Paludisme grave : pourquoi doit-on développer des modèles in vitro sur le terrain ? - Conférence de la 8e édition du Cours international « Atelier Paludisme » - RAZAKANDRAINIBE Romy - Madagascar - romy@pasteur.mg

1. 1

2. Malaria’s facts
-About 3.3 billion people are at risk of malaria
-Every year: about 250 million malaria cases
2 and nearly one million deaths

3. Why studying malaria ?
1. Epidemiological transition of malaria
• Urbanization =>↓ rate of transmission
• Increased drug consumption
2. Antiparastic treatment: insufficient to control CM
Target : reduce mortality rates
=> Propose new strategies that can both eliminate
parasites and protect from the pathogenic
mechanisms induced by the parasite

4. Malaria complication: severe malaria
Severe malaria: characterized by cerebral malaria,
metabolic acidosis, renal failure, pulmonary oedema,
anemia, hypoglycemia, shock, and jaundice
Cerebral malaria (CM): the pathogenesis is
heterogenous and the neurological complications
are often part of a multisystem dysfunction.
=>CM characteristics : sequestration and Cerebral oedema
and/or micro-hemorrhages are features of endothelial
alteration in CM

5. Sequestration and Heamorrhages

6. Defined CM
Taylor et al, Nat Med. 10: 143‐145, 2004
In patients, CM-associated brain damage can be studied on only
post-mortem specimens and is the end-point of a fatal syndrome.
However, it is not known whether these alterations also are present
at the first stages of the disease.

7. Issues
• Post-mortem analyses = Study of severe malaria pathology at the
terminal stage
• Informative changes early in the disease process are inaccessible !!
• The mouse models for CM do not recapitulate human disease completely
• Understanding the pathogenesis of CM is important = can be achieved
through a combined approach involving ex vivo studies using patient
samples (blood cells, plasma), in vitro modelling of the interactions
between the various cells and their released mediators
7

8. Modelling

9. Pathogenic mechanisms hypothesis
• Mechanical theory: sequestration ?
• Immunopathology theory: BBB alteration, effects of the
immune system and mediators ?
• Combination of both ?
• The fine mechanisms of this complex syndrome remain
incompletely understood.
9

10. Blood brain barrier (BBB)
courtesy of Ronan Jambou
10

11. Endothelial cells and P. falciparum infection
Jambou et al. Plos pathogen (2010)
al.
11 Immunology unit (IPM)

12. Gap of knowledge
• The fate and effects of Infected Red Blood Cells
uptake in endothelial cell are unknown
• Studies use up today P. falciparum lab strain
=> How about wild strain (large diversity)?
12

13. Objectives
To set up an in vitro model of BBB to study the effects
immune cells and the fate of red blood cells infected
with field isolates
of P. falciparum internalized in endothelial cells
13

14. Endothelial cells as APC
• Endothelial cells express MHC II in tissue culture (Leeuwenberg et al. 1988)
1988)
• Endothelial cells are able for cross Presentation (Rock el al . 2005)
• Antigen presentation by a continuous human microvascular
endothelial cell line (HMEC-1), to human T cells. (Bosse D, et al. 1993)
• Endothelial cells have capacity to costimulate T cells in vitro (Briscoe
DM et al . 1997)
• Presence of Immunoproteasome (Mishto et al.2006)
Endothelial cell act as an antigen presenting cell (APC)
14

15. Specific objectives
FIELD
Patient Healthy Immune villagers
Parasited red blood cell Immune cells
?
Antigen presentation
internalization
APOPTOSIS
JUNCTION OPENING
Analyze, on the field, the interaction of P. falciparum-sensitized endothelium
and immune cells and its role in the pathology
Analyzing BBB alteration induced by immune cells from healthy premunized donors

16. Methodology
In vitro model: Human Brain Endothelial Cells (HBEC-D3)+astrocytes
Co-cultivation with P. falciparum late stage (field isolate)
(positive magnetic selection: Miltenyi column and magnet)
Analysis of endothelium permeability Analysis of adhesion molecule expression
(Lucifer yellow diffusion) (qPCR)
assessment of Cytokine
CM model +Immune cells from health patients
production
Analysis of calcium flux
Quantification of adhesion Detection of apoptosis
(Fluo4-AM) (Tunnel assay, caspase 3,7,9 )
assay,
PKH labelling and fluorimetry
Fluorometry Flow cytometry
Immunology Unit

17. Analysis of permeability

18. • Quantification of gene (required for antigen presentation)
expression changes by RT-qPCR
Tm CT values
HPRT 82,4 HPRT 21,71 CD80
CD83 86,6 CD83 27,83
CD86 83,2 CD86 26,01
CD80 82,9 CD80 32,93
CD83
CD86
HPRT
Run profile set up

19. Conclusion and perspectives
In vitro modelling in field: strengthening SOUTH
research capacity, hypothesis generation
Development of new therapeutic strategy to protect
BBB
the identification of antigens presented by the
endothelial cells to the immune system=> new
vaccine candidate

20. THANK YOU FOR YOUR ATTENTION
•Parteners
•University of Sydney (vascular immunology Unit : G. GRAU, V. COMBES, J. WHEWAY)
•Epidemiology unit – Institut Pasteur Madagascar
•Malaria Unit - Institut Pasteur Madagascar
•Dr COURAULT P.O ( HBEC-D3) Cochin Institut Paris