Dengue Hemorrhagic Fever: Pathology and...

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Dengue Hemorrhagic Fever: Pathology and Pathogenesis Judith F. Aronson, M.D. Department of Pathology The University of Texas Medical Branch Galveston TX Dengue Hemorrhagic Fever is the most severe manifestation of human infection by the mosquito-borne flavivirus Dengue. Dengue virus is an enveloped virus, with a single stranded, positive sense RNA genome that encodes three structural genes (E, PrM, C) and seven nonstructural genes. There are four antigenically distinct serotypes of Dengue virus (DEN1-4). Geographic expansion of the range of dengue serotypes and the Aedes aegypti vector has been accompanied by dramatically increasing numbers of Dengue fever and DHF cases. DHF is distinguished from classic Dengue Fever (DF) by the presence of vascular leak, manifesting as hemoconcentration, hypoproteinemia, serous effusions, and in the most severe cases, shock. DHF has been classified into four grades based on clinical indicators, with grades III and IV representing Dengue shock syndrome (DSS). DHF occurs most commonly in children and is associated with secondary infection by a heterologous Dengue serotype. DHF is generally associated with higher viremia titers than DF. Thrombocytopenia is a constant feature of dengue infections, but the mechanism of this is not clear. DIC is seen in only a few instances of grades III-IV DHF. Plasma leak coincides with defervescence and clearance of viremia, suggesting immunopathological mechanism of endothelial injury as opposed to direct effects of virus.

The pathology of fatal DHF has been well described in large autopsy series. Hemorrhages of the pleura, epicardium, gastrointestinal mucosa and skin are present, and serous effusions and edema of retroperitoneal soft tissues are prominent. Histopathologic manifestations are dominated by the liver lesion, which consists of variable degrees of hepatocellular necrosis, primarily midzonal. Other features of the associated hepatitis, such as presence of Councilman bodies and Torres bodies are reminiscent of Yellow Fever. Spleens show atrophy of the white pulp, both T and B cell areas, along with increased numbers of reactive lymphocytes in the red pulp, correlating with the presence of atypical lymphocytes in the peripheral blood. Capillaries and arterioles in several organs show endothelial swelling, minimal perivascular inflammation and edema, and rare apoptotic endothelial cells. In general, histopathologic changes do not explain the profound microvascular insufficiency characteristic of this disease.

Dendritic cells and cells of the mononuclear phagocyte system are important early targets of infection. Immature Langerhans cells are permissive for infection and are likely the earliest target after infection by the bite of an infected mosquito. Antibody dependent enhancement of monocyte infection has been demonstrated in primary unfractionated cultures of human peripheral blood leukocytes and splenocytes infected with various DEN isolates. In tissues obtained at autopsy or biopsy, immunohistochemistry demonstrates viral antigen in hepatocytes, Kupffer cells, splenic macrophages, and, focally, in endothelial cells.

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DHF is believed to be immunologically driven. The Halstead hypothesis states that secondary infection by a different Dengue strain results in antibody dependent enhancement of mononuclear phagocyte infection. Secondary dengue infections are also associated with generation of cross-reactive T cell responses originating from T memory cells. Severe disease is associated with immunological activation markers, such as sIL-2R, IL-2, and activated immunophenotype of peripheral blood monocytes. The degree of liver injury correlates not with viremia, but with markers of immune activation. Mechanisms of endothelial injury are likely multiple and include direct viral effects and indirect effects of cytokines and other mediators. Overproduction of inflammatory cytokines, such as IFNγ, TNFα, MCP-1, and IL-8 has been documented in serum of DHF patients. Monocyte/macrophages and activated T cells are among the probable sources of these mediators. Antibodies generated against the viral NS1 protein cross-react with microvascular endothelial cells and may initiate endothelial injury. Infected endothelial cells show altered expression of VEGF receptors and matrix metalloproteinases, which participate in the regulation of endothelial permeability. The viral protein NS1 interacts with the complement inhibitory protein clusterin, suggesting alterations in complement regulation. The identification of of cross-reactive anti-E antibodies that bind plasmin peptides suggest possible interference with fibrinolysis/coagulation systems. Any explanation of vascular leak syndrome in DHF must take into account the relatively sparse infection of microvascular endothelial cells and paucity of frank endothelial damage in fatal human cases. Because animal models that recapitulate the natural history and pathology of DHF are not available, significant gaps remain in understanding the kinetics and sites of viral replication and their relationship to plasma leakage syndromes.

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Reference List

1. Basu, A. and U. C. Chaturvedi. 2008. Vascular endothelium; the battlefield of dengue viruses. FEMS Immunology & Medical Microbiology 53:287-299

2. Bhamarapravati, N., P. Tuchinda, and V. Boonyapaknavik. 1967. Pathology of Thailand haemorrhagic fever: a study of 100 autopsy cases. Annals of Tropical Medicine and Parasitology 61:500-510

3. Durbin, A. P., M. J. Vargas, K. Wanionek, S. N. Hammond, A. Gordon, C. Rocha, A. Balmaseda, and E. Harris. 2008. Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever. Virology 376:429-435

4. Halstead, S. B. and P. Simasthien. 1970. Observations related to the pathogenesis of dengue hemorrhagic fever. II. Antigenic and biologic properties of dengue viruses and their association with disease response in the host. Yale J Biol Med 42:276-292

5. Huerre, M. R., N. T. Lan, P. Marianneau, N. B. Hue, H. Khun, N. T. Hung, N. T. Khen, M. T. Drouet, V. T. Q. Huong, D. Q. Ha, Y. Buisson, and V. Deubel. 2001. Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children. Virchows Archiv 438:107-115

6. Jessie, K., M. Y. Fong, S. Devi, S. K. Lam, and K. T. Wong. 2004. Localization of Dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization. Journal of Infectious Diseases 189:1411-1418

7. Kou, Z., M. Quinn, H. Chen, W. W. Rodrigo, R. C. Rose, J. J. Schlesinger, and X. Jin. 2008. Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells. Journal of Medical Virology 80:134-146

8. Leong, A. S., K. T. Wong, T. Y. Leong, P. H. Tan, and P. Wannakrairot. 2007. The pathology of dengue hemorrhagic fever. Seminars in Diagnostic Pathology 24:227-236

9. Limonta, D., G. Torres, A. B. Perez, and M. G. Guzman. 2009. Apoptosis in tissues from fatal dengue shock syndrome. Journal of Clinical Virology 40:50-54

10. Wu, S.-J. L., G. Grouard-Vogel, W. Sun, J. R. Mascola, E. Brachtel, R. Putvatana, M. Louder, L. Filgueira, M. A. Marovich, H. K. Wong, A. Blauvelt, G. S. Murphy, M. L. Robb, B. I. Innes, D. L. Birx, C. G. Hayes, and S. Frankel. 2000. Human skin Langerhans cells are targets of dengue virus infection. Nature Medicine 6:816-820

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Bullet Points and keywords

• Dengue hemorrhagic fever is an acute febrile syndrome with vascular leak, caused by the widely distributed, mosquito born flavivirus Dengue. DHF is most commonly seen in children experiencing a secondary infection with a heterologous serotype of dengue virus. Seroepidemiologic and immunologic studies suggest that pre-existing, non-neutralizing antibody enhances infection of target mononuclear phagocytes and increases virus replication.

• The pathology of fatal DHF has been well described in human autopsy series, but information on the kinetics and location of viral replication in tissues during natural infection is sparse. Animal models that faithfully recapitulate the all aspects of the natural history and pathology of DHF are not available.

