Research Article Elevated Dengue Virus Nonstructural ... · (DF) to the life-threatening syndromes of dengue hemor-rhagic fever (DHF) and dengue shock syndrome (DSS), showing manifestations

Research ArticleElevated Dengue Virus Nonstructural Protein 1Serum Levels and Altered Toll-Like Receptor 4 ExpressionNitric Oxide and Tumor Necrosis Factor Alpha Productionin Dengue Hemorrhagic Fever Patients

Denise Maciel Carvalho1 Fernanda Gonccedilalves Garcia1

Ana Paula Sarreta Terra1 Ana Cristina Lopes Tosta1 Luciana de Almeida Silva2

Luacutecio Roberto Castellano3 and David Nascimento Silva Teixeira1

1 Institute of Health Sciences Laboratory of Cellular Activation Federal University of Triangulo Mineiro38025-350 Uberaba MG Brazil2Institute of Health Sciences Department of Infectious and Parasitic Diseases Federal University of Triangulo Mineiro38025-350 Uberaba MG Brazil3Human Immunology Research Group Technical Health School Federal University of Paraiba (UFPB)58051-900 Joao Pessoa PB Brazil

Correspondence should be addressed to David Nascimento Silva Teixeira silvateixeiramednetcombr

Received 18 August 2014 Revised 18 November 2014 Accepted 25 November 2014 Published 11 December 2014

Academic Editor Jean-Paul Gonzalez

Copyright copy 2014 Denise Maciel Carvalho et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Background During dengue virus (DV) infection monocytes produce tumor necrosis factor alpha (TNF-120572) and nitric oxide (NO)which might be critical to immunopathogenesis Since intensity of DV replication may determine clinical outcomes it is importantto know the effects of viral nonstructural protein 1 (NS1) on innate immune parameters of infected patients The present studyinvestigates the relationships between dengue virus nonstructural protein 1 (NS1) serum levels and innate immune response (TLR4expression and TNF-120572NO production) of DV infected patients presenting different clinical outcomes MethodologyPrincipalFindings We evaluated NO NS1 serum levels (ELISA) TNF-120572 production by peripheral blood mononuclear cells (PBMCs) andTLR4 expression onCD14+ cells from37 dengue patients and 20 healthy controls Early in infection increased expression of TLR4 inmonocytes of patients with dengue fever (DF) was detected compared to patients with dengue hemorrhagic fever (DHF)MoreoverPBMCs of DHF patients showed higher NS1 and lower NO serum levels during the acute febrile phase and a reduced response toTLR4 stimulation by LPS (with a reduced TNF-120572 production) when compared to DF patients ConclusionsSignificance DuringDV infection in humans some innate immune parameters change depending on the NS1 serum levels and phase and severity ofthe disease which may contribute to development of different clinical outcomes

1 Introduction

Dengue virus (DV) infects 50ndash100 million people worldwideevery year and an additional 25 billion people are at high riskliving in dengue endemic areas [1ndash3] In Brazil dengue fever(DF) has been a serious health problem and in 2013 fromthe 2351703 cases reported in America approximately 61occurs in Brazil [2] Dengue has various clinical presentationsand clinical illness range from a self-limited dengue fever

(DF) to the life-threatening syndromes of dengue hemor-rhagic fever (DHF) and dengue shock syndrome (DSS)showing manifestations such as increased vascular perme-ability hepatomegaly decreased platelet counts hemorrhageand plasma leakage with the risk of fatal hypovolemic shock[4 5]

Regardless of numerous studies the immunopathologicalmechanisms involved in the development of severe dengueare not fully understood and various controversial results are

Hindawi Publishing CorporationJournal of Tropical MedicineVolume 2014 Article ID 901276 9 pageshttpdxdoiorg1011552014901276

2 Journal of Tropical Medicine

being published around the world [5] Antibody-dependentenhancement [6] inappropriate T cell [7 8] ldquotsunamirdquocytokine response [9 10] and host genetic factors [11] areamongst the postulated causes leading to severe dengue

