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Malaria’s facts

--About 3.3 billion people are at risk of malariaAbout 3.3 billion people are at risk of malaria--Every year: about 250 million malaria cases Every year: about 250 million malaria cases

and nearly one million deathsand nearly one million deaths

Why studying malaria ?

1. Epidemiological transition of malaria• Urbanization => ↓ rate of transmission• Increased drug consumption

2. Antiparastic treatment: insufficient to control C M

Target : reduce mortality rates

=> Propose new strategies that can both eliminate parasites and protect from the pathogenic mechanisms induced by the parasite

Severe malariaSevere malaria : characterized by : characterized by cerebral malariacerebral malaria , , metabolic acidosis, renal failure, pulmonary metabolic acidosis, renal failure, pulmonary oedemaoedema , , anemia, hypoglycemia, shock, and jaundiceanemia, hypoglycemia, shock, and jaundice

Cerebral malaria (CM)Cerebral malaria (CM) : the pathogenesis is : the pathogenesis is heterogenousheterogenous and the neurological complications and the neurological complications are often part of a are often part of a multisystemmultisystem dysfunction.dysfunction.

=>=>CM CM characteristicscharacteristics : : sequestrationsequestration andand Cerebral Cerebral oedemaoedemaand/or and/or micromicro --hemorrhageshemorrhages are features of endothelial are features of endothelial alteration in CMalteration in CM

Malaria complication: severe malaria

Sequestration and Heamorrhages

Defined CM

In patients, CM-associated brain damage can be studie d on only post-mortem specimens and is the end-point of a fat al syndrome. However, it is not known whether these alterations also are present at the first stages of the disease.

Taylor et al, Nat Med. 10: 143‐145, 2004

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

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Modelling

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.

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Blood brain barrier (BBB)

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courtesycourtesy ofof Ronan Jambou Ronan Jambou

EndothelialEndothelial cellscells andand P. falciparumP. falciparum infectioninfection

11 ImmunologyImmunology unit (IPM)unit (IPM)

Jambou et Jambou et alal . . PlosPlos pathogenpathogen (2010)(2010)

Gap of knowledge

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•• The fate and effects of Infected Red Blood Cells The fate and effects of Infected Red Blood Cells uptake in endothelial cell are unknown uptake in endothelial cell are unknown

•• Studies use up today Studies use up today P. falciparumP. falciparum lab strainlab strain=> How about wild strain (large diversity)?=> How about wild strain (large diversity)?

Objectives

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To set up an To set up an in vitroin vitro model of BBB to study the effects model of BBB to study the effects immune cells and the fate of red blood cells infec ted immune cells and the fate of red blood cells infec ted

with field isolates with field isolates of of P. falciparumP. falciparum internalized in endothelial cells internalized in endothelial cells

Endothelial cells as APC

•• Endothelial cells express MHC II in tissue culture Endothelial cells express MHC II in tissue culture ((LeeuwenbergLeeuwenberg et al. 1988et al. 1988))

•• Endothelial cells are able for cross Presentation Endothelial cells are able for cross Presentation (Rock el al . 2005)(Rock el al . 2005)

•• Antigen presentation by a continuous human Antigen presentation by a continuous human microvascularmicrovascular

endothelial cell line (HMECendothelial cell line (HMEC--1), to human T cells. 1), to human T cells. ((BosseBosse D, et al. D, et al. 1993)1993)

•• Endothelial cells have capacity to Endothelial cells have capacity to costimulatecostimulate T cells T cells in vitroin vitro (Briscoe (Briscoe

DM et al . 1997)DM et al . 1997)

•• Presence of Presence of ImmunoproteasomeImmunoproteasome ((MishtoMishto et al.2006)et al.2006)

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Endothelial cell act as an antigen presenting cell (APC)

FIELDFIELD

PatientPatient HealthyHealthy Immune Immune villagersvillagers

Specific objectives

internalizationinternalization

AntigenAntigen presentationpresentation

ParasitedParasited redred bloodblood cellcell Immune Immune cellscells

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APOPTOSISAPOPTOSIS

JUNCTION OPENINGJUNCTION OPENING

Analyze, Analyze, on the fieldon the field , the interaction of , the interaction of P. falciparumP. falciparum--sensitized endothelium sensitized endothelium and immune cells and its role in the pathology and immune cells and its role in the pathology

Analyzing BBB alteration induced by immune cells from healthy Analyzing BBB alteration induced by immune cells from healthy premunizedpremunized donorsdonors

Methodology

In vitro model: HHuman uman BBrain rain EEndothelial ndothelial CCellsells (HBEC-D3)+astrocytes

Co-cultivation with P. falciparum late stage (field isolate)(positive magnetic selection: Miltenyi column and magnet)

AnalysisAnalysis ofof endotheliumendothelium permeabilitypermeability((Lucifer Lucifer yellowyellow diffusiondiffusion ))

AnalysisAnalysis ofof adhesionadhesion moleculemolecule expressionexpression((qPCRqPCR))

CM CM modelmodel +Immune +Immune cellscells fromfrom healthhealth patientspatients

AnalysisAnalysis ofof calcium fluxcalcium flux(Fluo4(Fluo4 --AM)AM)

FluorometryFluorometry

Quantification Quantification ofof adhesionadhesionPKH labelling PKH labelling andand fluorimetryfluorimetry

DetectionDetection ofof apoptosisapoptosis(Tunnel (Tunnel assayassay , , caspasecaspase 3,7,9 ) 3,7,9 )

FlowFlow cytometrycytometry

assessment of Cytokineassessment of Cytokine

productionproduction

ImmunologyImmunology Unit Unit

Analysis of permeability

•• Quantification Quantification ofof genegene ((requiredrequired for for antigenantigen presentationpresentation) )

expression changes by expression changes by RTRT--qPCRqPCR

HPRT 21,71

CD83 27,83

CD86 26,01

CD80 32,93

HPRT 82,4

CD83 86,6

CD86 83,2

CD80 82,9

CT values

HPRTHPRT

CD86CD86CD86CD86

CD83CD83

CD80CD80Tm

Run profile set upRun profile set up

�� In vitro In vitro modellingmodelling in field: strengthening SOUTH in field: strengthening SOUTH research capacity, hypothesis generation research capacity, hypothesis generation

�� Development of new therapeutic strategy to protect Development of new therapeutic strategy to protect BBBBBB

�� the identification of antigens presented by the the identification of antigens presented by the endothelial cells to the immune system=> new endothelial cells to the immune system=> new vaccine candidatevaccine candidate

Conclusion and perspectives

THANK YOU FOR YOUR THANK YOU FOR YOUR ATTENTIONATTENTION

•Parteners

•University of Sydney (vascular immunology Unit : G. GRA U, V. COMBES, J. WHEWAY)

•Epidemiology unit – Institut Pasteur Madagascar

•Malaria Unit - Institut Pasteur Madagascar

•Dr COURAULT P.O ( HBEC-D3) Cochin Institut Paris