Transition écologique du paludisme et implications thérapeutiques - Conférence du 7e édition du Cours international « Atelier Paludisme » - JAMBOU Ronan - Australie - rjambou@med.usyd.edu.au
Bulimia nervosa ( Eating Disorders) Mental Health Nursing.
Transition écologique du paludisme et implications thérapeutiques
1. Transition écologique du paludisme et implication
thérapeutique
Ronan Jambou MD, PhD
Vascular Immunology Unit
Vascular
Immunology
Unit
2. PLAN
1. Who is ill
Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
• Histamine and BBB
• Effector role of platelets
• Microparticules
• Direct effect of Infected red blood cells
3. PLAN
1. Who is ill
Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
• Effector role of platelets
• Histamine and BBB
• Microparticules
4. 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
5. 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.
7. Area of Dakar
17°
W 16° 15° 14° 13° 12°
16°
N
Seasonal
transmission 15°
Dakar
SENEGAL
4 M inhabitants 14°
= 1/3 Senegal
13°
3.5% surface
8. What’s a seasonal transmission
250
Incidence of Malaria in a small village Gouye Kouly
Gouly couly
08/2004-08/2006
200
épisodes non febriles
épisodes
épisodes fébriles
150
accés
nombre
100
50
0
8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8
2004 2005 2006
mois et années
9. In West Africa
Prevalence and risk of Malaria are
highly variable
They summarize all the parameters
of the transmission
risk is not the same everywhere
and for everyone
10. Adaptation of strategies
Change in drug strategy
Change in drug strategy ITN implementation
ITN implementation
WHO Africa report 2006
11. Expected decrease of Rural Malaria transmission
Kilifi _ Kenya Malaria J 2007
Kilifi _ Kenya Malaria J 2007 Swaziland WHO Report 2006
Swaziland WHO Report 2006
12. 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 la ria 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)
13. From children to adults
0,4
0,3
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
septembre octobre novembre decembre 0
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
14. « most of the cases occur among children under 5
years » : still true ?
WHO African report 2006 : data 2005
Nb Cases Cases ratio Countries
countries = or > 5y < 5y Deaths
ratio >1
(>= 5y / <5)
total 25 19.5 13.4 1.4
West Africa 13 9.5 4.9 1.88 9/4
Central Africa 6 3.7 3.2 1.12 3/0
East – southern 9 6.2 5.3 1.2 5/3
15. CM is associated with delay in efficient treatment
Cerebral Mild Mal Mild Mal.
malaria Hospital dispensary
N° patients 34 42 102
Mean age (SD) 14.5 (10.3) 15.5 (13) 13.7 (11)
Sex ratio 1 1.5 0.8
Patients with temperature
> 40°C (%) 14,7% 12,5% 14 %
Hemoglobin (mean g/L) 9,7 12,2 10,6
Parasitemia (paras. / µL) 2656 1276 10436
patients treated before
consultation 27,5 % 38,5 % 18,4 %
patients consulting before 4 days
after beginning of the symptoms 35,3% 31,7% 57,8%
Self treatment // Delay
Self treatment Delay
16. CM is associated with drug resistance
DHFR CRT exon 2
45%
90%
40%
35% 80%
30% 70%
25% 60%
20%
50%
15%
10% 40%
5% 30%
0% 20%
Wild 1 mutat. 2 mutat. 3 mutat.
10%
0%
MM n= 28
CVIET CVIET/CVMNK CVMNK
CM n= 16
codon 51 59 108
wild N C S MM n= 102
mutant I R N CM n= 37
one + - +
two + + +++ CM
triple + + + MM
17. CM is associated to multi-infection and specific isolates
25 A 0,82
100 2,5 He
0,8
90 20
0,78
80 2
15 0,76
70
0,74
60 1,5 10
0,72
50 0,7
5
40 1 0,68
30 0 0,66
20 0,5 Fann HPD Gued All
10
0 0 Fstat
Fstat
Cerebral Mild Mal Mild Mal.
Malaria (hosp) (dispens) 15
GUED n=129
10 HPD n=59
5
Percent of multi-infection
Axis 2
0
n° isolates / patients -5
10
-15
-15 -5 5 15
PCA
PCA Axis 1
18. Which mechanisms ?
host
pathogen
tox Infectious pathology
Adaptation of the treatment
Healthcare supply
tox Immunopathology
models additive treatments
19. Which mechanisms ?
host
pathogen
tox Infectious pathology
Adaptation of the treatment
Healthcare supply
tox Immunopathology
additive treatments
20. PLAN
1. Who is ill
Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
• Role of platelets and TNF
• Histamine and BBB
• Microparticules
• Direct effect of IRBC
23. ? Which effectors
DC
Mφ
NK
?
inflammatory
Plasmodium
CD4 CTKs ↑ Mo
?
