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Transition écologique du paludisme et implication
                  thérapeutique

                 Ronan Jambou MD, PhD



          Vascular Immunology Unit




             Vascular
           Immunology
                 Unit
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
PLAN
1. Who is ill
      Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
  •     Effector role of platelets
  •     Histamine and BBB
  •     Microparticules
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
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.
Transition écologique du paludisme et implications thérapeutiques
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
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
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
Adaptation of strategies

Change in drug strategy
Change in drug strategy             ITN implementation
                                    ITN implementation




WHO Africa report 2006
Expected decrease of Rural Malaria transmission

Kilifi _ Kenya Malaria J 2007
Kilifi _ Kenya Malaria J 2007   Swaziland WHO Report 2006
                                Swaziland WHO Report 2006
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)
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
« 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
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
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
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
Which mechanisms ?

             host
pathogen

           tox              Infectious pathology



                          Adaptation of the treatment
                             Healthcare supply



                    tox      Immunopathology



                          models      additive treatments
Which mechanisms ?

             host
pathogen

           tox              Infectious pathology



                          Adaptation of the treatment
                             Healthcare supply



                    tox      Immunopathology



                             additive treatments
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
sequestration
Hunt & Grau

24: 491-499, 2003

Schofield & Grau

5: 722-735, 2005




mechanisms?
?        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
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
TNF: a central mediator in immunopathology
anti-TNF antibody
                                     VASCULAR LESIONS :
                                       septic shock (*)
                                       cerebral malaria
TNF                                                  (*) Beutler & Cerami, 1985

                     adhesion
                     molecules         TISSUE LESIONS :
                                        graft-versus-host
  microvessel                          pulmonary fibrosis
(brain, lung, ...)                         granuloma
                                             arthritis
                                               DTH
                                 Grau et al., Immunol Rev; 112: 49-70 (1989)
                                      Int Rev Exp Pathol 34: 159-171 (1993)
Cytokines and pathology: approaches

Pathogen

S



    R
                            Δ pathology?   observation

S
                 cytokine
           + mini-pumps               ?    induction

S
           +
               antibodies,
                inhibitors
                                      ?    inhibition
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
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
Accumulation of platelets in a
TNF-mediated pathology (LPS shock)
Platelets: the conventional view
Prevention of LPS-induced mortality
          by anti-platelet antibodies
       pretreatment        LPS   survivors/total (%)

none                        -       10/10 (100)
none                        +         4/41 (10)
normal mouse IgG            +          0/6 (0)
normal rabbit IgG           +          0/6 (0)
mouse anti-platelet mAb     +       10/12 (83%)
rabbit anti-platelet IgG    +        7/9 (77%)
Prevention of hemorrhagic necrosis
               by anti-platelet or anti-CAM mAbs
                  anti-platelet    anti-CAM
                                              51Cr-erythrocytes        (% cpm)
- 24 h (id)     - 2 h (ip)     0 h (id)
                                          0     25           50   75    100   125

  PBS          none            TNF
  LPS          none            PBS
  LPS          normal IgG      TNF
  LPS          anti-CD11a      TNF                            *
  LPS          anti-CD54       TNF                   *
  LPS          anti-platelet   TNF                       *
Platelet-EC interactions


                           stroke

                             metastasis

                             INFLAMMATION
                             THROMBOSIS
microvessel                 angiogenesis

                                Mol. Pathol. 50: 175-185 (1997)
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
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
PLAN
1. Who is ill
      Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
  •     Effector role of platelets
  •     Histamine and BBB
  •     Microparticules
  •     IRBC
Vascular permeability

                               pathogens         stimuli
Major role in:
                        EC
Diabetes
Dengue fever
Age degenerescence
ARDS
Myocardial infarction
Tumour angiogenesis                  Junction
                         VVO                        pinocytosis
Brain injury                       remodelling
Histamine induces vascular congestion
          Guido Majno (1961)




vein

                                    arteriole
Histamine induces opening of intercellular junctions
                 J cell Biol (1969)
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
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
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
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
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 ?
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
Mosquitos bites induce mast cells activation
             JI Demeure (2005)
Monitoring of cells monolayer impedance to measure junction permeability
                   ECIS™: Electric Cell-substrate Impedance Sensing




                                            HBEC-D3
IRBC, NRBC
                                            Histamine, TNF
                                                                      washing

                          Spreading: 5 days                   1.5 h                  24 h
HBEC


                                   Attachment and spreading of the cells              TEER
                       20000
   Resistance (ohms)




                       15000


                       10000


                        5000


                           0
                               0          20             40                60   80          100
                                                          Time (hours)
Histamine or platelet supernatant induce opening of junctions


