Breaking of the wall : role of allergy and histamine release

Immunology Unit Institut Pasteur de Madagascar

Immunology Unit Institut Pasteur de Madagascar

Ronan Jambou MD, PhD

Vascular

Immunology

Unit

Expected decrease of Rural Malaria transmissionExpected decrease of Rural Malaria transmission

Swaziland WHO Report 2006Swaziland WHO Report 2006Kilifi _ Kenya Malaria J 2007Kilifi _ Kenya Malaria J 2007

Kilifi _ East Africa Mwangi et al JID 2005Kilifi _ East Africa Mwangi et al JID 2005

Changes in transmission modulate Malaria profil Changes in transmission modulate Malaria profil

53%32Saharevo, Madagascar: ≤1 b.i.a./human/year

50%25Pikine, Sénégal: ≤1 b.i.a./human/year

41%62Ndiop, Sénégal: 10-30 b.i.a./human/year

23%43Dielmo, Sénégal: 100-300 b.i.a./human/year

10%25Danané, Côte d'Ivoire: ≥ 300 b.i.a./human/year

% withinadluts

Total Nb of attacks 60 years old

53%32Saharevo, Madagascar: ≤1 b.i.a./human/year

50%25Pikine, Sénégal: ≤1 b.i.a./human/year

41%62Ndiop, Sénégal: 10-30 b.i.a./human/year

23%43Dielmo, Sénégal: 100-300 b.i.a./human/year

10%25Danané, Côte d'Ivoire: ≥ 300 b.i.a./human/year

% withinadluts

Total Nb of attacks 60 years old

Ann

ual N

bof

mal

aria

atta

cks

0

1

2

3

4

5

6

0 5 10 15 20 25 30 35 40 45 50

Age (years)

60A

nnua

l Nb

of m

alar

ia a

ttack

s0

1

2

3

4

5

6

0 5 10 15 20 25 30 35 40 45 50

Age (years)

60

West Africa / Madagascar (meta-analysis)West Africa / Madagascar (meta-analysis)

No clear change in mortalityNo clear change in mortality

Schofield & Grau 2005

Hunt & Grau 2003

sequestration inflammation

(oedema)pathology

Major role in: Major role in: Major role in: ECECEC

Vascular permeability

� Diabetes� Dengue fever� Age degenerescence� ARDS� Myocardial infarction� Tumour angiogenesis� Brain injury

� Diabetes� Dengue fever� Age degenerescence� ARDS� Myocardial infarction� Tumour angiogenesis� Brain injury

VVO Junction remodelling

pinocytosis

pathogenspathogenspathogens stimulistimulistimuli

Histamine

Histamine induces vascular congestion

Guido Majno (1961)

vein

Histamine

HRF/TCTPbasophiles

cytokine network (IL4, IL2..) eicosanoid pathway

endothelial cells responseto inflammation

platelets / neutrophils

DC response to TH2 typeproduction of IgE

vascular permeability

Cerebral malaria ??

( HR1, HR2, HR3, HR4, HR5 )

IgE AntigenAllergy / helminths

Histamine can modulate endothelial and immune cells func tions

vWFPlatelet + IRBC

adhesion

PfTCTP is expressed by late trophozoites

PF

TC

TP

T

rans

crip

tRQ

2 8 14 20 3226 38 44 50 hours

0,51

1,52

2,53

3,54

4,5

05

10152025303540

P0 P6 P12 P18 P30P24 P36 P42 P48

- synchronisation + sampling every 6h over 54h = « sampli ng time »

- use of thin smear and timing genes (P David) to define th e « parasite time » (gene8h= MAL8P1.4, gene 12h=PFI1735c- normalisation of mRNA on average of (N1= PFC0255c and N 2=PFA0570), then attime with less mRNA (T44 = 1 for PfTCTP)

Parasite cycle time

Sampling time

gene T8h

gene T12h

Tim

ing

gene

s Pf TCTP

- 38% identity, 53% similarity with hTCTP

- mimics hTCTP in vitro on histamine release

- 0.1 to 1µg/ml in serum from patients with malaria

- totally conserved in 350 P falciparum field isolates => target human cells ??