• Major pathologic findings are hemorrhages, edema, and midzonal, paucicellular necrosis in the liver. Large reactive lymphocytes are seen in peripheral blood and lymphoid tissues. There are small foci of mild perivascular inflammation, edema, and endothelial swelling in microvasculature of many organs. Important target cells include dendritic cells, mononuclear phagocytes, hepatocytes, and focally, endothelial cells.

• Suggested mechanisms of vascular leak include overproduction of pro-inflammatory cytokines by activated T cells and monocyte/macrophages, direct viral effects on regulation of endothelial permeability, alterations in regulation of complement and fibrinolytic systems, and antiviral antibody that cross-reacts with endothelial cells.

Keywords: Dengue virus, hemorrhagic fever, cytokines, endothelium, immunopathology, hepatitis

Importance of microvesiculation in the immunopathology in cerebral malariaImportance of microvesiculation in the immunopathology in cerebral malaria

Georges E. R. Grau, M.D., Ph.D.

Vascular Immunology UnitThe University of Sydney

Vascular Immunology UnitThe University of Sydney

AustraliaAustralia

i ti li ti l microparticlesmicroparticles: :

d d b d d b i i l tii i l ti ffproduced by produced by microvesiculationmicrovesiculation ofofplasma membranesplasma membranes

Approach to cerebral malaria Approach to cerebral malaria pathogenesispathogenesis

cytokines / receptorscytokines / receptors

plateletsplatelets

microparticlesmicroparticles

Overview /Overview / microparticlesmicroparticles (MP)(MP)Overview / Overview / microparticlesmicroparticles (MP) (MP)

•• what are they?what are they?

•• do they change in CM?do they change in CM?

•• what happens if they are blocked?what happens if they are blocked?

•• what do they do to their targets?what do they do to their targets?y gy g

Hunt & Grau

Schofield & Grau

5: 722-735 2005

24: 491-499, 2003

5: 722-735, 2005

Hunt & Grau

D Schofield & Grau

24: 491-499, 2003

5: 722-735, 2005

mechanisms?mechanisms?mechanisms?mechanisms?

TNFα or TNFβ (lymphotoxin)?

Grau et al Science 237: 1210 1987Grau et al., Science 237: 1210, 1987Engwerda et al., J Exp Med 195: 1371, 2002Rae et al .,FASEB J 18: 499, 2004

Effector cellsEffector cells Hunt & Grau, 2003

monocytemonocyteCD8CD8++ T cellT cell

Effector cellsEffector cells

mTNF mLTα1β2 mLTα2β1

LTβR

sLTsLTαα33 Togbe et al. PLoS One 2008

LTβR

TNFR2

Unknown Unknown targettarget

ICAM-1upregulation

Effects?

targettargetcellcell

p gEndothelial cellEndothelial cell

MRI assessment of brain swelling in mice with CM

Copyright ©2005 Society for Neuroscience

Penet, M.-F. et al. J. Neurosci. 2005; 25: 7352-7358

Intravascular platelet binding in Intravascular platelet binding in CM: CM: p gp gIHC IHC and and LLigandigand--IInduced nduced BBinding inding SSites ites

von zur Mühlen et al., J Clin Invest (2008)

LIBS: detection of MRILIBS: detection of MRI--invisible lesionsinvisible lesions

von zur Mühlen et al., J Clin Invest (2008)

In vivo In vivo arguments in arguments in favourfavour of a of a pathogenicpathogenicrolerole of of plateletsplatelets in in microvascularmicrovascular pathologypathology

platelets sequester in the organs where lesions platelets sequester in the organs where lesions will occur

- cerebral malaria- pulmonary fibrosis- Shwartzman reaction, LPS shock

DTH- DTH

anti-LFA-1 mAb blocks platelet sequestration and pathology

platelet depletion prevents microvascular platelet depletion prevents microvascular damage and mortality

Res Immunol. 1993; 144: 355-63

ModellingModelling humanhuman cerebral cerebral malaria malaria Immunostaining

1

in vitroin vitrog

(patient brain)

2BRAIN endothelialcell

cell isolation

cell

PRBC co cultures3

Mo

WBC

co-cultures

PLTPLT

PRBC

platelets

PLT

ECEC

VascularImmunology

Unit

Platelets bind to TNFPlatelets bind to TNF--activated activated endothelium and potentiate its apoptosisendothelium and potentiate its apoptosis

TNF TGF-β release

indirect killing

direct killingTNF TGF β releasedirect killing

death

endothelial activation/sensitisation

Wassmer SC et al., J Immunol 176: 1180-4, 2006

Platelets bind to TNFPlatelets bind to TNF--activated activated endothelium and potentiate its apoptosisendothelium and potentiate its apoptosis

OEDEMA HAEMORRHAGEHAEMORRHAGE

Wassmer SC et al., J Immunol 176: 1180-4, 2006

2 levels of complexity2 levels of complexity

cell-cell interactions cell-derived microparticles

Mo Mo+

VascularImmunology

Unit

MicroparticleMicroparticle production: membrane production: membrane vesiculationvesiculation

Zwaal et al, Blood 96

VVeesiculationsiculation: : microparticlemicroparticle (MP) production(MP) production

TNFA23187

LPS PSPS

MP

LPSADP

PSPS

<1 μm surface AgsMP <1 μm surface Agsfrom the cell

of origin

b b l itl it

proadhesive and procoagulating properties

ΔΔ membrane membrane polaritypolarityΔΔ phospholipidphospholipid asymmetryasymmetry

cell surfacecell surface

MP MP phenotypephenotype isis the image of the image of itsits‘‘mothermother cellcell’’

ICAM-1ICAM 1

Brain ECBrain EC

EMPEMP

VascularImmunology

Unit

non stim. TNF

BothBoth TNF and TNF and LTLTαα enhanceenhance MP production MP production byby humanhuman endotheliumendothelium

restingHUVEC

+ TNF

by by humanhuman endotheliumendothelium

resting

Combes et al., J. Clin. Invest. 104: 93-102, 1999

+ TNF

C ,

HBEC

A B C

EMP20 µ 10 µ 5 µ

Wassmer et al., IAI, 74: 645-653, 2006

DramaticDramatic increase increase of pof plasma endothelial lasma endothelial i i li i l ii M l iM l i hildhild i hi h CMCM

Control acute phasef llp < 0.0001

microparticlesmicroparticles in in MalawianMalawian childrenchildren withwith CMCM

EMP

0.24 %

follow up

125

150

175

ma

p = 0.01p = 0.005

p 0 000

SS LOG 75

100

125P

/ µl p

las

MP

Cerebral malaria

025

50

EMP

EM

SS LOG

2.32 % 60N 138 80 37 35 27 1348Malaria - + + + + + + +CM - - + + - - + +SMA - - - - + + + +

+ EMP correlate with TNF in CM patients (acute phase)SS LOG

Combes et al, JAMA 291: 2542-4, 2004

Are Are microparticlesmicroparticles pathogenicpathogenic ??

