1
Malaria’s facts             -About 3.3 billion people are at risk of malaria              -Every year: about 250 million m...
Why studying malaria ?1. Epidemiological transition of malaria• Urbanization =>↓ rate of transmission• Increased drug cons...
Malaria complication: severe malariaSevere malaria: characterized by cerebral malaria,  metabolic acidosis, renal failure,...
Sequestration and Heamorrhages
Defined CM                                           Taylor et al, Nat Med. 10: 143‐145, 2004In patients, CM-associated br...
Issues    • Post-mortem analyses = Study of severe malaria pathology at the      terminal stage    • Informative changes e...
Modelling
Pathogenic mechanisms hypothesis    • Mechanical theory: sequestration ?    • Immunopathology theory: BBB alteration, effe...
Blood brain barrier (BBB)                                 courtesy of Ronan Jambou10
Endothelial cells and P. falciparum infection                                               Jambou et al. Plos pathogen (2...
Gap of knowledge     • The fate and effects of Infected Red Blood Cells       uptake in endothelial cell are unknown     •...
Objectives     To set up an in vitro model of BBB to study the effects      immune cells and the fate of red blood cells i...
Endothelial cells as APC     • Endothelial cells express MHC II in tissue culture (Leeuwenberg et al. 1988)               ...
Specific objectives                                                 FIELD                                   Patient       ...
Methodology      In vitro model: Human Brain Endothelial Cells (HBEC-D3)+astrocytes              Co-cultivation with P. fa...
Analysis of permeability
• Quantification of gene (required for antigen presentation)  expression changes by RT-qPCR       Tm                      ...
Conclusion and perspectivesIn vitro modelling in field: strengthening SOUTHresearch capacity, hypothesis generationDevelop...
THANK YOU FOR YOUR ATTENTION•Parteners•University of Sydney (vascular immunology Unit : G. GRAU, V. COMBES, J. WHEWAY)•Epi...
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Paludisme grave : pourquoi doit-on développer des modèles in vitro sur le terrain ?

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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

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Paludisme grave : pourquoi doit-on développer des modèles in vitro sur le terrain ?

  1. 1. 1
  2. 2. Malaria’s facts -About 3.3 billion people are at risk of malaria -Every year: about 250 million malaria cases2 and nearly one million deaths
  3. 3. Why studying malaria ?1. Epidemiological transition of malaria• Urbanization =>↓ rate of transmission• Increased drug consumption2. Antiparastic treatment: insufficient to control CMTarget : reduce mortality rates=> Propose new strategies that can both eliminate parasites and protect from the pathogenic mechanisms induced by the parasite
  4. 4. Malaria complication: severe malariaSevere malaria: characterized by cerebral malaria, metabolic acidosis, renal failure, pulmonary oedema, anemia, hypoglycemia, shock, and jaundiceCerebral 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. 5. Sequestration and Heamorrhages
  6. 6. Defined CM Taylor et al, Nat Med. 10: 143‐145, 2004In patients, CM-associated brain damage can be studied on onlypost-mortem specimens and is the end-point of a fatal syndrome.However, it is not known whether these alterations also are presentat the first stages of the disease.
  7. 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 mediators7
  8. 8. Modelling
  9. 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. 10. Blood brain barrier (BBB) courtesy of Ronan Jambou10
  11. 11. Endothelial cells and P. falciparum infection Jambou et al. Plos pathogen (2010) al.11 Immunology unit (IPM)
  12. 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. 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 cells13
  14. 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. 15. Specific objectives FIELD Patient Healthy Immune villagers Parasited red blood cell Immune cells ? Antigen presentation internalization APOPTOSIS JUNCTION OPENINGAnalyze, on the field, the interaction of P. falciparum-sensitized endotheliumand immune cells and its role in the pathologyAnalyzing BBB alteration induced by immune cells from healthy premunized donors
  16. 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 productionAnalysis 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. 17. Analysis of permeability
  18. 18. • Quantification of gene (required for antigen presentation) expression changes by RT-qPCR Tm CT valuesHPRT 82,4 HPRT 21,71 CD80CD83 86,6 CD83 27,83CD86 83,2 CD86 26,01CD80 82,9 CD80 32,93 CD83 CD86 HPRT Run profile set up
  19. 19. Conclusion and perspectivesIn vitro modelling in field: strengthening SOUTHresearch capacity, hypothesis generationDevelopment of new therapeutic strategy to protectBBBthe identification of antigens presented by theendothelial cells to the immune system=> newvaccine candidate
  20. 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

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