Monocytes and dendritic and endothelial cells seem to bethe main targets of DV in vivo and in vitro and DV antigenscan be identified inmacrophages of infected patients and alsoon endothelial cells of dead DHF patients [12ndash14] Thus it isapparent that interactions between monocytes and endothe-lial cells leading to a vascular damage play a key role in thepathophysiology of dengue diseaseMonocytesmacrophagescan produce various mediators in response to DV infectionand it is possible that dysregulation of innate and bystanderimmune activation may play a role in aggravating diseaseAmong the mediators produced by activated monocytestumor necrosis factor alpha (TNF-120572) and nitric oxide (NO)might be key molecules A positive association betweenhigh soluble TNF receptor levels and the severity of DHFwas described [15] Single-nucleotide polymorphism analysisidentified TNF-120572 polymorphisms at the TNF-308A allele tobe a possible risk factor for development of hemorrhagicdisease in patients infected with DV [16 17] Using a mousemodel a direct relationship between TNF-120572 and denguehemorrhage was identified because TNF-120572 deficiency greatlydiminished hemorrhage development [18] Moreover pro-duction ofNOcan affect systemic vascular resistance and leadto hypotension shock and death if not corrected NO levelsare increased in many infectious diseases When DVs werecocultured with human Kupffer or spleen cells increasedproduction of NO was reported [19] and elevated levels ofNO were found in DF patients [20] DVs were susceptible toa NO donor treatment and viruses were detected at higherrates in infected cells after iNOS inhibition indicating thatNO might play an important role in controlling monocytesDV infection [21] Thus it seems that TNF-120572 and NO wouldbe involved not only in generating severe symptoms [22 23]but also in the elimination of viruses [24ndash26]

TNF-120572 and NO are produced in response to toll-likereceptor 4 (TLR4) stimulation Toll-like receptors (TLRs) areimportant inmicrobial recognition [27] and they are involvedin the generation of antiviral molecules and proinflammatorycytokines which probably exert immunopathological func-tions [27] Although the implications of TLRs functions inviral infections have been investigated [28] the knowledgeabout dengue is restricted de Kruif et al [29] evaluatedTLR gene-expression profiling of childrenwith severe dengueinfectionsThe authors demonstrated mainly that TLR7 genetranscription was upregulated while TLR2 were downreg-ulated indicating the in vivo role of particular TLRs withdifferent disease-severity parameters

TLR4 is recognized as a LPS receptor [30 31] and a previ-ous study showed an interaction among DV LPS and CD14at themembrane of primary humanmonocytesmacrophages[32] The bacterial lipopolysaccharide (LPS) a ligand of theCD14-TLR4 complex was able to block DV and modulatevirally induced cytokine production by human monocytesand macrophages So based on that we asked if there isa regulatory role for the LPS receptor TLR4 on cytokineproduction during the acute phase of human DV infection

DV genome is a single-stranded positive sense RNAwhich codes for 10 gene products including structuralproteins capsid (C) premembrane (prM) envelope (E) andnonstructural proteins NS1 NS2A NS2B NS3 NS4A NS4Band NS5 [33 34] Due to the fact that intensity of DVreplication might influence clinical outcomes it is importantto investigate the impact of some viral proteins on innateimmune parameters of DV infected patients Among theseproteins the NS1 glycoprotein is a singular glycoproteinsince it does not form part of the virion structure but isexpressed on the membrane of infected cells NS1 circulatesat high levels in the sera of patients during the acute phaseof illness is a well-known early diagnostic marker and maybe involved on the pathophysiology of DV infection [35]Preliminary evidence has shown that NS1 is involved in viralRNA replication [35] but an association between NS1 levelsperturbation of innate immune response and severe diseaseis still unknown In addition toll-like receptor regulation onmonocytes can also theoretically be elicited by proteins suchas viral NS1 Thus the aim of this study was to investigate therelationships between in vivo secreted levels ofNS1 and innateimmune response parameters (TLR4 expression and TNF-120572NO production) in infected dengue patients with differentclinical outcomes