EExpxpeerimentalrimental cerebral malaria cerebral malaria EExpxpeerimentalrimental cerebral malaria cerebral malaria

CECERREEBRAL BRAL

((murinemurine modmodeel)l)((murinemurine modmodeel)l)

Infection: CECERREEBRAL BRAL PHASEPHASE

Infection: Plasmodium

berghei ANKA anaemiaanaemia,,hyperparasitaemiahyperparasitaemia

eendothndotheelial activationlial activation,hyperparasitaemiahyperparasitaemia

cytoadherencecytoadherencedays0 21147

death (CMdeath (CM--resistant)resistant)

death (CMdeath (CM--susceptible)susceptible)

MP MP levelslevels are are higherhigher atat the time of CM in the time of CM in ggsusceptible susceptible micemice

Swiss C57BL/6

500 p = 0.001

300

400

asm

a p = 0.01

200

300

P / μ

l pla

0

100MP

PbA - + - +

Enzymes Enzymes controllingcontrolling phospholipidphospholipidmovementsmovements

Outer leaflet

PE

PSPS

Inner leaflet

amino-phospholipid floppaseamino phospholipidtranslocase

floppase

scramblase

ApproachApproach to to genesgenes

hypothesis-drivenhypothesis-drivenypyp

non hypothesis-drivennon hypothesis-driven

SERENDIPITYSERENDIPITY

non hypothesis drivennon hypothesis driven

SERENDIPITYSERENDIPITY

ATP ATP BindingBinding Cassette Transporter A1Cassette Transporter A1

NBD1 NBD2 COOHH2N

Human Pathology

Deleted segment in KO mice

(H t l N t C ll Human Pathology(Tangier Disease)

(Hamon et al. Nat Cell Biol. 2 : 399, 2000)

ABCA1 gene deletion prevents CMABCA1 gene deletion prevents CMg pg p

cerebralcerebral20

mia

(%)

100

cerebralcerebralphasephase

ABCA1/

ABCA10

10

Para

sita

em

50

75ABCA1+/+

(DBA/1)ABCA1-/-(DBA/1)va

l (%

) +/+ -/-

25

50

C57BL/6

(DBA/1) (DBA/1)

Surv

iv

0 7 14 21

0

0 7 14 21Time after infection by PbA (days)

UUpon pon PbAPbA infectioninfection: : higherhigher levelslevels ofof

microparticle quantitation in plasma

pp ggpplasma lasma microparticlesmicroparticles in ABCA1in ABCA1+/++/+ micemice

cellular origin of microparticles

400

0.027

c opa t c e qua t tat o p as a

Plateletsasm

a 300

400

Platelets

EC

MP

/ µl p

l

100

200

ABCA1

0+/+ +/+ -/- -/-

PbA - -+ +Combes et al., Am J Pathol 166: 295-302, 2005

DownDown--regulation of brain inflammation regulation of brain inflammation i PbAi PbA i f d ABCA1i f d ABCA1 // ii

ICAM-1 LFA-1 GPIIb-IIIa

in PbAin PbA--infected ABCA1infected ABCA1--//-- micemice

+/+ +/+ +/+

-/- -/- -/-

Microparticles fromMicroparticles from

l t th th f PbA i f t d ABCA1 KO i

Microparticles from Microparticles from PbAPbA--infected CMinfected CM--susceptible mice are:susceptible mice are:

WBCMP

more procoagulant than those from PbA-infected ABCA1-KO mice

me

MP

+20

25 0.02

l clo

tting

tim

Normal mouse plasma 10

15

ctio

n co

ntro

Clotting time 0

5

% re

duc

ABCA1 +/+ -/-/ /

MicroparticlesMicroparticles fromfrom

f f f C S

MicroparticlesMicroparticles fromfromPbAPbA--infectedinfected CMCM--susceptible susceptible micemice are:are:

more inflammatory than those from non infected CM-S mice

4000.004

300

400

g/m

l)

<0.0001WBCMP

100

200TN

F (p

gMΦ TNFTNF ??

Medium LPSMP NI MP PbA

0

C bl k dCan one block MP production?

Yes, by gene deletion : ABCA1 knock-out mice

but how about pharmacologically?

Blocking EC activation?activation?

Stabilising Stabilising membrane PS?

Stabilisingmembranes?membranes?

b l b h h l dStabilising membrane phospholipids

• Rationale: cell activation = modification of membrane lipidsp

• Maintaining membrane asymmetry may• Maintaining membrane asymmetry may stabilise lipids

• Code: THP-22

THP 22 f b i d h li l llTHP-22 treatment of brain endothelial cells

Wash

THP-22 (0 – 5 mM) THP-22 (0 – 5 mM)

TNF / LT (100 ng/ml)

12h 24h time

MP quantificationIn culture supernatantIn culture supernatant

THP-22 treatment abolishes human brain endothelial vesiculation in vitro

2.0

0.0002*** *** 0.00100.0002 **

n ber

1 0

1.50.0034

**0.0072

**

esic

ulat

ion

l MP

num

b

0.5

1.0

inde

x of

ve

old

of b

asa

0.0THP 22 (mM) 0 1 2 5 0 1 2 5 0 1 2 5

fo

THP-22 (mM) 0 1 2 5 0 1 2 5 0 1 2 5

TNF 100 ng/ml LT 100 ng/ml

THP-22 treatment prevents CM developmentp p

80

100 PbA + VehiclePbA + THP-22

rviv

al

parasitaemia

20

40

60

Perc

ent s

u

5 0

7.5

10.0

%

NS

-5 00 7 14 21 28

0.0

2.5

5.0

PbA

%

0i.p. THPi.p. THP--22 treatment22 treatment

80

100

urvi

val

PbATHP-22

+ +- +

per os!per os!

20

40

60

Perc

ent s

u per os!per os!

-5 00 7 14 21 280p.o.THPp.o.THP--22 treatment22 treatment Penet et al., Proc Natl Acad Sci USA 105:1321-6, 2008

Can Can wewe modulatemodulate IsIs thisthisMP production?MP production?

Is Is thisthisprotective protective

againstagainst CM?CM?

Blocking EC

againstagainst CM?CM?in vitroin vitro in vivoin vivo

LMP 420* ddg

activation?

Stabilising

LMP-420* nd+ nd

Stabilising membrane PS? THP-22# yes+ +

Stabilisingmembranes? CTC, DiA nd+ nd

(*) Wassmer, Cianciolo, Combes & Grau, PLoS Med 9: e245, 2005(#) Penet et al., Proc Natl Acad Sci USA 105:1321-6, 2008

What are the downstream effects of MP?What are the downstream effects of MP?

In vitroIn vitro modellingmodelling ofof humanhuman CMCMIn vitro In vitro modellingmodelling of of humanhuman CMCM

PRBCPMP

+/- TNF

90 ’overnight 90 ’

Brain EC

CytoadherenceMorphological featuresPhenotypic changesPhenotypic changes

PMPPMP bindbind to andto and activateactivate HBECHBECPMPPMP bindbind to andto and activateactivate HBECHBECPMP PMP bindbind to and to and activateactivate HBECHBECPMP PMP bindbind to and to and activateactivate HBECHBEC

Ø PMP

Ø PMP PMP

ICAM-1

TNF

PMPHBEC

FI

DAPI

MF

VCAM-1IgG1

PKH67

MFI

How ?How ?How ?How ?PMP PMP adhereadhere to and to and penetratepenetrate in in brainbrain ECECPMP PMP adhereadhere to and to and penetratepenetrate in in brainbrain ECEC

Brain ECAdherent

PMPconsequences ?

Internalised PMP mechanisms ?