Plasmo-DB

Allergic response in seasonal transmission area

« The background »

15°

14°

13°

16°N

Dakar

17°W 16° 15° 14° 13° 12°

SENEGAL

Area of Dakar

Seasonal transmission

4 M inhabitants = 1/3 Senegal

3.5% territory

An arabiensis

Gouly Couly

Longitudinal study: Gouly couly Longitudinal study: Gouly couly

Nov. 04Baso, IgE

Follow up

EnrolmentJun 04

Jun 05Baso, IgE

july 04IgE

EndJun 06

Rainy season Rainy seasondry season dry seasondry season

Drug resistance study

CD203Surface marker

activation of basophiles

CD63granule marker

Release

Low

high

medium

0

20

40

60

80

100

9-19 20-39 40-80

0

2

4

6

8

10

Total IgE ug/L

9-19 20-39 40-80

0

0,5

1

1,5

july 04 Nov 04 July 05

IgE

ug/

mL

0

0,5

1

1,5

2

2,5

IgE

ug/

mL

0

5

10

15

20

25

30

1 2 3

IgE

ug/

mL

% of subjects with > 0.4µg/l

0

5

10

15

20

25

30

35

40

45

9-19 20-39 40-80

% Ig

E_P

f pos

itive

nov_04Jun_05

% of subjects with IgE-Pf

No difference according to age for

high IgE level

Stability of IgE level« High is high »

No change in [IgE] according to season

Higher percent of Pf-IgE during dry

season

IgE responses : Gouly couly IgE responses : Gouly couly

Basophiles responses : Gouly couly Basophiles responses : Gouly couly

standard antigens

flmpD farinaeD pteronyssinus

0102030405060708090

9-19y 20-39y 40-80y

Per

cent

res

pond

ers

nov jun

Percent of responders maximum during rainy

season

Pf can induce response for 10% of villagers during rainy season

High response for salivary glandsHemozoin

Pf ghostSalivary glands

0

10

20

30

40

50

60

70

80

9-19y 20-39y 40-80y

nov jun nov jun nov jun

Per

cent

res

pond

ers

Mosquitoes can trigger basophiles

activation

Pf antigens

Mosquitos bites induce mast cells activationJI Demeure (2005)

Mosquitos bites induce mast cells activationJI Demeure (2005)

No difference according to age for

high IgE level

Stability of IgE level« High is high »

Higher percent of Pf-IgE during dry

season

Percent of responders maximum during rainy

season

Pf can induce response for 10% of villagers during rainy season

High response for salivary glands

Mosquitoes can trigger basophiles

activation

IgE responsesIgE responsesBasophiles responsesBasophiles responses

Anti-IgE present“consummed” ?

Players are there !

Basophyle activation during Cerebral malaria

« Is it a relevant question ??? »

Area of Dakar

Patients

Severe malaria (SM) cases were recruited at the Int ensive Care Unit (CHNU)

Mild malaria (MM) matched cases were recruited at t he Saint-Martin Dispensary

Healthy matched subjects attending the laboratory for routine examination

Mild malaria have = lower basal level of CD203c exp ression

Basal level of CD203c expression

Cerebral malaria = higher reactivity to IgE-indepen dent stimulation

IgE independant activation of basophiles

HZ hemozoin (malaria pigment)

Severe malaria=

higher reactivity to IgE-dependent

stimulation+

No over-stimulation

A

B

Basophiles response to anti-IgE in healthy subjects

Ex vivo priming with Pf- or h-TCTP increases IgE dependant response and induces over stimulation

hTCTP

Pf-TCTPAnti-IgE 1micro/ml

Basophiles response to anti-IgE in healthy subjects

Modulation by Pf-TCTP

(horizontal lines represent median for positive individuals only)

ELISA with Pf-specific monoclonal antibodies

Pf-TCTP / anti Pf-TCTP

Cerebral malaria = higher level of Pf-TCTP

PfTCTP in serum and response to anti-IgE

Stimulation with anti-IgE 0 (PBS), 0.1, 1 or 10 µg/mL

Presence of PfTCTP is associated with higher IgE depe ndant response

Anti-PfTCTP in serum and response to IgE

Presence of anti-PfTCTP is associated with protection against excessive IgE dependant response

Summary

Cerebral malaria is associated with higher basophile response to both IgE independent and dependant

response, without overstimulation

rPf-TCTP enhances basophile response during CM and induces overstimulation in healthy subjects

Presence of PfTCTP in serum enhances IgE dependant response; presence of anti-Pf-TCTP decreases this

response.

