dengue virus induces thrombomodulin expression in

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Dengue virus induces thrombomodulin expression inhuman endothelial cells and monocytes in vitro

Lien-Cheng Chen a, Huey-Wen Shyu b, Hui-Min Lin c, Huan-Yao Lei d,Yee-Shin Lin d, Hsiao-Sheng Liu d, Trai-Ming Yeh c,*

a

Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan, ROCb Department of Medical Technology, Fooyin University, Kaohsiung, Taiwan, ROCc Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan, ROCd Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan, ROC

Accepted 25 February 2009

KEYWORDSCoagulation;Hemorrhage;

Inflammation

Summary Objectives: Dengue virus (DV) infections can cause severe life-threatening dengue

hemorrhagic fever/dengue shock syndrome (DHF/DSS). However, the mechanism to cause

hemorrhage in DV infections remains poorly understood. Thrombomodulin (TM), expressed

on the surface of endothelial cells and monocytes, is very important in regulation of coagula-tion and inflammation. Therefore, the effect of DV on the TM expression was studied in vitro

using both endothelial cells and monocytes.

Methods and results: The expression of TM in human endothelial cell line, HMEC-1, monocytic

cell line THP-1 and peripheral blood mononuclear cells derived from human blood was in-

creased after DV infection, UV-inactivated DV or recombinant DV envelop protein domain III

stimulation as demonstrated by flow cytometry and immunofluorescent staining. Western blot

analysis further confirmed only DV but not enterovirus 71 infection of HMEC-1 cells increased

TM protein expression. In addition, RT-PCR analysis showed the increase of TM mRNA as well as

other protein C activation-related molecules in DV stimulated HMEC-1 in a dose-dependent

manner.

Conclusion: These results suggest that DV stimulation of human endothelial cells and mono-

cytes can increase the expression of TM, which may contribute to the anticoagulant properties

of cells during DV infection.

2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Dengue virus (DV) infection which is transmitted by mos-quitoes Aedes aegypti and Aedes albopictus is prevalent inover 100 countries especially in tropical and subtropicalareas and threatens the health and life of more than 2.5

* Corresponding author. Tel.: 886 6 2353535x5778; fax: 886 6236 3956.

E-mail address: [email protected] (T.-M. Yeh).

0163-4453/$36 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.jinf.2009.02.018

www.elsevierhealth.com/journals/jinf

Journal of Infection (2009) 58, 368e374
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billion people.1,2 According to disease severity, DV infec-tion may generally, result in mild dengue fever (DF) orsevere life-threatening dengue hemorrhagic fever/dengueshock syndrome (DHF/DSS).3e5 Most DF patients will bespontaneously recovered. However, in some cases, espe-cially during secondary infection with different serotypesof DV, DHF/DSS may develop. Even though antibody depen-dent enhancement (ADE) has been proposed to explain why

DHF/DSS occurs mostly in secondary infection with differ-ent serotype of DV,6 the mechanism of hemorrhage inducedby DV infection is not yet fully understood.

Thrombomodulin (TM) is a 75 kDa transmembrane gly-coprotein expressed mainly on the surface of vascular en-dothelium. However, TM has also been detected in humanmonocytes and a variety of other tissues.7,8 TM acts asa vascular endothelial cell receptor for thrombin to acti-vate protein C (PC).9 Activation of PC by the thrombin-TM complex is further enhanced when PC is bound tothe endothelial cell protein C receptor (EPCR).9,10 Acti-vated PC dissociates from EPCR and binds to proteinS on appropriate cell surfaces where it inactivates factorsVa and VIIIa, and thereby inhibits further thrombin

generation. Therefore, TM plays an important role in theanti-coagulant state of endothelium. On the other hand,circulating high levels of soluble TM may be associatedwith ongoing endothelial damage or ongoing inflammationduring disease course.11 An elevated serum level of solu-ble TM has been reported in dengue patients.12e14 Inaddition, DHF/DSS patients have higher concentrationsof soluble TM than those with DF.12e14 However, it is un-clear whether the increased soluble TM in dengue patientsis due to the increase of TM expression in DV-infectedendothelial cells or due to the increase of endothelialdamage in dengue patients. In this study we tried to ex-plore this question by using live and UV-inactivated DV

(UV-DV) to stimulate human microvascular endothelialcell line (HMEC-1) and human acute monocytic cell line(THP-1) in vitro to understand the effect of DV on theexpression of TM in these cells. The results indicatedthat DV antigen stimulation can increase TM expressionon HMEC-1 and THP-1, which may contribute to theanti-coagulant state of these cells during DV infection.