NKT CD8 Mo
endothelium
Which interaction
? between blood cells
Schofield & Grau
5: 722-735, 2005
24. PLAN
1. Who is ill
Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
• Role of platelets and TNF
• Histamine and BBB
• Microparticules
• Infected red blood cells
26. Cytokines and pathology: approaches
Pathogen
S
R
Δ pathology? observation
S
cytokine
+ mini-pumps ? induction
S
+
antibodies,
inhibitors
? inhibition
27. TNF plays a major role in sequestration
sol
or
PRBC binding
mem mem
TNF LT ICAM-1 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
28. TNF-dependent pathology is also platelet-dependent
Pulmonary fibrosis
Nature. 1990 Mar 15;344(6263):245-7.
Cerebral malaria
Science. 1987 Sep 4;237(4819):1210-2.
Shwartzmann reaction
J Leukoc Biol. 1993 Jun;53(6):636-9.
DTH J Exp Med. 1991 Mar 1;173(3):673-9
34. In vitro evidence for a role of platelets in
PRBC-EC bridging
PRBC
PLT
PLT
PRBC
PLT
PLT
EC
Wassmer et al., J Infect Dis 189: 180-9, 2004
Wassmer et al., J Immunol 176: 1180-1184, 2006
35. Other roles of platelets in malaria: clumping
• aggregation of PRBC
• formation of giant clumps
• role in sequestration and in “sludging”
clump
Pain et al., Proc Natl Acad Sci USA 98: 1805-10,
2001
36. PLAN
1. Who is ill
Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
• Effector role of platelets
• Histamine and BBB
• Microparticules
• IRBC
37. Vascular permeability
pathogens stimuli
Major role in:
EC
Diabetes
Dengue fever
Age degenerescence
ARDS
Myocardial infarction
Tumour angiogenesis Junction
VVO pinocytosis
Brain injury remodelling
40. Histamine can modulate endothelial and immune cells functions
Allergy / helminths IgE Antigen
platelets / neutrophils basophyls 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 Histamine Increases
to inflammation Cerebral malaria ?? during malaria
41. 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
42. 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
43. IgE responses : Gouly couly
100
10 1,5 Low
80
8
IgE ug/mL
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
1
nov_04
No difference 0,5
45 Jun_05
40 according to age for 0
35 high IgE level
% IgE_Pf positive
30
25 high
20 Stability of IgE level 30
15 25
IgE ug/mL
10 20
5
Higher percent of 15
0 Pf-IgE during dry 10
9-19 20-39 40-80 5
season
0
% of subjects with IgE-Pf 1 2 3
44. IgE increase in CM and can induce IL4 production
IgE+anti-E
IgG+anti-E
IgG+anti-G
IgE Deposition in brain microvessels Immunoglobulin E (IgE) containing
and on parasitized erythrocytes from complexes induce IL-4 production in
cerebral malaria patients human basophils: effect on Th1/Th2
Yoshimasa maeno, AJTMH 2000, balance in malaria
M.A. Nyakeriga, Acta Tropica 2002
Pathology ? TH2 response ?
45. Basophiles responses : Gouly couly
standard antigens
nov jun
90
Percent responders
80 flmp
70 D farinae
60 D pteronyssinus
50
40 Percent of responders
30 maximum during rainy
20 season
10
0
9-19y 20-39y 40-80y Pf can induce response
for 10% of villagers
during rainy season
80 nov jun nov jun nov jun
70 High response for
Pf antigens
Percent responders
Hemozoin
60 salivary glands
Pf ghost
50 Salivary glands
40
30
20 Mosquitoes can
10 trigger basophiles
0 activation
9-19y 20-39y 40-80y
52. Histamine increases VCAM but not ICAM expression
18
16
Percent of positive cells
14
12
10
8
6
4
2
0
control Histamine100 Histamine100 Histamine_10 Histamine_1
+ antiH1H2
Histamine 100µM, 10µM, 1µM
Cimetidine 20µM,
diphenylhydramine 20µM,
53. Anti_H1 increases survival of P berghei infected mice
100
Percent survival
Control
75 Citicholine
diphenyhydramine
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 Beghdadi et al 2008
9. 3
54. 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
Induction of idiopathic allergy ?
56. PfTCTP is expressed by late trophozoites
40
Timing genes
35 gene T8h Pf TCTP
30
gene T12h
25
- 38% identity, 53%
20
15 similarity with hTCTP
10
5 - mimics hTCTP in vitro
0 on histamine release
P0 P6 P12 P18 P24 P30 P36 P42 P48
Sampling time - 0.1 to 1µg/ml in serum
from patients with malaria
4,5 - totally conserved in 350
Transcript RQ
4
3,5
P falciparum field isolates
PFTCTP
3 => target human cells ??
