Cerebro-Vascular Diseases Georges E. R. Grau , M.D., Privat-Docent - - PowerPoint PPT Presentation

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Cerebro-Vascular Diseases Georges E. R. Grau , M.D., Privat-Docent - - PowerPoint PPT Presentation

Feb 21, 2024 590 likes •1.06k views

An Animal Replacement Alternative for the Investigation of Cerebro-Vascular Diseases Georges E. R. Grau , M.D., Privat-Docent Discipline of Pathology Marie Bashir Institute Overview experimental approaches main disease studied our

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An Animal Replacement Alternative for the Investigation of Cerebro-Vascular Diseases

Georges E. R. Grau, M.D., Privat-Docent

Discipline of Pathology

Marie Bashir Institute

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Overview

  • experimental approaches
  • main disease studied
  • our co-culture model system
  • other clinical applications
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  • “Le fait qu’on se soucie des animaux

aujourd’hui est un signe que l’humanité progresse”

  • “The fact that we care about animals

nowadays is a sign that mankind is progressing” Boris CYRULNIK

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“All models are wrong, but some are useful”

George E.P. Box (1919-2013)

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Overview

  • experimental approaches
  • main disease studied
  • our co-culture model system
  • other clinical applications
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Endothelial cells (EC)

characterisation

culture isolation

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Endothelial cells

Also: a strategic location

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Approches expérimentales in vivo

perfusion de cytokines

mécanismes des lésions

  • bservation : immunohistopathologie

intervention : modulation de la réponse immune blocage de molécules d’adhérence (mAbs, souris KO) déplétion en leucocytes ou plaquettes

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Overview

  • experimental approaches
  • main disease studied
  • our co-culture model system
  • other clinical applications
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Major life-threatening complication: a diffuse encephalopathy due to untreated infection with Plasmodium falciparum

Mult ltisyst isystem m dys ysfunc function tion

up to 30% mortality rate

Coma

Neurological sequelae Seizures

Disori sorientation entation

Delirium

Severe metabolic acidosis

Cerebral Malaria (CM)

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Petechial haemorrhages engorgement of capillaries + enlargement of perivascular spaces brain

  • edema

↑↑↑ pro-inflammatory cytokines (TNF, IFN-g, LT)

enlarged perivascular spaces red blood cells leucocytes

Experimental cerebral malaria

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Current approaches for the study of CM

P

In vitro - modelling of CM lesion In vivo – animal models Ex vivo – post-mortem histopathology on human brain tissue Clinical studies in endemic areas

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CM is a strictly T-cell dependent pathology

cerebral m alaria

7 days

S

live PbA

anti-CD4 m Ab

+

  • +
  • S

S S

thymectomy

S

BM graft (Tdepl.)

CD4+ T cells CD8+ T cells

850R thymectomy BM graft (Tdepl.) 850R thymectomy BM graft (Tdepl.) 850R

R

CD4+ T cells CD8+ T cells cerebral malaria

+

  • +
  • I

Grau et al., J Immunol 137: 2348, 1986

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TNF is an essential mediator in CM

 high serum levels during CM  its neutralisation prevents CM

  • antiserum
  • mAb
  • pXF

 induces CM in resistant mice  absence of CM in

  • transgenics for sTNFR
  • TNF knock-outs

CM anti-TNF

Grau et al., Science 237: 1210-12, 1987

II

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“Sans technique, le genie n’est rien qu’une sale manie”

Georges BRASSENS

“Without technique, genius is nothing more than a lousy habit”

  • RESPECT
  • CARE
  • MINIMUM BURDEN
  • …
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Overview

  • experimental approaches
  • main disease studied
  • our co-culture model system
  • other clinical applications
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BRAIN endothelial cell

Mo

PRBC WBC platelets

Modelling human cerebral malaria in vitro

Immunostaining (Malawian patient) 1 cell isolation 2 co-cultures 3

PLT PLT PLT

EC

PRBC

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PRBC PLT

PLT PLT PLT

EC PRBC

In vitro evidence for a role of platelets in PRBC-EC bridging

Wassmer et al., J Immunol 176: 1180-1184, 2006

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brain endothelial cell platelet

which molecules ?