Allergic response is enhanced during CMRelevant with higher histamine detection

Histamine and brain endothelial cell interaction« to treat or not to treat »

Opening the junction Opening the junction

(Vandenbrouke et al 2008)

Histamine

HistamineNRBC IRBC180

200

220

240

260

280

300

320

340

360

380

400

420

440

Sur

face

(pi

xel)

P < 10-5

P < 10-6

NS

Histamine 100µM

IRBC

ECIS: Electric Cell-substrate Impedance Sensing

Monitoring of cells monolayer impedance to measure jun ction permMonitoring of cells monolayer impedance to measure jun ction perm eabilityeability

HBEC-D3

HBEC1.5 h

washingHistamine, TNF

Spreading: 5 days 24 h

IRBC, NRBC

TEERAttachment and spreading of the cells

0

5000

10000

15000

20000

0 20 40 60 80 100

Time (hours)

Res

ista

nce

(ohm

s)

Histamine or platelet supernatant induce opening of junctions

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

29 29.5 30 30.5 31 31.5 32

stimulationHistamine

Control medium

hours

Resistanceohm

LPS

PLT SN

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

23 28 33 38 43

stimulationMedium change

Histamine

Control medium

hours

Resistanceohm

LPS

PLT SN

H1 receptor mediates histamine effect on HBECsra

tio a

t T0

-0,35

-0,3

-0,25

-0,2

-0,15

-0,1

-0,05

0

0,05

0,1

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

his100-anti_H1

histamine100

TNF

hist100-antiH2

control

Anti H1 inhibits histamine effect on HBEC-D3 monola yer

Histamine 100 µM

Histamine induce calcium mobilization thru HR1Histamine induce calcium mobilization thru HR1

Histamine 1 µM

-100

0

100

200

300

400

500

0 5 10 15 20 25

Time (min)

Ca

(nM

)

Histamine 10 µM

Histamine

Time (min)

0.6

1.6

2.6

3.6

0 20 40 60Time (min)

Nor

mal

ised

Rat

io

Diphenylhydramine (20µM)

Histamine

Cimetidine (40µM)

0.6

1.6

2.6

3.6

4.6

0 20 40 60 80

Nor

mal

ised

Rat

io Histamine

(Pre-incubation 40 min)

H1

H2

Histamine increases VCAM but not ICAM expression

0

2

4

6

8

10

12

14

16

18

controlHistamine100Histamine100

+ antiH1H2

Histamine_10 Histamine_1

Per

cent

of p

ositi

ve c

ells

*

Histamine 100µM, 10µM, 1µMCimetidine 20µM, diphenylhydramine 20µM,

*Flow cytometry analysis after trypsinization of the HBEC-D3

Anti H1/H2 inhibits histamine effect on VCAM1 expres sion

The murine model of CM

Score Observation

0 No discernible clinical signs

1 Hunched posture, slightly

ruffled fur

2 Very ruffled fur, incipient

motor impairment

3 Very ruffled fur, severe

motor impairment such

as ataxia, hemiplegia

and paraplegia,

convulsions, fitting and

the Wooley white sign

(CBA only).

4 Very little movement, cold

to the touch

neurological

phase

cerebral

malaria

0 7 14

Blood collected

Platelet-free plasma

hyperparasitaemia

severe

anaemia

21days

39.

128.

4237.

246.

5.

DPHCiticoline ControlDay post-PbA

infection

ControlCiticholineDiphenyhydramine (anti-H1)

0 7 140

25

50

75

100

Day post-PbA infection

Per

cent

sur

viva

l

Anti_H1 increases survival of P berghei infected mice

Beghdadi et al 2008

Conclusion Conclusion

Allergy : 20% of population with high IgE level = st ableSeasonal variation of basophiles activation => Impact of mosquitoes bites on histamine release

Anti-histamine = can improve the treatment of CM ?