2,5
2
1,5
1
0,5
2 8 14 20 26 32 38 44 50 hours
Parasite cycle time
- 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)
57. Plasmodium can induce histamine release
hTCTP PfTCTP
Histamine
10 µg/ml 100 µg/ml
histamine release by basophils induced by rTCTP is IgE dependant
(MacDonald et al 2001)
58. Conclusion 2
20% of population with high IgE level = stable
Seasonal variation of basophiles activation
Impact of mosquitoes bites on histamine release
Histamine modulates endothelial cells thru H1R
Histamine induces rapid opening of intercellular junction
responsive for edema
PfTCTP poorly active on HBEC ( ongoing .. Hypoxia) => HRF
Anti-histamine = new way
to improve the treatment of CM ?
59. PLAN
1. Who is ill
Rural Malaria / Urban Malaria
2. « Parasite » hypothesis
3. « BBB alteration » hypothesis
• Effector role of platelets
• Histamine and BBB
• Microparticules
• IRBC
62. TNF enhances MP production by endothelium
Electron microscopy Release in culture
resting + TNF
250
200
HUVEC
EMP / 10 3 cells
150
100
50
EMP 0
0 10 100 1000
TNF (ng/ml)
Combes et al., J. Clin. Invest. 104: 93, 1999
63. Dramatic increase of plasma endothelial
plasma
microparticles in Malawian children with CM
acute phase
p < 0.0001 follow up
175 p = 0.01
150 p = 0.005
EMP / µl plasma
125
100
75
50
25
0
N 60 48 138 80 37 35 27 13
Malaria - + + + + + + +
CM - - + + - - + +
SMA - - - - + + + +
Combes et al, JAMA 291: 2542-4, 2004
64. Citicoline can protect against CM
First trial in human (Dakar HPD 2007) Protection of mice injected with PBA
100 Control
Citicoline
75 Artesiminin
%survival
CTC + Artesiminin
50
25
0
0 7 14 21
Day post-PbA infection
Qunine + CTC 1.5g/day Injection 1g/kg day 4 to end
no significant improvement of mortality
at this dose
65. PLAN
1. Who is ill
Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
• Effector role of platelets
• Histamine and BBB
• Microparticules
• IRBC
66. What happens during IRBC/endothelial cells contacts
NKT
Mo
Treg host cells
CD8
brain
endothelial
cell P. falciparum-
membranes infected red cells
67. Co-cultures of parasitised red blood cells (PRBC) and
human brain endothelial cells (HBEC)
PRBC-PKH26
Calcein AM
Microscopy
O/N 1 h 30 0 / 1 h / 2 h / 4h TEER
HBEC
Q PCR
Flow cytometry
TNF unbound cell washing
removal
INCUBATION
AutoMACS® - purified PRBC
70. PRBC-PKH26 HIS + anti human-IgG
ON 1 h 30 0/1h/2h confocal
HBEC
microscopy
TNF washing washing
AutoMACS® - purified PRBC
[HIS : pool of 10 hyper-immune sera
from African adults with malaria]
71. HBEC: D3 + IRBC-PKH26-calcein
before washing 30 min co-culture 40 min co-culture
HZ
Ration : 50 RBC/1 HBEC Adhesion Beginning of transfer
72. 1 h 30 min co-culture
PKH26
(after washing)
Diffusion of membrane compounds
HZ
calcein Merge
HBEC: D3 + IRBC: 3Ci
73. calcein
HBEC+ IRBC
4 h co-culture
Diffusion of
membrane
PKH26 and
cytosolic
compounds
74. 5.5 h co-culture HBEC / parasitised RBC
HBEC + PRBC-PK26 HBEC + PRBC + HIS Merge
HBEC
PRBC
76. Conclusion 3
Antigen presentation Dakar
Trogocytosis-like
antibodies T cells
IRBC adhesion and transfer
start as soon as 30 min
Y
Y Y
Adhesion of IRBC
Membrane transfer
Recycling MP=0
presentation
Structure of Increase of VCAM
Early endosomes TLR , Coagulation
parasite/HBEC
Interface and
proteins (SNARE , Ca ++
Vamp) Cell activation
Modulation of genes
expression
HBEC Opening of junctions
Phosphorylation +++
Apoptosis ?
Src activation ?
77. Ongoing
Africa
T and B cells
Antigen presentation
antibodies T cells
Field isolates Y
Y Y
Adhesion of IRBC
Recycling
presentation
Structure of the interface
proteins involved ? Cell activation (Src, Rac1)
Opening of junctions
78. University Sydney Institut Pasteur de Institut Pasteur (PF5)
V Combes Dakar F Nato
A Sanchez Perez D Aldebert P Beguin
F Elassaad L Marrama
MJ Jambou R Paul
GE. Grau Y Seck Hopitaux de Dakar
ML Varela B Diop
IMTSSA- Le Pharo JC Moreau
F Diène-Sarr
S Pelleau
Ibrahima Dia
D Parzy