2 Methodology

21 Study Group and Case Classification DV infectedpatients (119899 = 37) 17 female and 20 male subjects wereenrolled in this study Twenty healthy individuals 10 femalesand 10 males were included as healthy controls (HCs) AllHCs tested negative for DV NS1 antigen and DV IgMIgGantibodies and had not been vaccinated against yellow fevervirus Dengue patients were enrolled from February 2010 toApril 2013 in UFTM University Hospital and two healthcarecenters located in the city of Uberaba Brazil Dengue caseswere classified as DF or DHF according to the 1997 WorldHealth Organization (WHO) guidelines [36] We applied theold guidelines since the new WHO guidelines published in2009 are more directly focused on clinical practice and arenot broadly used in research [37]

22 Blood Samples Collection andProcessing After 2nd (acutephase) and 9th day (beginning of convalescence phase)from the commencement of symptoms twenty milliliters ofheparinized peripheral venous blood (PB) and five milliliterswithout anticoagulant (serum) were collected from DVinfected patients and noninfected HCs The heparinizedblood collected was used for isolation of peripheral bloodmononuclear cells (PBMCs) and serum was stored at minus80∘Cuntil further use

23 Serological Diagnosis of Dengue Dengue-specific IgMand IgG were detected using a capture ELISA (PanBio)whereas Dengue NS1 was detected using the immunochro-matographic kit (PanbioQueenslandAustralia) according tomanufacturerrsquos instructions

Journal of Tropical Medicine 3

24 NS1 Serum Levels Serum samples were collected attwo different phases (acute and convalescence) from allpatients and were subjected to diagnostic assays accordingto manufacturerrsquos instructions The qualitative presence ofNS1 was determined using the Platelia NS1 Ag enzymeimmunoassay (Bio-Rad Laboratories Marnes-la-CoquetteFrance) as indicated by the manufacturer For quantitativemeasurements the same kit was used but with amodified pro-tocol also designed by the same manufacturer According toBio-Rad technical support version 052008 (Platelia DengueNS1 Ag quantitative detection of dengue virus NS1 antigenin human serum or plasma by enzyme immunoassay) dueto its high sensitivity about ninety percent (90) of positivesera tested in routine with Platelia Dengue NS1 Ag qualitativeare showing optical densities (OD) exceeding the assay rangelinearity (ODgt 30)The quantitative protocol uses a differentconjugate dilution (1 5000) and a formula for calculation ofNS1 units based on recombinant human NS1 (positive con-trol) andNS1 cut-off control kit calibrators OD readingsThisprotocol applies to samples that have been confirmed positivewith the traditional protocol with anOD gt 30 (or close to themaximum OD readable with the plate reader) Calculationsfor samples tested with diluted conjugate 1 5000 NS1 AgBio-Rad units per milliliter (BRUmL) are calculated bymultiplying the OD of the sample tested with 1 5000 dilutedconjugate by 150 as follows

Sample NS1 (BRUmL) = Sample OD times 150R4m (1)

where R4m is themean value of theODs of the cut-off controlduplicates (R4)

Results are expressed in Bio-Rad units per milliliter(BRUmL)

25 Isolation and Stimulation of PBMCs PBMCs were iso-lated from heparinized blood samples of HCs and DVinfected patients by density gradient centrifugation usingFicoll-Hypaque (Histopaque 1077 Sigma Aldrich ChemicalCo St Louis MO) PBMCs were cultured at 1 times 106 cellsmLin 24-well polystyrene tissue culture plates at 37∘C in 5CO

2

using RPMI 1640 medium (BioWhittaker Walkersville MD)supplemented with 10 heat-inactivated fetal bovine serum100UmL penicillinstreptomycin (Sigma Aldrich ChemicalCo) and 1 of L-glutamine (Sigma Aldrich Chemical Co)The PBMCs were stimulated for 24 h in the presence orabsence of a TLR4 agonist 10 120583gmL of ultrapure lipopolysac-charide (LPS InVivoGen USA) PBMC culture supernatantswere harvested after 18 h of cell culture and stored at minus80∘Cuntil analysis Aliquots of PBMCs were suspended in 1mLof solution destined for freezing (90 inactivated fetal calfserum (FCS Gibco Invitrogen) plus 10 dimethyl sulphox-ide (DMSO Sigma Chemical Co St Louis MO)) and storedinitially at minus80∘C for 24 hr before introduction into liquidnitrogen and aliquots were cryopreserved for later flowcytometer studies