MPMP--target interactions: 3 patternstarget interactions: 3 patternsg pg p

“compartmentalised”: micropinocytosis?“surface”“cytosolic”

Some PMP are internalised in Some PMP are internalised in lysosomeslysosomesSome PMP are internalised in Some PMP are internalised in lysosomeslysosomesyyyy

PMPPKH67 green

Lysosomes Lyso-tracker red

90 min incubation37◦C

VascularImmunology

Unit

PlateletPlatelet MP MP alsoalso bindbind to PRBC and to PRBC and transfertransferplateletplatelet antigensantigens to to theirtheir surfacesurface

PMPSN

TL PKH26

ran

ge

0.18 %0.00 %5

0.88 %

Acr

idin

e or

14.5 %PKH67 Merge

5 µm

0.21 %0.00 %

CD41

PMP PMP dramaticallydramatically increaseincrease RBC RBC bindingbinding to ECto EC

6 0G RSG RP /N Im

p

p = 0.0002nRBCPRBC60

4 0

5 0

6 0 G RP /N I

adhé

rent

s / c

ham

p = 0,0076p = 0.0076

PRBC

40

50

60

BC

/ fie

ld

1 0

2 0

3 0

Nom

bre

de G

R a

10

20

30

Bou

nd R

B

RBC TNF RBC (RBC + PMP) TNF (RBC + PMP)PMP RBC TNF PMP RBC0

N

TNF ‐ + ‐ + ‐ +

MPP 1ère condition∅MPP MPP 2ème conditionPMP on PRBC∅MPP PMP on EC

‐ +‐ +TNF ‐ +0

11

2 1 2

HBEC-5i2 1 2

HBEC-5i

Membrane fusion / Ag transfer?Membrane fusion / Ag transfer?

Ag (R)

Membrane fusion / Ag transfer?Membrane fusion / Ag transfer?

Ag (R)MP

+ ?cell

morphology?functions?signalling?

DifferentialDifferential labelling of membrane labelling of membrane versusversusggcytosoliccytosolic elementselements for the for the coco--culturescultures

PRBC-PKH26 Calcein AM

HBEC 1 h 30

Calcein AM

0 / 1 h / 2 hO/N

(D3 line)

microscopy

unbound cell

removal

TNF washingpyremoval

INCUBATION

30 min co30 min co cultureculture 40 min co40 min co cultureculture90 min co90 min co--cultureculture(before washing)(before washing)

HBEC: D3 cell line + IRBC: 3Ci

30 min co30 min co--cultureculture 40 min co40 min co--cultureculture (before washing)(before washing)

Beginning of transferAdhesion

PKH261 h 30 min1 h 30 min coco--culture culture

(after washing)(after washing)(after washing)(after washing)Diffusion of membrane compounds

MergeMergecalcein gg

HBEC: D3 + IRBC: 3Ci

4 h4 h coco--cultureculture

HBEC: D3 + IRBC: 3Ci

PKH26calcein

Diffusion of both membrane and cytosolic compounds

Could this transferred membrane material i l d P f l i i ?

PRBC-PKH26

include P. falciparum antigens? HIS + anti human-IgG

HBEC 1 h 30 confocal microscopy

0 / 1 h / 2 hONmicroscopy

washingTNF washing

AutoMACS® - purified PRBC

[HIS l f 20 h i[HIS : pool of 20 hyper-immune sera from African adults with malaria]

Intracellular localisation

Surface localisation

Roles of microparticles during CMRoles of microparticles during CM

Antigen transfer

PLT

PMPPRBC TNF

ADHESION + EC DAMAGE EC DAMAGE

?

Adhesion molecules

MP as players of pathogenesisMP as players of pathogenesis

• Plasma TNF is high in CMScience 1987, NEJM 1989

• TNF enhances endothelial MP release in vitroJ Clin Invest 1999

• High plasma MP found in patients with CMJAMA 2004

• ABC A1 gene knock-out mice – Do not show a rise in MP– Are fully protected against the cerebral pathology

Am J Pathol 2005

• Treatment with PTTH reduces MP and prevents CM.Proc Natl Acad Sci USA 105:1321-6, 2008

MP in cerebral malaria:MP in cerebral malaria:CONCLUSIONSCONCLUSIONS

• Are dramatically elevated at the time of CM

• Are pro-inflammatory and procoagulant

• Their blockade prevents lesions (in vitro: H; in vivo: M)

• Can enhance binding of RBC to brain EC

• Can transfer antigens to EC and alter EC functions

Summaryy

cytokines / receptorscytokines / receptorscytokines / receptorscytokines / receptors- TNF-β, not TNF-α, is required- TNFR2 and LTβR are required

plateletsplatelets

TNFR2 and LTβR are required

- accumulate at the site of lesions, beforethese become MRI-detectable

ti i t i d th li l lt ti

microparticlesmicroparticles

- participate in endothelial alterations

- downstream and upstream events

upstream downstream

kinetics?cytoskeleton?

fusion?internalisationcytoskeleton?

rafts? adhesivenessinternalisation

ag transfers

target cell

activated cell

procoag.

microparticlesnovel

signalling?

Δ permeability

signalling?trogocytosis?

pathology?

VascularFatima EL-ASSAAD

ImmunologyUnit

Araz BOGHOSSIAN

Jacqueline THEODORA

Duang Dao NANTAKOMON

Dorothée FAILLE

Discipline of Pathology

Joel Pankoui MFONKEU

Pauline GOH, PhD The University of SydneyThe University of Sydney

Sponsored Sponsored by:by:

Angeles SANCHEZ-PEREZ, Ph D

Jean-Marie MATHYS, PhD

ARCARC

NH & MRCNH & MRC

Valéry COMBES, PhD

Ronan JAMBOU, MD, PhD

The Cooper FoundationThe Cooper Foundation

Georges E. GRAU, MD, PhD

Bacterial Sepsis

United States and Canadian Academy of PathologyBinford-Dammin Society of

Infectious Disease PathologistsMarch, 2009

Daniel Remick, M.D.Department of Pathology & Laboratory Medicine

Boston University School of MedicineBoston Medical Center

DisclosuresSupported by:

R01 GM050401 – PI R01 ES0113538 - PIP01 GM067189 - PIT32 NIAIDU54 GM62119

Outline for the Sepsis TalkSIRS – Sepsis - Cytokines

Real sepsis – not endotoxemia

Legitimate animal model of sepsis

6@6, Six at Six

Early deaths, late deaths SIRS CARS

Multiplexing for more data

Using the changes to alter therapy

SIRS - Systemic Inflammatory Response Syndrome

2 or more of the following are neededTemperature >38° or < 36 °C

Heart rate > 90 beats/minute

Respiratory rate >20 or PaCO2 < 32mm Hg

WBC >12K or < 4K/mm3, or >10% bandsBone Chest 1992;101:1644

SIRS - Definition Too Broad?

• Sweating - hyperthermia• Heavy breathing -

tachypnea• Heart racing - tachycardia

The definitions are too broad and encompass a wide range of different activities, not all of which are pathologic

The Problem of Sepsis

• Reviewed discharges of 750,000,000 patients

• Between 1979 and 2000 the incidence of sepsis increased 8.7% per year

• Gram positive more frequent than Gram negative

• Mortality is greatest among black men

Martin, NEJM 2003:348:1546

Sepsis – The Movie

1999 Movie with George ClooneyMark Wahlberg Ice Cube

Cytokines and Sepsis

• Explosive release of cytokines is responsible for the organ injury and mortality in sepsis

The 4 Pillars of SupportCytokines Cause Sepsis

LPS

rele

ases

cyt

okin

es

Cyt

okin

e in

fusi

on =

se

psis

Sep

sis

have

hig

h cy

toki

nes

Blo

ck c

ytok

ines

be

tter s

urvi

val

Cytokine Inhibition for Rx of Sepsis

• In animal modelsBlocking TNF improves surivalBlocking IL-1 improves survival

Proposed patient studies

Block TNF or IL-1

Save lives

So Simple

Cytokine inhibitors for Sepsis

• Based on multiple preclinical trials using animal models of sepsis, large scale clinical trials were initiated

• All trials enrolled patients meeting the SIRS criteria

• All patients received routine clinical care for sepsis (antibiotics, fluids, organ support)