Materials and methods

Preparation of virus stock and virus titration

Dengue virus serotype 2 (strain PL0046) was propagated in

C6/36 cells. Briefly, monolayers of C6/36 were inoculatedwith the virus at multiplicity of infection (MOI) of 0.1 andincubated at 26 C, 5% CO2 for 5 days. The culture mediumwas harvested and cell debris was removed by centrifuga-tion at 900g for 10 min. After further centrifugation at16,000 g for 10 min, the virus supernatant was collectedand stored at 80 C until use. Virus titer was determinedby plaque assay using BHK-21 cell line. Briefly, a 10-fold se-rial dilution of virus was added to BHK-21 monolayer andthen incubated at 37 C, 5% CO2 for 5 days. Plaque numberswere counted after crystal violet staining. UV-DV was con-ducted in a Stratagene UV-stratalinker apparatus using2500 mJ of UV radiation. Inactivation of virus was confirmed

by showing no plaque after UV irradiation. In addition, EV71was propagated in Vero cells which were maintained inDMEM (Gibco BRL Life Technologies, Grand Island, NY) sup-plemented with 10% heat-inactivated FCS (Gibco) as previ-ously described.15

Cell culture

Human microvascular endothelial cell line (HMEC-1),obtained from the Centers for Disease Control andPrevention (CDC, Atlanta), was grown at 37 C i n 5 %CO2 in MCDB 131 medium (Gibco) containing 10% FCS,1% L-glutamine, 1% penicillin, 1 mg/ml hydrocortisone(SigmaeAldrich, St Louis, MO), and 10 ng/ml epidermalcell growth factor (EGF) (Gibco). Only cells with lessthan 25 passages were used. The monocytic cell line(THP-1) was grown at 37 C in 5% CO2 in a RPMI 1640medium with 10% heat-inactivated FCS. Human PBMCfrom normal blood donors who had no antibody againstDV were isolated by Histopaque-1.077 lymphocyte sepa-ration medium (SigmaeAldrich).

Viral infection and ADE

HMEC-1 cells (1 106) were cultivated in 12-well tissue-culture plates for overnight and then infected with DVat MOI as indicated in triplicate for 2 h at 37 C. Unboundviruses were removed by washing with medium. Negativecontrols were mock-infected with medium instead of vi-rus. Positive controls were incubated with 6 mM phorbol-12-myristate 13-acetate (PMA, SigmaeAldrich). THP-1cells were infected with DV at the MOI of 10 with or with-out the presence of mouse monoclonal antibody againstpre M (70-21) (1 mg).16

Immunofluorescent staining and flowcytometric assay

After infection for 24 h, cells were fixed with 4% parafor-maldehyde for 30 min, then permeabilized with 0.5% TritonX-100 for 10 min. Cells were then washed with PBS andblocked with 0.05% BSA in PBS. Fixed cells were stainedwith primary antibody at 4 C for 1 h. Mouse anti-humanTM antibody (Santa Cruz Biotechnology, Santa Cruz, CA)or rabbit anti-human TM antibody (Santa Cruz Biotechnol-ogy) was used as primary antibodies. After being washed,the cells were incubated with secondary antibody. FITC-

conjugated goat-polyclonal anti-mouse IgG antibody(1:200 dilution; Jackson Immuno Research, West Grove,PA), Alexa 488-conjugated goat-polyclonal anti-rabbit IgGantibody (1:200 dilution; Molecule Probes, Eugene, OR) orAlexa 594-conjugated goat-polyclonal anti-rabbit IgG anti-body (1:200 dilution; Molecule Probes) were used as sec-ondary antibodies. The cells were observed undera fluorescent microscope (Olympus, Japan) after counter-stained with Evans blue or analyzed on a FACSCaliburflow cytometer (BD Immunocytometry Systems, San Jose,CA) using the software WinMDI with a minimum of 10,000events collected. Each experiment was done in triplicateand a representative result was shown.