26 Cell Viability Determination The viability of PBMCs inculture and after LPS stimulation was quantified by their

ability to reduce 3-(45-dimethylthiazol-2-yl)-25-diphenyl-tetrazoliumbromide (MTT) to formazan precipitate in dupli-cate wells MTT (Sigma-Aldrich) at a final concentration of5mgmLwas added to each well 4 h before the termination ofthe experiment Formazan dye was dissolved by incubationin DMSO (Merck Berlin Germany) and its concentrationwas determined spectrophotometrically at an absorbancewavelength of 560 nm

27 TNF-120572 Quantification PBMC culture supernatants wereharvested after 18 h of culture and tumor necrosis factor alpha(TNF-120572) concentration was measured by ELISA according tothe manufacturerrsquos protocol (BD-Bioscience USA)

28 Serum Nitric Oxide Measurement The concentration ofNO in serum samples was indirectly measured by deter-mining both nitrate and nitrite levels Total serum NOwas measured by utilizing a nitric oxide (NO

2

minusNO3

minus)assay kit (Sigma Aldrich USA) following the manufacturerrsquosinstructions This assay determines nitric oxide based on theenzymatic conversion of nitrate to nitrite by nitrate reductaseThe reaction is followed by a colorimetric detection of nitriteas a product of the Griess reaction based on the diazotizationreaction in which acidified NO

2

minus produces a nitrosatingagent which reacts with sulfanilic acid to yield the diazoniumion This ion is then combined to N-(1-naphthyl) ethylene-diamine to form the chromophoric azo derivative whichabsorbs light at 540 nm Protein interference was avoidedby treating samples with zinc sulfate and centrifugationfor 10min at 2000timesg Samples were spectrophotometricallyquantified using a Turner microplate reader at 540 nm(PROMEGA USA) NaNO

2was used as a standard and a

curve of nitrite concentration against its OD was plotted

29 Flow Cytometry PBMCs flow cytometry was performedby incubating 50 120583L containing 5 times 105 PBMCs with optimalconcentrations of monoclonal antibodies anti-CD14-FITCand anti-TLR4-PE for 30min at room temperature in thedark Cells were washed twice (300timesg for 5 minutes) andsuspended in 500 120583L of PBS with 1 of fetal calf serum(FCS) and 01 sodium azide for data acquisition Beforeeach experiment the instrument was checked for stabilityand reproducibility using Calibrite beads (Becton DicksonSan Jose CA) Control tubes include cells incubated withmedium alone (control of background fluorescence) andcells incubated with FITC and PE conjugated mouse isotypecontrol antibodies (control of nonspecific binding) Duringacquisition a hundred thousand events were counted persample

210 CytometryAnalysis Strategy Theexpression of theTLR4was defined by using gates on contour plots CD14+ cells wereobtained by drawing a gate of fluorescence versus specificgranularity (SSC parameter) The cells contained in thisgate named region 1 (R1) were further analyzed in a PE-fluorescence channel representing the TLR4 expression


Table 1 Demographic and clinical information of dengue patients and healthy controls enrolled in the study

Patients data Healthy controls (HC)(119899 = 20)

Dengue fever (DF)(119899 = 26)

Dengue hemorrhagic fever (DHF)(119899 = 11)

Age (mean plusmn SD) 37 plusmn 14 31 plusmn 11 39 plusmn 22Male (119899 = 30) 10 14 06Females (119899 = 27) 10 12 05Platelets (times103mm3) 271 plusmn 36 152 plusmn 82 68 plusmn 27

211 Statistical Analyses All data analyses were carried outusing the applicative GraphPad Prism software (Graph PadCA) As the numeric variables had nonparametric distribu-tion Mann-Whitney and Kruskal-Wallis tests were used tocompare two or more groups respectively The significanceset was adjusted to 119875 lt 005