Treatment of Human SepsisMonoclonal antibody to TNF

0

5

10

15

20

25

30

35

Placebo 7.5 mg/kg 15 mg/kg

Mor

talit

y at

28

days

994 total patients No improvement in survival Abraham JAMA 1995;273:934

Treatment of Human SepsisTNF soluble receptors TNF:Fc

0

10

20

30

40

50

60

Placebo .15 mg/kg .45 mg/kg 1.5 mg/kg

Mor

talit

y at

28

days

141 total patientsIncrease in mortality with therapy Fisher NEJM 1996;334:1697

Treatment of Human SepsisIL-1 receptor antagonist

05

10152025303540

Placebo IL-1ra

Mor

talit

y at

28

days

696 total patients No improvement in survival OpalCrit Care Med 1997;25:1115

• Patients with rheumatoid arthritis evaluated

• 144 publications reviewed • Trimmed to 9 studies• 5014 patients• Careful review and extraction of the data

JAMA 2006:2275 Bongrartz et al

TNF inhibitors may increaseRisk of Infection

IndividualPublications

Combined ResultsOdds Ratio:2.0 (1.3-3.1)

JAMA 2006:2275 Bongrartz et al

Risk of Infection with α-TNF Ab

Central Hypothesis

Not everyone is the same

Not All People

Are Identical

Not All Septic

Patients Are

Identical




6@6, Six at Six




What about real patients?

Patient with Acute Sepsis

• 24 year old male familial adenomatous polyposis

• 5/27 Proctocolectomy, pancreatitis post-op, also hyponatremia

• 7/24 Admitted to an outside hospital, CT scan shows dilated loops of bowel

• Possible stricture, residual polyps, doing well

Patient with Acute Sepsis, cont’d

• 7/29 Acute abdominal pain– X-ray shows free air– Emergent exploration – necrotic bowel,

free stool in abdomen– Rx with pressors, antibiotics, aggressive

fluid support• 7/30 Cardiac arrest, resusciated• 7/31 1:30AM Pronounced Dead

Graphic Photos to Follow

Serosa

Mucosa

Serosa

Subarachnoid Hemorrhage

Profile of “Typical Septic Patients”27 patients

Hotchkiss J. Immun. 2001, 166:6952

• Age 62, range 18 to 92• Days septic 12.6, range 1 to 77• Co-Morbid Conditions

– Renal failure with hemodialysis 10%– Diabetes – 10%– Liver disease – 10%– Heart disease10%

Sepsis Therapy

• Antibiotic Therapy• Fluid Resuscitation• Organ support

– Dialysis– Ventilator– Blood replacement

Other modulators areADDED TO STANDARD RX

Failed Sepsis Therapies

Glucocorticoids Ibuprofen (COX inhibitor)α-endotoxin antibodies PAF antagonistBradykinin antagonist IL-1 raα-TNF antibodies TNF-SR

What has worked?

Activated Protein C

Significant ↑ in survival

NEJM 2001:344:699

July August Sept

Stock price of Eli Lilly, June - Sep 2000Activated Protein C

Sepsis trials reported

Prozac off patent early

aPC for Adults with Severe Sepsisand Low Risk of Death

NEJM September 29, 2005 353:1332Abraham et. al.

Severe Sepsis: sepsis induced dysfunction of at least 1 organLow risk of death: APACHE < 25 or single organ failure

Required by FDA after post-hoc data analysis

2640 patients enrolledStudy terminated early

MortalityPlacebo 17.0%aPC 18.5%

Increased Incidence of Bleeding Events

Serious Bleeding Events Day 1-6during infusion

Placebo aPC0

5

10

15

20

25

30

35

# of

Eve

nts

Serious Bleeding Events Day 1-28

Placebo aPC0

10

20

30

40

50

60

# of

Eve

nts

Bleeding Events Leading to Transfusion

Placebo aPC0

102030405060708090

# of

Eve

nts

aPC should not be used for septic patients with a low risk of death




6@6, Six at Six




LipopolysaccharideNot even close

• Lethal LPS• Non-Lethal LPS• Lethal CLP• Non-Lethal CLPCollect 20 μl blood over first 24 hours

TNF

0 2 4 6 9 120

1000

2000

3000

4000

5000

6000 LPS Lethal

LPS Non-lethal

CLP Lethal

TNF

(pg/

ml) CLP Non-lethal

Hours Post-treatment

A Better Model of Sepsis

Cecal Ligation and Puncture

Standard CLP Protocol

• Isoflurane Anesthesia• Fluid resuscitation at the time of surgery• Analgesia (buprenorpherine)• Antibiotics twice a day x 5 days• Fluid resuscitation twice a day x 5 days• Repeated peripheral blood sampling 20 μl

Steps in CLP

1) Open skin and peritoneum

2) Exteriorize Cecum

3) Ligate below ileocecal valve

4) Puncture twice

5) Close peritoneal cavity, running stitch orinterrupted sutures

6) Insert minimetterssubcutaneously

7) Close skin withwound glue

CLP Lethality

Time (Days)0 1 2 3 4 5 6 7 8 9

% S

urvi

vors

0

20

40

60

80

100

Sham

25G

21G

18G

(n=6)

(n = 19)

(n = 18)

(n = 6)

Ebong, S.Infection & Immunity 1999:67 pg 6603

Temperature Profile

Time (Hrs)

Tem

pera

ture

(

o C)

25

30

35

40

Sham25G21G18G

12 24 48

*

*

*

*

*

* = p < 0.05

A: Sham

0

10

20

30

40

50

60

C: 21G

Time (Days)0 1 2 3 4 5 6 7 8

0

10

20

30

40

50

60

B: 25G

Act

ivity

Cou

nts/

5 m

inut

es

0

10

20

30

40

50

60

Gross motor activity and return to diurnal rhythm.

Survival proportions

6 71 2 3 4 5 60

50

100treatednot treated

*p=0.0038

day

Perc

ent s

urvi

val

Treatment: Imipenem 25 mg/kg in LR withD5W twice/day x 5 days




6@6, Six at Six




Overall Survival 21 G CLP

0 3 6 9 12 15 18 210.0

0.2

0.4

0.6

0.8

1.0

DAYS

Surv

ival

N= 69

50% mortality – what predicts?

Interleukin 6? Biomarker for mortality

Over 30 papers show:

⇑ IL6 = ⇓ Survival in human septic patients

Experiment, sacrifice mice at different time points after CLP of increasing lethality

Collect peritoneal fluid and plasma

Local vs Systemic IL-6 after CLP

Plasma IL-6, ng/ml

1e+2 1e+3 1e+4 1e+5 1e+6 1e+7

Per

itone

al IL

-6, n

g/m

l

1e-1

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

1e+7

1e+8

1e+9

Objective

• Using the cecal ligation and puncture model of sepsis, can we define parameters which will predict outcome?

• Can these parameters be defined in sufficient time to initiate a therapeutic intervention?