Clinical trial

Cerebral malaria is associated with hyper reactivity of basophiles , without auto-regulationPfTCTP enhances / anti-TCTP decrease, this response

Rapid opening of intercellular junction thru H1R Anti-H1 can inhibit this effect

University SydneyV Combes

A Sanchez PerezF ElassaadMJ Jambou

GE. Grau

IMTSSA- Le PharoS PelleauD Parzy

Institut Pasteur de Dakar

D AldebertS Pelleau

L Marrama F Diène-Sarr

R PaulIbrahima Dia

B Diop JC Moreau

Institut Pasteur (PF5)

F Nato, P Beguin

Institut Pasteur de Madagascar

RomyTsiry

Emma

Thanks for your attention

A B C

IFNg IL10 IFN/IL10

Severe malaria = imbalanced pro/anti-inflammatory r esponse

IFNg / IL10 balance

Histamine release and HRFsHistamine release and HRFs

Human TCTP (IgE-dependent HRF)

-Gene locus 13q12-q14

- induces histamine release by basophiles of atopic p atients

- in vitro : increase reactivity of basophiles to other stimul us (IgE, ..etc)

- induce proliferation of B cells and activation of e osinophiles

Pf TCTP

- 38% identity, 53% similarity with hTCTP

- mimics hTCTP in vitro on histamine release

- 0.1 to 1µg/ml in serum from patients with malaria

- totally conserved in 350 P falciparum field isolate s => target human cells ??

TCTP

- found in all eukaryotes , with two conserved motifs (microtubules binding domain)

- control cells proliferation, division, and apoptose => overexpression in cancer

Induction of idiopathic allergy ?

Relative quantity

2

1

-5

20

6

60

ICAM-1 VCAM-1IL-1b

IL-6IL-8

TNFb TLR-2

PfTCTP down regulates expresion of TRL2 and Vcam1 in HMEC

8h of stimulation

Moderate direct effect of recombinant PfTCTP on endothelial cells- Vcam and TRL2 repression- no effect on permeability - no effect on microparticules productions

sequestration

Severe Malaria

NewbornfeverdehydrationconvulsionMetabolic disorders= impaired consciousness

ChildSevere anaemia +++High parasitaemia +++Cerebral pathology

AdultRespiratory distresscerebral malarialow parasiteamia= delay in treatment

29

15 17

53

61

54 55

85 82

42

76

3737

47

74

Monde Afrique Asie Amériquelatine/Caraïbes

Régions plusavancées

1950 2000 2030 (projections)

Source : Nations Unies, Perspectives de la population dans le monde, Edition 2003 (scénario moyen), 2004.

From rural to urban: trends of the population From rural to urban: trends of the population

Major challenge for the next 10 years

Changes of risks Changes in control strategies

Prevalence of malaria in consultations

0

0,05

0,1

0,15

0,2

0,25

0,3

septembre octobre novembre decembre

00,10,20,30,40,50,6

septembre octobre novembre decembre

00,050,1

0,150,2

0,250,3

0,350,4

sept oct nov dec

part of class of age in the malaria cases

prevalence of severe malaria.

0

200

400

600

800

1000

1200

1400

M F M F M F M F M F0-1 an 1- 4ans 5-14 ans 15- 49 ans 50ans &+

consultations by age

0-1 y1- 4 y5-14 y

15- 49 y> 50 y

From children to adults From children to adults

brain endothelial cells

TNF plays a major role in sequestrationTNF plays a major role in sequestration

Lucas et al., Eur J Immunol 27: 1719, 1997Lou, Lucas & Grau Clin Microbiol Rev 14: 810, 2001

Stoelcker et al., Infect Immun 70: 5857, 2002

monocyte bindingmonocyte bindingmonocyte bindingmonocyte bindingICAMICAMICAMICAM----1 1 1 1 upregulation upregulation upregulation upregulation

platelet bindingplatelet bindingplatelet bindingplatelet binding

memTNF

TNFR2TNFR2

sol or

memLT

PRBC bindingPRBC bindingPRBC bindingPRBC binding

Functional consequence of TNFR2 upregulation (Functional consequence of TNFR2 upregulation ( mouse modelmouse model , PbA infection), PbA infection)