3 Results

31 Study Group Characterization In this study 57 patientswere enrolled 37 of which were positive for DV infectionFrom the 37 dengue positive cases 11 were classified as DHFpatients The demographic and clinical information of the37 dengue patients enrolled in this study are summarized inTable 1

32 NS1 Quantitation in Patientrsquos Serum NS1 antigen wasfound circulating from the second day after the onset offever to day 9 NS1 circulation levels varied among individ-uals during the course of the disease ranging from 22 to600 BRUmL of serum During the acute febrile phase DHFpatients display higher NS1 serum levels than DF patientsMoreover during the beginning of convalescence phase NS1levels were also higher onDHF than inDF patients (Figure 1)

33 Nitric Oxide SerumLevels andClinical Outcomes Duringthe acute febrile phase of DV infection we observed asignificant increase in NO serum levels on DF patientsrsquoserum and a decrease in DHF patients when compared tohealthy controls (Figure 2) During the convalescence phaseno differences could be detected among groups

34 Nitric Oxide and NS1 Serum Levels When analyzingthe distribution of NS1 serum levels among all DV infectedpatients we observed a clear division between two patientgroups (data not show) One group displayed low levelsof serum NS1 (below 100 times 103 BRUnitsmL) and theother displayed high levels of NS1 (equal or above 100 times103 BRUnitsmL) Thus besides the clinical form and basedon the fact that we establish a cut-off we also analyzedpatientrsquos data according to their NS1 content (lt100 times103 BRUnitsmL or ge100 times 103 BRUnitsmL) We show thatpatients with NS1 serum levels ge100 BR times 103 BRUnitsmLdisplayed reduced NO serum levels when compared topatients with NS1 levels below 100 times 103 BRUnitsmL(Figure 3)

0

100

200

300

400

500

DefervescenceAcute phase

BRUtimes103m

L

lowast

lowast

DF DHF DF DHF

Figure 1 Dengue virus nonstructural protein 1 (NS1) serum levelsof patients with dengue fever (DF 119899 = 26) and dengue hemorrhagicfever (DHF 119899 = 11) during the acute phase and convalescencemeasured by ELISA and expressed as median BRU times 103mL in abox plot Equivalent symbols (lowast ) represent statistically significantdifferences (119875 lt 005) between groups

35 Stimulation of TLR4 TNF-120572 Production and NS1 Levelsand Clinical Outcomes Production of TNF-120572 after TLR4stimulation with LPS was also investigated DHF patientspresent a lower response to TLR4 stimulation than DFpatients (Figure 4) Figure 5 shows that dengue patients withhigher NS1 serum levels displayed a reduced TLR4 responseto LPS (with a lower TNF-120572 production) than the group ofpatients with less NS1 content (lt100 times 103 BRUnitsmL)

36 TRL4 Expression on CD14+ Cells Subsequently to thealterations detected on TLR4 response to its agonist LPS inDHF patientrsquos cells we attempt to investigate if this reducedresponse was due to modulations on TLR4 expression onmonocytes In fact TLR4 expression was downmodulatedon DHF monocytes during the acute phase of the diseasewhen compared to CD14+ cells obtained from DF patients(Figure 6(b)) High levels of serum NS1 were also associatedwith a reduced TLR4 membrane expression on these cells(Figure 6(c))

4 Discussion

It is possible that during DV infection protection or patho-genesis is determined at the edge of innate and adaptive


Acute phase

0

10

20

30

40

50Convalescence

0

10

20

30

40

HC DFDHF HCDF DHF

lowast

lowast

lowast

NO

2minusN

O3minus

(120583M

L)

NO

2minusN

O3minus

(120583M

L)

Figure 2 Nitric oxide serum levels (120583M of NO2

minus

NO3

minus) of patients with dengue fever (DF 119899 = 26) dengue hemorrhagic fever (DHF119899 = 11) and healthy controls (HC 119899 = 20) measured by Griess reaction after reduction of serum samples with nitrate reductase Results wereexpressed as median using a box plot Equivalent symbols (lowast ) represent statistically significant differences (119875 lt 005) between two groups