Plasma IL-6 6 h after CLP

IL-6 AT 6 HRS

DEAD ALIVE0

500

1000

1500

2000

2500

3000

3500

P=<.0001

STATUS AT 72 HRS

PG/M

L

IL-6 AT 6 HRS

DEAD ALIVE0

1000

2000

3000

4000

5000

6000

STATUS AT 72 HRS

PG/M

L

Shock, 2002:17 pg 463

Plasma IL-6 at 6 hours

0 1 2 3 4 50

20

40

60

80

100

P=0.0001>2000 pg/ml n=20

DAYS

Perc

ent S

urvi

val

<2000 pg/ml, n=49

Plasma IL-6 at 6 hoursPrediction of mortality at 3 days after CLP

Sensitivity = 91%Specificity = 90%

IL-6 Knockout Mice

• C57 BL\6 background (previous work was BALB\c or ICR)

• Complete lack of IL-6 production• CLP protocol used, with varying needle

sizes

Infect Immun 2005:73 pg 2751

21 Gauge X 2Punctures

0 2 4 6 8 10 12 14 16 18 20 22 24 26 280

25

50

75

100KNOCKOUT n=9WILD TYPE n=9

p=.96

Days

Perc

ent s

urvi

val

25Gauge X 2Punctures

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

25

50

75

100 KNOCKOUT n=18WILD TYPE n=18

p=.40

Days

Perc

ent s

urvi

val

25Gauge X 1Puncture

0 2 4 6 8 10 12 14 16 18 20 22 24 26 280

25

50

75

100

KNOCKOUT n=34

WILD TYPE n=37

p=.44

Days

Perc

ent s

urvi

val

Sham

0 2 4 6 8 10 12 14 16 18 20 22 24 26 280

25

50

75

100

KNOCKOUT n=9WILD TYPE n=9

p=1

Days

Perc

ent s

urvi

val

0 2 4 6 8 10 12 14 16 18 20 22 2424

26

28

30

32

34

36

WILD TYPEKNOCK OUTA

Hours

Deg

rees

Cel

cius

2 8 12 2024262830323436 WILD TYPE

KNOCK OUTSB

Hours

* * * * *

Deg

rees

Cel

cius

Body Temperature

ScorecardCLP Sepsis

28 day mortality 60% 40%Responds to Rx Yes YesEffect of Age Death DeathSource of infection Peritoneum Lung

Weight Change = death = deathFailed α-TNF Yes YesMalaise Yes YesFever No YesIL-6 predicts death Yes YesLymphocytes In death In death

Neutrophils In death In death

Recap before we get lost

• Sepsis is bad for you and your next of kin• Heterogeneity exists in the individual

response to sepsis• CLP reproduces many of the features of

sepsis• Early inflammatory markers predict early

deaths




6@6, Six at Six




Why do Septic Patients Die?• Too Much Inflammation

– Need to cool things down

• Too Little Inflammation– Need to heat things up

Hypothesis: The cause of death is different in early (5 days) vs late sepsis

Test the SIRS ⇒ CARS

• Is there evidence for this hypothesis–CLP performed on ICR mice–Necrotic cecum resected on day 3–Routine parameters monitored for

28 days

Plasma IL-6 Levels

-2 0 2 4 6 8 10 12 14 16 18 20-10000

0

10000

20000

30000

40000

50000Dead in 4 days (n=21)Alive for 20 days (n=30)

Day

IL-6

(pg/

ml)


Plasma IL-6 Levels

-2 0 2 4 6 8 10 12 14 16 18 20-10000

0

10000

20000

30000

40000

50000 Dead after 4 days (n=7)

Day

IL-6

(ug/

ml)


Plasma IL-6 Levels

0 5 10 15 20-1000

1000

3000

5000

7000

9000

(=12700)(n=10)

Day

IL-6

pg/

ml


Day 15 Survivor

Day 20 Non-Survivor

Day 10 Non-Survivor

Peritoneal Lavage


Peritoneal CFUs in Chronic Sepsis

Resected Surviving Dying

1

1000

1000000

1.0×1009

1.0×1012

1.0×1015

1.0×1018

CFU

/ml


Early (5 days) vs Late Deaths

Early Late

IL-6 Always high

Variably high

Bacteria ± present Always present




6@6, Six at Six




Where do we go from here?

• IL-6 is just one of many markers• Careful evaluation of multiple markers• Real time evaluation to direct moment by

moment therapy

Multiplex Immunoassay

• Attempt to create a protein chip to quantitate inflammatory mediators

• Essentially a sandwich ELISA on a chip• Initial trials with 16 cytokines

Shock 2004:21 pg 26Proteomics 2005:5 pg 4712J Immunol 2005:175 pg 3282J Immunol 2007:179 pg 623

Why not beads?Cost

CostCost

Principle of Protein Chip

Glass Slide

Ab TNF TNF

Biotin

TNF

BiotinCy5-SA

TNF

BiotinCy5-SA

1) Block2) Add sample 3) Detection

Antibody

4) StreptavidinCoupled to Fluorochrome Cy5

5) Readfluorescence

96 well plate single well

Antibodies spottedIn quadruplicate

Location of the spotIndicates the What is beingmeasured

IL-6 TNF

Correlation Between I. ELISA &Micro array

2 4 6

2

4

6

R2 =1.0

I. ELISA(log)

Mic

ro a

rray

(log)

Cytokines Presently on Protein Chip

• IL-1, IL-1RA, IL-1SR II, • IL-4, IL-6, IL-8, IL-10, IFN-γ, • MCP-1, MIP-1α, MIP-1 β, RANTES, • TNF-α, TNF-SR I, TNF- SR II, • βNGF

Early and Late Deaths

• What are the differences in plasma biomarkers for early vs late deaths?

• Do these have sufficient predictive power?• Is there any value added to measuring the

biomarkers?

28 DAY SURVIVAL

0 4 8 12 16 20 24 280

20

40

60

80

100

n=90

DAYS AFTER CLP

Perc

ent s

urvi

val

survival at day 28= 38%survival at day 14= 49%survival at day 5= 57%

39 DIED 17 DIED

“CHRONIC DEATHS”“ACUTE DEATHS”

