiles of atopic patients
- in vitro : increase reactivity of basophiles to other stimulus (IgE, ..etc)
- induce proliferation of B cells and activation of eosinophiles
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 ??
Induction of idiopathic allergy ?

45. PfTCTP down regulates expresion of TRL2 and Vcam1 in HMEC
60 8h of stimulation
Relative
quantity
20
6
2
1
-5
ICAM-1 IL-6 TNFb TLR-2 VCAM-1
IL-1b IL-8
Moderate direct effect of recombinant PfTCTP on endothelial cells
- Vcam and TRL2 repression
- no effect on permeability
- no effect on microparticules productions

46. sequestration

47. Severe Malaria
Newborn
fever
dehydration Adult
convulsion Respiratory distress
Metabolic disorders cerebral malaria
= impaired consciousness low parasiteamia
= delay in treatment
Child
Severe anaemia +++
High parasitaemia +++
Cerebral pathology

48. From rural to urban: trends of the population
85 82
76 74
61
54 55
53
47
42
37 37
29
15 17
Monde Afrique Asie Amérique Régions plus
latine/Caraïbes avancées
1950 2000 2030 (projections)
Source : Nations Unies, Perspectives de la population dans le monde, Edition 2003 (scénario moyen), 2004.

49. From children to adults
0,3
0,4
0-1 y
0,25 0,35 1- 4 y
0,2
0,3 5-14 y
0,25 15- 49 y
0,15
0,2 > 50 y
0,1 0,15
0,05
0,1
0,05
0
septembre octobre novembre decembre
0
sept oct nov dec
Prevalence of malaria in consultations
prevalence of severe malaria.
1400
0,6
1200
0,5 1000
0,4 800
0,3 600
0,2 400
0,1 200
0 0
septembre octobre novembre decembre M F M F M F M F M F
0-1 an 1- 4ans 5-14 ans 15- 49 ans 50ans &+
part of class of age in the malaria cases
consultations by age
Changes of risks Changes in control strategies
Major challenge for the next 10 years

50. TNF plays a major role in sequestration
sol
or
PRBC binding
mem mem
TNF LT ICAM-1
ICAM- monocyte binding
upregulation
platelet binding
TNFR2
brain endothelial cells
Functional consequence of TNFR2 upregulation (mouse model, PbA infection)
Lucas et al., Eur J Immunol 27: 1719, 1997
Lou, Lucas & Grau Clin Microbiol Rev 14: 810, 2001
Stoelcker et al., Infect Immun 70: 5857, 2002