0

10

20

30

40

50lowast

lowast

NS1 lt 100 times 103 NS1 ge 100 times 103

NO

2minusN

O3minus

(120583M

L)


minus

NO3

minus) of dengueinfected patients (119899 = 37) during the acute phase of the diseasepresenting low (NS1 lt 100 times 103 BRUmL) and high (NS1 ge 100 times103 BRUmL) dengue virus nonstructural protein 1 (NS1) serumlevels measured by Griess reaction after reduction of serum sampleswith nitrate reductase Results were expressed as median using abox plot Equivalent symbols (lowast) represent statistically significantdifferences (119875 lt 005) between groups

immunity controlled by cytokines produced as a consequenceof DV interactions with its main targets human monocytesand endothelial cells [12ndash14] Besides that it is important toknow the impact of viral proteins such as NS1 on innateimmune parameters of DV infected patients However adefined connection between viral replication and increasedvascular permeability in DHF development is still subject ofspeculation

We are able to detect higher serum levels of soluble NS1in DHF patientsrsquo serum when compared to DF (Figure 1)NS1 concentrations must be a key point since high levels ofthis protein could affect innate immune response and mayinfluence later development of different clinical forms Onestudy demonstrated that NS1 levels in human sera appear to

NS

0

100

200

300

400

500

(pg

mL)

lowast

lowast

HC DF DHF HC DF DHF+ LPS

Figure 4 Tumor necrosis factor alpha (TNF-120572) production (pgmL)by 1 times 106 peripheral blood mononuclear cells (PBMCs) frompatients with dengue fever (DF 119899 = 26) and dengue hemorrhagicfever (DHF 119899 = 11) during the acute phase of the disease andhealthy controls (HC 119899 = 20) PBMCs were not stimulated (NS) orstimulated for 18 hours with 10120583gmL of lipopolysaccharide (+ LPS)and TNF-120572 production measured by ELISA Results were expressedas median using a box plot Equivalent symbols (lowast) representstatistically significant differences (119875 lt 005) between groups

be significantly higher in patients who developed DHF ratherthan DF [38] During DV infection mouse and humansdevelop antibodies against NS1 that cross-react with endothe-lial cell epitopes inducing a nitric oxide dependent apoptosis[39] Results from our work and data from other studiesstrengthen the fact that intensity of DV replication in theearly times of infection may influence clinical outcomes butpathogenesis of endothelial dysfunction related to vascularleakage syndrome is not a fully understood phenomenon

Among important innate immune parameters that couldbe affected by dengue NS1 levels NO and TNF-120572 seem to bevery important molecules Nitric oxide a gaseous moleculeis a product of enzymes called NO synthases (NOS) [40]


NS0

100

200

300

400

500

(pg

mL)

LPS

lowast

lowast

NS1 lt 100 times 103

NS1 ge 100 times 103

Figure 5 Tumor necrosis factor alpha (TNF-120572) production (pgmL)by 1times 106 peripheral bloodmononuclear cells (PBMCs) fromdengueinfected patients (119899 = 37) during the acute phase of the diseasepresenting low (NS1 lt 100 times 103 BRUmL) and high (NS1 ge 100 times103 BRUmL) NS1 serum levels PBMCs were not stimulated (NS) orstimulated for 18 hours with 10120583gmL of lipopolysaccharide (LPS)and TNF-120572 production measured by ELISA Results were expressedas median using a box plot Equivalent symbols (lowast) representstatistically significant differences (119875 lt 005) between groups

that are classified into three isoforms specifically endothelialNOS (eNOS) inducible NOS (iNOS) and neuronal NOS(nNOS)Themain physiologic function of eNOS-derivedNOis vasodilatation iNOS can be found in some cell types suchas monocytesmacrophages as well as endothelial cells andis expressed when cells are activated with molecules suchas LPS or interferon gamma (IFN-120574) [41] NO produced bymacrophages and endothelial cells plays an important role inregulating the diameter of blood vessels inhibiting leukocyteadhesion and platelet aggregation [40 41]