0 C 6 24 48 720

10

20 MCP-1

bdl

ng/m

l

0 C 6 24 48 720

10

20

30

40 MIP-1

bdl

ng/m

l

0 C 6 24 48 720

1

2

3

4TNFα

bdl

ng/m

l

0 C 6 24 48 720

5

10

15 TNF SRI

ng/m

l

0 C 6 24 48 7205

10152025 TNF SRI

ng/m

l

0 C 6 24 48 720

5

10 IL-10

bdl

ng/m

l

0 C 6 24 48 720

50100150200

250IL-1ra

bdl

ng/m

l

HOURS AFTER CLP

0 C 6 24 48 720

5

10 IL-6R

ng/m

l

0 C 6 24 48 7205

10152025 EOATAXIN

ng/m

l

0 C 6 24 48 720

5

10

15 IL-1β

bdl

ng/m

l

DEAD BY DAY 5DEAD AFTER DAY 5LIVED 28 DAYS

bdl BELOW DET. LIMIT

KINETIC PROFILES OF CYTOKINES IN ACUTE PHASE OF SEPSIS

PRO-INFLAMMATORY CYTOKINES

CHEMOKINES

ANTI-INFLAMMATORY CYTOKINES

0 C 6 24 48 720

25

50

75 KC

bdl

ng/m

l

HOURS AFTER CLP

0 C 6 24 48 720

20406080

100 IL-6ng

/ml

HOURS AFTER CLP

IL-6

6 @ 6

Dead by d

ay 5

Dead af

ter day

5Aliv

e at d

ay 28

01020304050607080

IL-6

ng/

ml

C 6 24 48 720

50

100

150 IL-6

HOURS AFTER CLP

*

*

bdl

ng/m

lEarly deaths

KC

C 6 24 48 720

20

40

60

80

100 KC

bdl

HOURS AFTER CLP

* *

*

ng/m

l

KC @ 6

Dead by d

ay 5

Dead af

ter day

5

Alive a

t day

28

0102030405060708090

KC

ng/

ml

Early deaths

IL-1 Receptor Antagonist

IL-1ra @ 6 hours

Dead by d

ay 5

Dead af

ter day

5Aliv

e at d

ay 28

0

25

50

75

100

IL-1

ra n

g/m

l

C 6 24 48 720

100

200

300 IL-1ra

bdl

HOURS AFTER CLP

*

**

ng/m

lEarly deaths

Receiver Operator Characteristic

Good BetterBetter

1 5 28141 5 280

20

40

60

80

100

EARLY DEATHS LATE DEATHS

n=34 n=22

mortality at day 5 = 41%mortality at day 28 = 62%

915

4 6

DAYS AFTER CLP

Perc

ent s

urvi

val

C 6 24 48 720

20

40

60 MIP-2

bdl

HOURS AFTER CLP

* *

ng/m

l

C 6 24 48 720.0

2.5

5.0 TNFα

bdl

HOURS AFTER CLP

* *

*

ng/m

l

C 6 24 48 720

2

4

6

8

10 TNF SRI

HOURS AFTER CLP

**

*

ng/m

l

Mortality PredictionROC Area under the curve

Best

Not soGood

6 h ROC

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

TNF-SR ITNF-SR II

IL-1TNF

IL-10IL-1ra

MCP-1MIP-2

KCIL-6

Early deaths

28 DAY SURVIVAL

0 4 8 12 16 20 24 280

20

40

60

80

100

n=90

DAYS AFTER CLP

Perc

ent s

urvi

val


39 DIED 17 DIED


Early deaths

Predicting Mortality

DeathDays

-1-2-3-4

Collect samples prior to deathMeasure plasma biomarkers presentbefore the subject dies

Early deaths

Predicting Mortality 24 hours prior to death

24 hours prior to deathROC

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

TNF-SR ITNF-SR II

IL-1TNF

IL-10IL-1ra

MCP-1MIP-2

KCIL-6

Early deaths

28 DAY SURVIVAL

0 4 8 12 16 20 24 280

20

40

60

80

100

n=90

DAYS AFTER CLP

Perc

ent s

urvi

val


39 DIED 17 DIED


Late deaths

Predicting Mortality

DeathDays

-1-2-3-4-5-6

Collect samples prior to deathMeasure plasma biomarkers presentbefore the subject dies

Late deaths

MIP-2 within 24h of death

0

1000

2000

3000

4000

5000

6000

7000

7 8 9 11 11 13 16 16 17 18 18 21 25 27

day of death

pg/m

l

C1C2SICK

120

MIP-2-chronic sepsis

ALIVE DEAD0

1000

2000

3000

4000

5000

6000

7000

120pg

/ml

Plasma Biomarkers in Chronic Sepsis MIP-2

J Immunol 2007:179 pg 623

Late deaths

Plasma Biomarkers in Chronic Sepsis IL-1RA

IL-ra within 24h of death

020000400006000080000

100000120000140000160000

7 8 9 11 11 13 16 16 17 18 18 21 25 27

day of death

pg/m

l

C1C2SICK

12000

IL-1ra-chronic sepsis

ALIVE DEAD0

50000

100000

150000

12000

n=14n=28

pg/m

l

Late deaths

TNF SR I within 24h of death

0

1000

2000

3000

4000

5000

6000

7000

7 8 9 11 11 13 16 16 17 18 18 21 25 27

day of death

pg/m

l

C1C2SICK1600

TNF SR I-chronic sepsis

ALIVE DEAD0

1000

2000

3000

4000

5000

6000

7000

1600n=28

n=14

Chronic Sepsis TNF-SRILate deaths

Plasma Biomarkers for Mortality in Chronic Sepsis

0.6 0.7 0.8 0.9 1.00.0 0.5 0.6 0.7 0.8 0.9 1.0

TNF SR IIHMGB-1

IL-6IL-10

αTNFMCP-1MIP-2

Body W.TNF SR I

IL-1ra

AUC value

Late deaths

Common BiomarkersAcute and Chronic Sepsis Deaths

24 hours prior to deathROC

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

TNF-SR ITNF-SR II

IL-1TNF

IL-10IL-1ra

MCP-1MIP-2

KCIL-6

0.6 0.7 0.8 0.9 1.00.0 0.5 0.6 0.7 0.8 0.9 1.0

TNF SR IIHMGB-1

IL-6IL-10

αTNFMCP-1MIP-2

Body W.TNF SR I

IL-1ra

AUC value

Acute Deaths Chronic Deaths




6@6, Six at Six




Using IL-6 as a guide to Rx

• High levels of IL-6 predict mortality• Measure IL-6 and use the levels to guide

therapy• Need to develop a rapid assay for IL-6• Non-specific inhibitor – high dose

glucocorticoids

6@6

> 26 ng/ml

< 26 ng/ml50% Rx DEX

50% Vehicle

50% Rx DEX

50% Vehicle

FLOW CHART OF DECISION TREE

1. Early Sepsis

2. Prospective stratification

Predicted to Die

Predicted to Live

Dex Therapy

1 2 3 4 5 6 70

20

40

60

80

100

TreatedNot Treated

n=44

n=44

No Stratification

Days

Perc

ent s

urvi

val

No stratific

ation

No Help

Predicted to liveSuggestion of harm

1 2 3 4 5 6 70

20

40

60

80

100

TreatedNot Treated

n=35

n=34

p=0.27

Predicted to Live

Days

Perc

ent s

urvi

val

Predicted to DieImprove survival

1 2 3 4 5 6 70

20

40

60

80

100

n=9

n=10

p=0.035

Predicted to Die

Not TreatedTreated

Days

Perc

ent s

urvi

val

Dex improves 28 day survival

1412345 7 280

20

40

60

80

100

TreatedNot Treated

n=9

n=10

Predicted to Die

Days

Perc

ent s

urvi

val

Conclusions• Sepsis is a heterogeneous disease

process• CLP is an adequate model of disease• Individual septic patients are optimally

treated with tailored, individual therapy• Much still needs to be done• Translational opportunities exist as we

move forward

The People Who Did the Work

Questions

Severe Febrile Shock and Hemorrhage: Pathology, Pathogenesis, and Diverse Microbial Etiologies

Yellow Fever and other Viral Hemorrhagic Fevers

Sherif R. Zaki, M.D., Ph.D. Chief, Infectious Disease Pathology Branch Division of Viral and Rickettsial Diseases Centers for Disease Control & Prevention

Atlanta, Georgia 30333.

Although the yellow fever virus is believed to have originated in Africa, the first recorded

outbreak was in Mexico in the seventeenth century. This was followed during the eighteenth

and nineteenth centuries by numerous outbreaks in the Caribbean, Central and South

America and the eastern part of the USA as far north as New York. Epidemics in more

temperate regions of the western hemisphere were the result of introductions through

seaports and of transport of mosquito vectors and viruses along commercial shipping routes.

At the beginning of the last century, yellow fever killed thousands yearly and was the first

“filterable agent” proven to be transmitted by an insect, giving birth to a whole new category

of viruses: the arboviruses. The work of the US Army Commission in Cuba, including

Walter Reed, William Gorgas and other coworkers, established that transmission of yellow

fever virus from humans to humans was by infected Aedes aegypti mosquitoes. Control

measures against this mosquito, along with immunization using a live attenuated virus

vaccine, effectively controlled urban yellow fever in the Americas. However, the disease

persisted sporadically in rural areas of both Africa and South America as a consequence of

sylvatic (jungle) cycles involving monkeys and forest-dwelling mosquitoes. In rural areas,

most yellow fever infections occur in people who visit or work in the forests of Africa and

South America. Periodically the virus is introduced into urban areas where the highly

domesticated mosquito Aedes aegypti occurs. This mosquito may become infected by feeding

on a viremic person who was infected in the forest, and secondary transmission can then

ensue. Urban epidemics have historically been explosive with many cases because

transmission is human to human via the Aedes aegypti mosquito, which feeds primarily on

humans.