Dengue virus induces TM expression 369


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Western blot

HMEC-1 cells were harvested at 24 h post DV or EV71 infec-tion at MOIZ 1. Cells were washed with cold PBS andscraped into the lysis buffer (50 mM Tris-HCl, pH 7.5,150 mM NaCl, 0.5 mM EDTA and 0.5% NP-40). Cell extractswere then mixed with an equal volume of 2 sample buffer(125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mer-

captoethanol and 0.2% bromophenol blue), separated bySDSePAGE and transferred onto nitrocellulose membranes.Blots were incubated with mouse anti-human TM antibody(1:500 dilution, Santa Cruz Biotechnology) or mouse anti-human b-actin antibody (SigmaeAldrich). Blots were thenincubated with horseradish peroxidase-conjugated goatanti-mouse IgG antibody (SigmaeAldrich), the secondaryantibody. Proteins were detected using the EnhancedChemiluminescence Western Blotting kit (Amersham Phar-macia Biotech, UK).

Reverse-transcription polymerase chainreaction (RT-PCR)

RNA was extracted by using an isolation Reagent (Trizole;Life Technologies Inc., Rockville, MD) and quantified at260 nm. Reverse-transcription (RT) was performed by usinga kit (Gibco) according to the manufacturers instructions.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)served as a control. The primers used to amplify GADPH,EPCR, protein S, tissue plasminogen activator (t-PA) andTM were listed in Table 1. A total reaction volume of20 ml contained 4 ml of RT product, 2.5 units of Taq DNApolymerase, 20 mM of dNTP, 0.1 mM of primer, and 1 TaqDNA polymerase buffer (Promega, Madison, WI). The reac-tion mixture was incubated in a thermocycler (Perkin-Elmer, Fremont, CA) programmed to pre-denature at95 C for 5 min, denature at 95 C for 30 s, anneal at 56 Cfor 45 s, and extend at 72 C for 1 min, for a total of 30 cy-cles. After the last cycle, the resulting mixture was incu-bated at 72 C for 7 min and cooled to 4 C.

Cloning and expression of recombinant E protein

The domain III region of DV E protein (E3 Ag) coding foramino acids 286e396 of the dengue E glycoprotein wasamplified by RT-PCR from PL0046 strain using Advantage IIpolymerase enzyme (Clontech, Palo Alto, CA) and thefollowing set of primers: forward primer, 50-TCAAAG-GAATGTCATAC-30 and reverse primer, 50-TTTCTTAAAC-

CAGTTGAG-30. The PCR product was gel-purified, digestedwith restriction enzymes Nde I and XhoI and ligated into theNde I/XhoI sites of the pET 21b expression vector (Novagen,Madison, WI) to produce pET21-EIII. The nucleotide se-quence was confirmed by using DNA sequence analysis.Briefly, BL21 (DE3) cells (Novagen) transformed with pET21-EIII were grown to A600 of 0.7 and induced with isopropyl-1-thio-D-galactopyranoside (IPTG) (SigmaeAldrich) to a finalconcentration of 5 mM at 37 C. for 8 h. Cells were lysedwith binding buffer (100 mM NaCl, pH 8.0). After centrifu-gation, the fusion protein was purified from a MagExtractorHis tag (Toyobo, Japan) according to the manufacturersinstructions.

Statistical analysis

Data are expressed as mean SD. Students t test was used

to analyze the significance of the difference between thetest and the control groups. Statistical significance wasset at P< 0.05.

Results

Both live and UV-DV increased TM expression inHMEC-1 cells

DV infection of HMEC-1 increased TM expression in a dose-dependent manner as demonstrated by indirect immuno-fluorescent staining (Fig. 1). In addition, cell morphologywas changed from round shape to irregularly protruding of

the cytoplasm in DV-infected HMEC-1 at MOIZ 10, whichmay be due to the cytopathic effect induced by DVinfection at high MOI.17 UV-DV stimulation also increasedTM expression in HMEC-1 cells. However, unlike DV infec-tion-induced TM expression which was more homoge-neously distributed in the cells, TM expression in cellsstimulated with UV-DV or PMA was distributed in granular-like pattern in the cytoplasm of cells.