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Tri-partite, quadri-partite cultures

platelets pRBC brain endothelial cells

T MF

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Vascular Immunology Unit

Modelling cerebral malaria in vitro: 2 levels of complexity

cell-cell interactions

Mo Mo

+ cell-derived microparticles Prog Neurobiol 91: 140, 2010

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i ii B A i iii ii

Dorothée Faille

Platelet MP (PMP) bind to and are internalised in brain EC

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Compartmentalisation of PMP in brain EC

PKH67 Merge WGA 10 µm 10 µm 1 µm

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PMP bind to and transfer platelet antigens on brain EC surface

PMP membrane PKH67 CD36 / GPIV

New surface phenotype for brain EC

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PMP PKH26 / calcein Control

PMP membrane and content have a different fate after contact with EC membrane

Membrane : PKH26 Content: calcein-AM

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PMP bind and transfer platelet antigens to PRBC

PMP SN CD41

0.88 %

Acridine orange

0.21 % 0.00 % 14.5 % 0.18 % 0.00 %

TL PKH67 PKH26 Merge 5 µm

Vascular Immunology Unit

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PMP enhance PRBC cytoadherence

  • n brain EC

None

 

PMP on EC PMP on RBC

 

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Are endothelial MPs immunomodulatory?

pRBC Brain endothelial cells

Immature T cell

Activated T cell

?

Membrane surface shedding

eMPs

Julie Wheway

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Overview

  • experimental approaches
  • main disease studied
  • our co-culture model system
  • other clinical applications
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Novel applications of our brain endothelium co-culture model

  • Multiple sclerosis (coll. Prof. S. Hawke)
  • Septic shock
  • Cryptococcal meningo-encephalitis

(coll. Prof. T. Sorrell)

  • Viral meningitides (coll. Prof. N. King)
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Multiple Sclerosis

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PBMCs Bottom well Top well Endothelial cell monolayer on the filter (3 μm Ø pores)

Trans-endothelial migration (TEM) in vitro model

Human brain microvascular endothelial cell line hCMEC/D3, on polycarbonate filters TNF + IFN-g stimulation

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Monocytes Input PBMCs

filter EC monolayer

“TOP”

“BOTTOM” “MEMBRANE”

Fingolimod reduces transmigration of PBMCs from MS patients across endothelium in a BBB in vitro model

B cells T cells

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Antibody panels for flow cytometric analysis

  • f leucocyte (PBMC) subsets.
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Conclusions / TEM in MS patients

  • PBMCs from non-treated MS patients adhere and

migrate more efficiently

  • Fingolimod
  • reduces TEM of T cells, B cells and monocytes towards

the levels of healthy controls

  • might act on leucocytes, additionally to its effect on

endothelial S1PR

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Septic Shock and the blood-brain barrier

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Vascular Immunology Unit

microparticles in sepsis

Mo

Target cell changes ?

LPS +/- TNF HBEC

Do LPS-induced monocytic MP (mMP) functionally differ from MP released from resting cells? Do mMP display pro-inflammatory / procoagulant properties ? What are the effects of mMP on endothelium integrity? MP

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Early endosomes Lysosomes 45 min 2 h 45 min 2 h

moMP: PKH67 / EEA-1 moMP PKH67 / LysoTracker

LPS-induced monocytic MP partially co-localise with endothelial lysosomes

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Beryl WEN

Effect of LPS-induced monocytic microparticles on endothelial integrity

Trans-endothelial electrical resistance (TEER)

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Cryptococcal Meningitis

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Flow chamber: to explore cells in movement

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Effect of TNF on binding of phagocytosed cryptococci to brain endothelium

Resting endothelium TNF-activated endothelium

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Mechanisms of cryptococcal passage

?

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Conclusion

Better knowledge Better treatment(s)

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Vascular Immunology Unit Valéry Combes Fatima El-Assaad Dorothée Faille Sharissa Latham Beryl Wen Anna Zinger Simon Hawke Georges E. Grau

The University of Sydney Australia

Alavita, Inc. /Stanford University

Anthony Allison Luis F. Fajardo

University of Genève, Switzerland

Christine Chaponnier

Université René Descartes, Institut Cochin, Paris, France

Pierre-Olivier Couraud Molecular Immunopathology Helen Ball Andrew Mitchell Nick Hunt Marie Bashir Inst. Westmead Julianne Djordjevic Tania Sorrell Viral Immunopathology Zheng Ling Nick King

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