Yellow fever illness varies from a subclinical infection to a fulminating disease terminating

in death. After an incubation period of 3-10 days, there is sudden onset of fever, chills,

headache and backache. Patients are usually severely ill, restless, with flushed face, swollen

lips, and congested tongues and conjunctivae. Many patients suffer from nausea and

vomiting and a bleeding tendency may be seen early on. A brief 1-2 day remission may occur

and is quickly followed by resumption of the febrile illness. The facial edema and flushing

are replaced by a dusky pallor, the gums become swollen and bleed easily, and there is a pro-

nounced hemorrhagic tendency with hematemesis, melena and ecchymoses. In spite of a high

fever, the pulse rate is slow and the blood pressure falls, resulting in renal failure with

albuminuria, oliguria and anuria. Death, when it occurs, is usually within 6-7 days of onset,

and is rare after 10 days of illness. The jaundice, which gives the disease its name, is

generally apparent only in convalescing patients. Mortality may be as occur in 20-50%. Most

patients with severe disease have leukopenia, thrombocytopenia, elevated hepatic enzymes

and coagulation defects. At autopsy the organs most affected are the liver, spleen, kidneys

and heart. Typically, midzonal necrosis is apparent in the liver, affecting cells around the

periphery of the lobule and sparing areas around the central vein. Acidophilic necrosis is

evident and Councilman inclusion bodies are usually present. Viral antigens, as detected by

immunohistochemistry, are usually confined to the liver in these fatal cases.

Treatment is supportive and confined to nonspecific measures, including maintenance of

fluid and electrolyte balances and replacement of any substantial amounts of blood lost

through hemorrhage. One dose of live, attenuated 17D vaccine provides complete protection

for 10 years and is notably free from reactions. Since 1937, this vaccine has protected about

44 million humans from yellow fever. However, since the late 1990s, close to 40 cases of

yellow fever vaccine-associated viscerotropic disease have been reported worldwide. The

risk of this adverse event is about three per million of doses administered and is highest

among people over 60 years old. Virus is widely distributed in various tissues in these cases

and is very distinct in cellular tropism as compared to that seen in naturally acquired disease.

It is hypothesized that it may be related to be due to genetic susceptibility.

Yellow fever is caused by a flavivirus and is classified as a hemorrhagic fever virus (VHF).

VHFs are a special group of viruses, belonging to four different families, transmitted to

humans by arthropods and rodents (Table 1). These viruses persist in nature through

zoonotic cycles, although in the case of dengue and sometimes yellow fever viruses, human-

to-human transmission through the bite of a mosquito vector is an important factor in disease

maintenance. Other hemorrhagic fever (HF) of infectious that must be included in the

differential diagnosis and excluded are malaria, rickettsial diseases, leptospirosis, shigellosis,

and typhoid fever. Characteristic pathologic features of yellow fever and other viral

hemorrhagic fevers are provided (Table 2). The presentation will highlight the clinical and

pathologic features of yellow fever and other viral hemorrhagic fevers (VHFs). The

presentation will prepare pathologists to recognize yellow fever and diagnose these various

infections. The differential diagnosis and anatomic pathologic approach to achieve an

etiologic diagnosis of these threatening diseases will be discussed.

TABLE 1. Hemorrhagic Fever (HF) Viruses

VIRUS

DISEASE NAME

CASE FATALITY

VERTEBRATE HOST

ARTHROPOD VECTOR

ARENAVIRUSES Junin Argentine HF 15-30% Rodents (Calomys

musculinus) None

Machupo Bolivian HF 15-30% Rodents (Calomys callosus)

Guanarito Venezuelan HF 15-30% Rodents (Zygodontomys brevicauda)

None

Sabia Brazilian HF 15-30% Presumably an unidentified rodent

None

Lassa Lassa fever ~ 15% Rodents (Mastomys) None BUNYAVIRIDAE Rift Valley fever Rift Valley fever ~ 50% Vertebrates (Sheep,

cattle) Mosquito, Aedes and others

Crimean Congo HF Crimean Congo HF

15-30% Vertebrates (Birds, hares, large ungulates)

Ticks, especially Hyalomma

Hantaan, Seoul, Puumala, and others

Hemorrhagic fever with renal syndrome (HFRS)

1-15% Rodents None

Sin Nombre, Black Creek Canal , and others

Hantavirus pulmonary syndrome (HPS) 50%

Rodents None

FILOVIRIDAE Marburg Marburg HF 25% Unknown Unknown Ebola Ebola HF 50-90% Unknown Unknown FLAVIVIRIDAE Yellow fever Yellow fever

20% Primates Mosquito,

especially Aedes Dengue Dengue HF,

dengue shock syndrome (DHF/DSS)

5% Primates, humans Mosquito, especially Aedes aegypti

Kyasanur Forest disease (KFD)

KFD 0.5-9% Rodents Ticks

Omsk hemorrhagic fever (OHF)

OHF 0.5-9 % Rodents Ticks

Table 3. Pathologic features in viral hemorrhagic fevers. DISEASE PATHOLOGIC FEATURES* Argentine HF Multifocal hepatocellular necrosis with minimal inflammatory response, interstitial

pneumonitis, myocarditis, and lymphoid depletion. Extensive parenchymal cell and reticuloendothelial infection, more than morphologic lesions would suggest.

Bolivian HF Venezuelan HF Lassa fever Rift Valley fever Widespread hepatocellular necrosis and hemorrhage, sometimes with midzonal

distribution, minimal inflammatory response, DIC, lymphoid depletion, and encephalitis. RVF antigens in very few individual hepatocytes.

Crimean Congo HF Widespread hepatocellular necrosis and hemorrhage with minimal or no inflammatory cell response and lymphoid depletion. Hepatic and endothelial cell infection and damage.

Hemorrhagic fever with renal syndrome (HFRS)

Retroperitoneal edema in severe HFRS, mild to severe renal pathologic changes. Congestion and hemorrhagic necrosis of renal medulla, right atrium of the heart, and anterior pituitary. Extensive endothelial infection mainly in renal and cardiac microvasculature.

Hantavirus pulmonary syndrome (HPS)

Large bilateral pleural effusions and heavy edematous lungs, mild to moderate interstitial pneumonitis, immunoblasts and atypical lymphocytes in lymphoid tissues and peripheral blood. Extensive infection of endothelial cells in pulmonary microvasculature.

Ebola HF Extensive and disseminated infection and necrosis in major organs such as liver,

spleen, lung, kidney, skin, and gonads. Extensive hepatocellular necrosis associated with formation of characteristic intracytoplasmic viral inclusions. Lymphoid depletion, microvascular infection and injury.

Marburg HF Similar to Ebola HF Yellow fever Midzonal hepatocellular necrosis; minimal inflammatory response. Councilman

bodies and microvesicular fatty change. Hepatocellular and Kupffer cell infection.

Dengue HF/DSS

Centrilobular and midzonal hepatocellular necrosis with minimal inflammatory response; Councilman bodies and microvesicular fatty change. Hyperplasia of mononuclear phagocytic cells in lymphoid tissues and atypical lymphocytes in peripheral blood. Widespread infection of mononuclear phagocytic and endothelial cells.

Kyasanur Forest Disease (KFD)

Focal hepatocellular degeneration, fatty change, and necrosis. Pulmonary hemorrhage, depletion of malpighian follicles, sinus histiocytosis, erythrophagocytosis, mild myocarditis, and encephalitis.

Omsk HF Little known; scattered focal hemorrhage, interstitial pneumonia, and normal lymphoid tissues

* These features represent the more characteristic pathologic findings in the different VHFs. More general findings seen to variable degrees in all HF are not listed in this table.

Dengue Hemorrhagic Fever: Pathology and...

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