DV induced TM expression in both HMEC-1 andTHP-1 cells

DV infection increased TM expression in HMEC-1 was also

confirmed by flow cytometry (Fig. 2). DV infection also in-duced TM expression in human monocytic cell line THP-1.In addition, in the presence of anti-DV pre M antibody whichcan enhance DV infection,16 TM expression was further in-creased as compared to DV infection alone (Fig. 2).

Recombinant DV E protein domain III induced TMexpression in HMEC-1, THP-1 cells and freshisolated monocytes

Since UV-DV increased TM expression in HMEC-1 cells and Eprotein domain III is the region binding to cells,18 we use re-combinant DV E protein domain III (E3 Ag) to incubate with

Table 1 Primers used for RT-PCR.

Gene Sequence

EPCR 50-CTGATCCTGACTGTCTATC-30

50-CCATCATACCTTTCGTGTT-30

Protein S 50-GCCTGGTTACTGTGGAGAAGGGC-3 0

50-CGGCAAGTTGTCTTTGAAGGTC-3 0

t-PA 50-TCAGCAAGGGAAATGGCTTGT-30

50-TCAGGGAGCTGAGGCAGG-3 0

TM 50-GAGGACGTGGATGACTGCAT-30

50-TCACAGTCGGTGCCAATGTG-3 0

GADPH 50-CCC TTC ATT GAC CTC-30

50-GTC ATC CAT GAC AAC TTT GG-30

370 L.-C. Chen et al.


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HMEC-1 and THP-1 cells. As shown in Fig. 3, TM expressionin both cell lines were increased in the presence of E3 Ag.Furthermore, using freshly isolated human monocytesfrom PBMC also demonstrated that TM expression was in-creased after E3 Ag stimulation (Fig. 3).

DV but not EV71 infection-induced TM expressionin HMEC-1 cells

To rule out the increase of TM expression is a generalphenomenon after virus infection of endothelial cells, wecompared TM protein levels after DV and EV71 infectionwhich as we have previously shown can also infect HMEC-1

cells.15 Western blot results of these experiments wereshown in Fig. 4. TM protein expression in DV-infected celllysate, as predicted, was significantly increased as

compared to that in un-infected control cell lysate. TM pro-tein expression in EV71-infected cell lysate, on the otherhand, was decreased as compared to un-infected controlcell lysate (Fig. 4). Similar amounts of actin were found inall the groups, indicating equivalent amounts of proteinwere loaded in each group.

Effects of DV infection on EPCR, protein S, t-PA andTM mRNA expression in HMEC-1 cells

In addition to TM, endothelial cells can express severalother molecules that are important to the activation ofprotein C and fibrinolysis such as EPCR, protein S and t-PA.

Therefore, the expression of these molecules in DV-infected HMEC-1 cells was analyzed by RT-PCR (Fig. 5). Aspreviously described,19,20 TM expression was increased ina dose-dependent manner after DV infection or UV-DV stim-ulation of HMEC-1 cells. Similar finding was found in theexpression of protein S which was also increased in thepresence of UV-DV. The expression of t-PA and EPCR; onthe other hand, was induced only after DV infection butnot UV-DV stimulation of HMEC-1 cells.

Discussion

Systemic inflammation induced during infection generally

shifts the hemostatic mechanisms in favor of thrombo-sis.21,22 However, infection with DV and certain viruses cantilt the hemostasis toward bleeding and cause viral hemor-rhage fever (VHF).23 Advances in our understanding of themechanism to cause hemorrhage by DV may, therefore,lead to the recognition of potentially useful treatmentagainst VHF.

Both endothelial cells and monocytes play importantroles in regulating hemostasis and both cell types aresusceptible to DV infection.17,24e26 DV infection of endothe-lial cells and monocytes induces cytokine production andmorphological changes. However, the effect of DV infectionon the expression of coagulation and fibrinolysis related

Figure 1 DV induced TM protein expression of HMEC-1. HMEC-1 cells were incubated with medium alone (C), PMA, DV infection or

UV-DV stimulation at MOIZ5 or 10 as indicated for 24 h. Cells were stained with FITC-conjugated anti-TM antibodies. The expres-

sion of TM was observed by fluorescent microscopy (400).