18000                                                                                                     Control medium
Resistance
   ohm                                                                                                    Histamine
                stimulation
16000                                                                                                     LPS
                                                                                                          PLT SN
14000


12000


10000


 8000
                                          18000                                                                      Control medium
                                          Resistance
                                             ohm     Medium                                                           Histamine
                                                                          stimulation
                                          16000        change                                                         LPS
 6000                                                                                                                 PLT SN
                                          14000



                                          12000


 4000                                     10000



                                           8000



 2000                                      6000



                                           4000



                                           2000                                                                    hours
    0
                                                                                                                            hours
        29      29.5          30   30.5                         31                           31.5        32
                                              0
                                                  23                 28                 33          38        43
H1 receptor mediates histamine effect on HBECs

                0,1



              0,05



                  0

                      1   2    3   4   5   6   7   8    10   11   12   13   14   16   17   18   19   20   21   22   23   24   25

              -0,05
ratio at T0




               -0,1



              -0,15



               -0,2



              -0,25



               -0,3
                                       his100-anti_H1
                                       histamine100
              -0,35
                                       TNF
                                       hist100-antiH2
                                       control
Histamine induce calcium mobilization thru HR1

                                                                                      3.6




                                                                  Normalised Ratio
             Histamine 100 µM
                                                                                                     Histamine
                                                                                      2.6

                                                                                      1.6

                                                                                      0.6
                                                                                            0             20         40          60
                                                                                                            Time (min)
                 Histamine 10 µM                                                                                        Cimetidine (40µM)
                                                                                      4.6




                                                                   Normalised Ratio
                                                                                                   Histamine
                                                                                      3.6

                                                                                      2.6
                                                                                                                                      H2
                                                                                      1.6

                                                                                      0.6
                                                                                            0        20        40         60     80
                                                                                                           Time (min)
                                                                                                                        Diphenylhydramine
                                                                                                                              (20µM)
                                                                                                   Histamine
                     Histamine 1 µM
          500
          400
          300
                                                                                                                                      H1
Ca (nM)




          200
          100
             0
          -100
                 0         5     10          15   20   25
                                                                                                (Pre-incubation 40 min)
                                Time (min)
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,
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
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 ?
Plasmodium can induce histamine release :
          TCTP/HRF protein
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)
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)
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 ?
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
Membrane vesiculation




                        From Zwaal et al.
Phosphatidyl-serine
Phosphatidyl-serine




Phosphatidyl-ethanolamine
Phosphatidyl-ethanolamine
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
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
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
PLAN
1. Who is ill
      Rural Malaria / Urban Malaria
2. « BBB alteration » hypothesis
  •     Effector role of platelets
  •     Histamine and BBB
  •     Microparticules
  •     IRBC
What happens during IRBC/endothelial cells contacts



                       NKT

                             Mo
                Treg                 host cells
                       CD8




 brain
 endothelial
 cell                               P. falciparum-
 membranes                          infected red cells
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
HBEC D3 + IRBC (3Ci)       merozoite

   1 h incubation



  PRBC




         hemozoin




                    HBEC
HBEC D3 + IRBC (3Ci)
    Engulfing ?
    knobs
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]
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
1 h 30 min co-culture
PKH26
                   (after washing)
              Diffusion of membrane compounds


                                      HZ




calcein           Merge




HBEC: D3 + IRBC: 3Ci
calcein
            HBEC+ IRBC

          4 h co-culture

           Diffusion of
           membrane
PKH26      and
           cytosolic
           compounds
5.5 h co-culture HBEC / parasitised RBC
HBEC + PRBC-PK26        HBEC + PRBC + HIS   Merge




                                                        HBEC




                                                        PRBC
IRBC but not NRBC induce opening of the junctions

                                                                               control
                                                                               NRBC
                                                                               IRBC
                                                                               Histamine 100

                     Stimulus                                                  NRBC + IRBC (vol/vol)                                                                 Stimulus
                      1,4                                                                                                                                  1,4




                                                                                                                        resistance ration (T/Tinjection)
                      1,2                                                                                                                                  1,2
resistance (ratio T/T0)




                          1                                                                                                                                      1




                      0,8                                                                                                                                  0,8




                      0,6                                                                                                                                  0,6




                      0,4                                                                                                                                  0,4




                      0,2                                                                                                                                  0,2




                          0                                                                                                                                      0

                              1   7   13   19   25   31   37    43   49   55   61   67   73   79   85   91   97   103                                                1   6   11   16   21   26   31   36   41   46   51   56   61   66   71   76   81   86


                                                               Time ( 0.1 hour)                                                                                                                            Time (0.1 hour)
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 ?
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
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