Figure 2 Flow cytometry analysis of TM expression in HMEC-1

and THP-1 cells. (A) HMEC-1 cells were mock-infected or in-

fected with DV (MOIZ 10) for 24 h. (B) THP-1 cells were

mock-infected or infected with DV (MOIZ10) with or withoutthe presence of anti-pre M antibody (70-21) (10 mg) for 24 h.

Cells were fixed, stained for TM protein and analyzed by flow

cytometry as described in the Materials and methods.



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molecules of these cells is still unclear. Previous studieshave demonstrated that the expression of t-PA and TMbut not plasminogen activator inhibitor 1 mRNA was in-creased in DV-infected endothelial cells.19,20 However, itis unclear whether the expression of TM protein is alsoup-regulated. In this study we demonstrated that DV in-duced TM expression in endothelial cells and monocytesat both mRNA and protein levels. DV infection-induced TMexpression is DV specific since EV71 which can also infectHMEC-1 could not induce TM expression but rather decreaseTM expression in HMEC-1 cells. In addition, that TMexpression in THP-1 cells was up-regulated in the presenceof anti-pre M mAb (70-21) which in turn can enhance DV in-fection,16 indicates that the up-regulation of TM expressionis DV specific. Furthermore, UV-DV and DV E protein domainIII (E3 Ag) could also up-regulate TM expression in both en-

dothelial cells and monocytes. Therefore, like Ebola virus,DV E protein may contribute to the hemostatic defect dur-ing DV infection by inducing TM expression on endothelialcells and monocytes.27

Previous studies have shown that the coagulation andfibrinolysis system is abnormally activated in denguepatients and the sera levels of protein C in dengue patientsare decreased compared to those in normal individ-uals.13,14,28 However, whether the decrease of protein Cin dengue patients sera is due to the increase of consump-tion or other mechanisms is unclear. In this study, in addi-tion to TM, we also noticed that the expressions of

several other protein C activation-related molecules suchas EPCR and protein S were increased in DV-infected endo-thelial cells by RT-PCR. The increased expression of TM,EPCR and protein S in DV-infected EC may enhance proteinC activation in dengue patients and result in the decreaseof protein C level in dengue patients. However, furtheranalysis of circulating activated protein C levels in denguepatients is required to test this hypothesis.

The precise mechanism to induce TM expression by DVis still unclear. Down-regulation of TM expression has beenfound in lipopolysaccharide or poly IC stimulated endo-thelial cells as well as herpes simplex virus-infectedendothelial cells.29e31 On the other hand, TM synthesiscan be up-regulated by cAMP or retinoic acid which in-duces transcription-dependent increase of gene expres-sion.32,33 In this study, we found that the expressions of

Figure 3 DV E protein domain III (E3 Ag) induced TM expression in HMEC-1, THP1 and monocytes. Cells were incubated alone orwith E3 Ag (250 ng/ml) for 24 h before being fixed, stained for TM protein and analyzed by flow cytometry.

Figure 4 Western blot analysis of TM protein in DV or EV71-

infected HMEC-1 cells. HMEC-1 cell were incubated with me-

dium alone or infected with DV or EV71 at MOIZ1. TM protein

expression in the cell lysates were analyzed by western blot as

described in the Materials and methods.

GADPH

t-PA

Protein S

EPCR

TM

DV MOI

Mock 1 10 UVDV

Figure 5 RT-PCR analysis of EPCR, protein S, t-PA and TM

mRNA expression in HMEC-1 cells. HMEC-1 cells were mock-

infected, infected with DV at MOIZ1 or 10 or UV-DV stimu-

lated as indicated for 24 h. RT-PCR was performed as described

in the Materials and methods.



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TM and protein S were induced either by DV infection orUV-DV stimulation, while the expression of EPCR and t-PA were induced only by DV infection, but not by UV-DVstimulation. Therefore, different mechanisms may be in-volved in the up-regulation of these molecules during DVinfection. Furthermore, we cannot rule out that the cyto-kines induced by DV infection may also contribute to theexpression of these molecules. It is known that cytokine

storm induced in dengue patients plays very importantroles in the immunopathogenesis of DHF/DSS.34e36 There-fore, further study to understand the mechanism involvedin TM expression in dengue infection both in vitro and invivo is required to reveal potential therapeutic targetsto prevent the hemostatic defect in DHF/DSS.

Acknowledgements

This work was supported by the grant NSC96-2628-B006-006-MY3 from the National Science Council, Taiwan.

References

1. Kyle JL, Harris E. Global spread and persistence of dengue.Annu Rev Microbiol 2008;62:71e92.

2. Henchal EA, Putnak JR. The dengue viruses. Clin Microbiol Rev1990;3:376e96.

3. Oishi K, Saito M, Mapua CA, Natividad FF. Dengue illness: clin-ical features and pathogenesis. J Infect Chemother 2007;13:125e33.

4. Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Micro-biol Rev 1998;11:480e96.

5. Rigau-Perez JG. Severe dengue: the need for new case defini-tions. Lancet Infect Dis 2006;6:297e302.

6. Halstead SB. Antibody, macrophages, dengue virus infection,

shock, and hemorrhage: a pathogenetic cascade. Rev InfectDis 1989;11(Suppl. 4):S830e9.

7. Boffa MC, Karmochkine M. Thrombomodulin: an overview andpotential implications in vascular disorders. Lupus 1998;7(Suppl. 2):S120e5.

8. McCachren SS, Diggs J, Weinberg JB, Dittman WA. Thrombomo-dulin expression by human blood monocytes and by humansynovial tissue lining macrophages. Blood 1991;78:3128e32.

9. Esmon CT. The protein C pathway. Chest 2003;124:26Se32S.10. Stearns-Kurosawa DJ, Kurosawa S, Mollica JS, Ferrell GL,

Esmon CT. The endothelial cell protein C receptor augmentsprotein C activation by the thrombin-thrombomodulin com-plex. Proc Natl Acad Sci U S A 1996;93:10212e6.

11. Aksoy MC, Aksoy DY, Haznedaroglu IC, Sayinalp N, Kirazli S,Alpaslan M. Thrombomodulin and GFC levels in Legge

Calvee

Perthes disease. Hematology 2008;13:324e

8.12. Butthep P, Chunhakan S, Tangnararatchakit K, Yoksan S,

Pattanapanyasat K, Chuansumrit A. Elevated soluble thrombo-modulin in the febrile stage related to patients at risk for den-gue shock syndrome. Pediatr Infect Dis J 2006;25:894e7.

13. Wills BA, Oragui EE, Stephens AC, Daramola OA, Dung NM,Loan HT, et al. Coagulation abnormalities in dengue hemor-rhagic Fever: serial investigations in 167 Vietnamese childrenwith Dengue shock syndrome. Clin Infect Dis 2002;35:277e85.

14. Sosothikul D, Seksarn P, Pongsewalak S, Thisyakorn U, Lusher J.Activation of endothelial cells, coagulation and fibrinolysis inchildren with Dengue virus infection. Thromb Haemost 2007;97:627e34.

15. Liang CC, Sun MJ, Lei HY, Chen SH, Yu CK, Liu CC, et al. Humanendothelial cell activation and apoptosis induced by enterovi-rus 71 infection. J Med Virol 2004;74:597e603.

16. Huang KJ, Yang YC, Lin YS, Huang JH, Liu HS, Yeh TM, et al. Thedual-specific binding of dengue virus and target cells for theantibody-dependent enhancement of dengue virus infection.

J Immunol 2006;176:2825e32.17. Bunyaratvej A, Butthep P, Yoksan S, Bhamarapravati N. Dengue

viruses induce cell proliferation and morphological changes of

endothelial cells. Southeast Asian J Trop Med Public Health1997;28(Suppl. 3):32e7.

18. Babu JP, Pattnaik P, Gupta N, Shrivastava A, Khan M, Rao PV.Immunogenicity of a recombinant envelope domain III proteinof dengue virus type-4 with various adjuvants in mice. Vaccine2008;26:4655e63.