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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
  • 22. Hunt & Grau 24: 491-499, 2003 Schofield & Grau 5: 722-735, 2005 mechanisms?
  • 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
  • 25. TNF: a central mediator in immunopathology anti-TNF antibody VASCULAR LESIONS : septic shock (*) cerebral malaria TNF (*) Beutler & Cerami, 1985 adhesion molecules TISSUE LESIONS : graft-versus-host microvessel pulmonary fibrosis (brain, lung, ...) granuloma arthritis DTH Grau et al., Immunol Rev; 112: 49-70 (1989) Int Rev Exp Pathol 34: 159-171 (1993)
  • 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
  • 29. Accumulation of platelets in a TNF-mediated pathology (LPS shock)
  • 31. Prevention of LPS-induced mortality by anti-platelet antibodies pretreatment LPS survivors/total (%) none - 10/10 (100) none + 4/41 (10) normal mouse IgG + 0/6 (0) normal rabbit IgG + 0/6 (0) mouse anti-platelet mAb + 10/12 (83%) rabbit anti-platelet IgG + 7/9 (77%)
  • 32. Prevention of hemorrhagic necrosis by anti-platelet or anti-CAM mAbs anti-platelet anti-CAM 51Cr-erythrocytes (% cpm) - 24 h (id) - 2 h (ip) 0 h (id) 0 25 50 75 100 125 PBS none TNF LPS none PBS LPS normal IgG TNF LPS anti-CD11a TNF * LPS anti-CD54 TNF * LPS anti-platelet TNF *
  • 33. Platelet-EC interactions stroke metastasis INFLAMMATION THROMBOSIS microvessel angiogenesis Mol. Pathol. 50: 175-185 (1997)
  • 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
  • 38. Histamine induces vascular congestion Guido Majno (1961) vein arteriole
  • 39. Histamine induces opening of intercellular junctions J cell Biol (1969)
  • 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
  • 46. Mosquitos bites induce mast cells activation JI Demeure (2005)
  • 47. Monitoring of cells monolayer impedance to measure junction permeability ECIS™: Electric Cell-substrate Impedance Sensing HBEC-D3
  • 48. IRBC, NRBC Histamine, TNF washing Spreading: 5 days 1.5 h 24 h HBEC Attachment and spreading of the cells TEER 20000 Resistance (ohms) 15000 10000 5000 0 0 20 40 60 80 100 Time (hours)
  • 49. Histamine or platelet supernatant induce opening of junctions 18000 Control medium Resistance ohm Histamine stimulation 16000 LPS PLT SN 14000 12000 10000 8000 18000 Control medium Resistance ohm Medium Histamine stimulation 16000 change LPS 6000 PLT SN 14000 12000 4000 10000 8000 2000 6000 4000 2000 hours 0 hours 29 29.5 30 30.5 31 31.5 32 0 23 28 33 38 43
  • 50. H1 receptor mediates histamine effect on HBECs 0,1 0,05 0 1 2 3 4 5 6 7 8 10 11 12 13 14 16 17 18 19 20 21 22 23 24 25 -0,05 ratio at T0 -0,1 -0,15 -0,2 -0,25 -0,3 his100-anti_H1 histamine100 -0,35 TNF hist100-antiH2 control
  • 51. Histamine induce calcium mobilization thru HR1 3.6 Normalised Ratio Histamine 100 µM Histamine 2.6 1.6 0.6 0 20 40 60 Time (min) Histamine 10 µM Cimetidine (40µM) 4.6 Normalised Ratio Histamine 3.6 2.6 H2 1.6 0.6 0 20 40 60 80 Time (min) Diphenylhydramine (20µM) Histamine Histamine 1 µM 500 400 300 H1 Ca (nM) 200 100 0 -100 0 5 10 15 20 25 (Pre-incubation 40 min) Time (min)
  • 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 ?
  • 55. Plasmodium can induce histamine release : TCTP/HRF protein
  • 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
  • 60. Membrane vesiculation From Zwaal et al.
  • 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
  • 68. HBEC D3 + IRBC (3Ci) merozoite 1 h incubation PRBC hemozoin HBEC
  • 69. HBEC D3 + IRBC (3Ci) Engulfing ? knobs
  • 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
  • 75. IRBC but not NRBC induce opening of the junctions control NRBC IRBC Histamine 100 Stimulus NRBC + IRBC (vol/vol) Stimulus 1,4 1,4 resistance ration (T/Tinjection) 1,2 1,2 resistance (ratio T/T0) 1 1 0,8 0,8 0,6 0,6 0,4 0,4 0,2 0,2 0 0 1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 Time ( 0.1 hour) Time (0.1 hour)
  • 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