19. Huang YH, Lei HY, Liu HS, Lin YS, Chen SH, Liu CC, et al. Tissueplasminogen activator induced by dengue virus infection ofhuman endothelial cells. J Med Virol 2003;70:610e6.

20. Jiang Z, Tang X, Xiao R, Jiang L, Chen X. Dengue virus regulatesthe expression of hemostasis-related molecules in human veinendothelial cells. J Infect 2007;55:e23e8.

21. Esmon CT. Crosstalk between inflammation and thrombosis.Maturitas 2004;47:305e14.

22. Esmon CT. Inflammation and thrombosis. J Thromb Haemost

2003;1:1343e

8.23. Schnittler HJ, Feldmann H. Viral hemorrhagic fevere a vascu-

lar disease? Thromb Haemost 2003;89:967e72.24. Huang YH, Lei HY, Liu HS, Lin YS, Liu CC, Yeh TM. Dengue virus

infects human endothelial cells and induces IL-6 and IL-8 pro-duction. Am J Trop Med Hyg 2000;63:71e5.

25. Halstead SB, Porterfield JS, ORourke EJ. Enhancement of den-gue virus infection in monocytes by flavivirus antisera. Am JTrop Med Hyg 1980;29:638e42.

26. Jessie K, Fong MY, Devi S, Lam SK, Wong KT. Localization ofdengue virus in naturally infected human tissues, by immuno-histochemistry and in situ hybridization. J Infect Dis 2004;189:1411e8.

27. Yang ZY, Duckers HJ, Sullivan NJ, Sanchez A, Nabel EG,Nabel GJ. Identification of the Ebola virus glycoprotein as the

main viral determinant of vascular cell cytotoxicity and injury.Nat Med 2000;6:886e9.

28. Mairuhu AT, Mac Gillavry MR, Setiati TE, Soemantri A, tenCate H, Brandjes DP, et al. Is clinical outcome of dengue-virus infections influenced by coagulation and fibrinolysis? Acritical review of the evidence. Lancet Infect Dis 2003;3:33e41.

29. Kim HK, Kim JE, Chung J, Kim YT, Kang SH, Han KS, et al. Lipo-polysaccharide down-regulates the thrombomodulin expres-sion of peripheral blood monocytes: effect of serum onthrombomodulin expression in the THP-1 monocytic cell line.Blood Coagul Fibrinolysis 2007;18:157e64.

30. Key NS, Vercellotti GM, Winkelmann JC, Moldow CF,Goodman JL, Esmon NL, et al. Infection of vascular endothelialcells with herpes simplex virus enhances tissue factor activity

and reduces thrombomodulin expression. Proc Natl Acad SciU S A 1990;87:7095e9.

31. Shibamiya A, Hersemeyer K, Schmidt Woll T, Sedding D,Daniel JM, Bauer S, et al. A key role for toll-like receptor-3(TLR3) in disrupting the hemostasis balance on endothelialcells. Blood 2008;113:714e22.

32. Yu K, Morioka H, Fritze LM, Beeler DL, Jackman RW,Rosenberg RD. Transcriptional regulation of the thrombomodu-lin gene. J Biol Chem 1992;267:23237e47.

33. Dittman WA, Nelson SC, Greer PK, Horton ET, Palomba ML,McCachren SS. Characterization of thrombomodulin expressionin response to retinoic acid and identification of a retinoic acidresponse element in the human thrombomodulin gene. J BiolChem 1994;269:16925e32.



7/7

34. Suharti C, van Gorp EC, Setiati TE, Dolmans WM,Djokomoeljanto RJ, Hack CE, et al. The role of cytokines inactivation of coagulation and fibrinolysis in dengue shocksyndrome. Thromb Haemost 2002;87:42e6.

35. Chaturvedi UC, Agarwal R, Elbishbishi EA, Mustafa AS. Cy-tokine cascade in dengue hemorrhagic fever: implications

for pathogenesis. FEMS Immunol Med Microbiol 2000;28:183e8.

36. Chen LC, Lei HY, Liu CC, Shiesh SC, Chen SH, Liu HS, et al. Cor-relation of serum levels of macrophage migration inhibitoryfactor with disease severity and clinical outcome in denguepatients. Am J Trop Med Hyg 2006;74:142e7.


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