NO is probably involved in hemorrhagic fevers and virus-induced shock when produced in high amounts [42] In ourstudy we found decreased NO serum levels during febrileacute phase in DHF compared to DF patients (Figure 2) Thereasons for the lower levels of NO in DHF patients are notclear to us but damage of endothelial cells during the acutephase of DV infection with a consequent failure of endothe-lial NO synthase could be involved On the other hand sinceNO is a ldquodouble-swordrdquo molecule [43] the increased levelsof NO in DF could play a role in decreasing viral load andaltering the evolution of the disease to its hemorrhagic formInterestingly patients with low NS1 serum levels during theacute phase of the disease also displayed higher NO serumlevels (Figure 3) The pattern of low serum NO levels in theDHF group during the acute febrile phase and higher serumNO levels during the beginning of convalescence phase mayalso be due to different eNOS and iNOS modulations in eachphase but this hypothesis needs to be tested A previous studyby Levy et al showed that distinct dengue serotypes yieldsimilar NO levels [44] suggesting that NO appears to beinvolved in the progression of dengue infection independent

of the DV serotype Taken together these data demonstratethat NOmight be contributing not only to protection but alsoto pathology of dengue infection depending on the amountof NO produced and the phase of the disease

Activated monocytesmacrophage also produce TNF-120572afterTLR4 stimulationThusmodulation of TLR4 expressionand responsiveness is an important issue to investigate duringhuman DV infection A correlation between high concen-tration TNF-120572 in blood and the severity of DHF has beenreported [15ndash17] TLR functions as receptors during dengueinfection are not yet clear and our data indicates a differentialregulation of TLR4 expression (Figure 6(b)) and responsive-ness (Figure 4) during the acute phase of this illness on cellsfrom DHF when compared to DF patients and that may bealso affected by NS1 serum levels (Figure 6(c)) Data from theliterature shows that DV cell entry in the monocyte cell lineTHP-1 is facilitated by antibodies activation of TLR-negativeregulators and downregulation of TLR4 and other genesassociated with TLR signaling [45] Besides that entry of DVvia Fc receptor during a virus-enhancing antibody complexinfection preferentially switches off the TLR4 dependentsignaling due to a significant collapse of the TLR4 pathway[46] The following downregulation of the proinflammatorycytokine productionmay provide a growth advantage for DVto propagate in host macrophages and for the development ofmore serious forms of the disease

The relationships between in vivo dengue virus nonstruc-tural protein 1 (NS1) levels and innate immune responseparameters (TLR4 expression and TNF-120572NO production)in infected dengue patients were investigated in our workResults obtained indicate that DHF patients displayed analtered innate immune response with low TLR4 expressionand reduced NO and TNF-120572 production during the acutephase of the disease when compared to DF patients Thusour data suggests that during this early acute phase patientswho develop DHFmay present a less efficient antiviral innateimmune response to DV that may promote an accumulationof huge amounts of circulating NS1 protein (Figure 1) whichin turnmay affect these correlated innate immune parameters(NO TNF-120572 and TLR4 Figures 3 5 and 6 resp)

A differential expression and responsiveness of TLR4 onCD14+ cells in DHF patients could be one relevant factorthat leads to different clinical outcomes but new studies arenecessary to understand the precise role of these pathways onDHF development in humans

5 Conclusions

During DV infection in humans DHF patients displayalterations on innate immune response (expression andresponsiveness of TLR4 on CD14+ cells and TNF-120572NOproduction) that are inversely correlated to NS1 serum levelsand phase and severity of the disease which may contributeto development of different clinical outcomes


1000

800

600

400

200

0

100 101 102 103 104

R1

SSC-

H

SSC

-hei

ght

FL1-H CD14 FITC

(a)

0

1000

2000

3000

4000

MFI

lowast

lowast

lowast

HC DF DHF

(b)

0

1000

2000

3000

4000

MFI


lowast

lowast

(c)

Figure 6 Toll-like receptor (TLR-4) expression of CD14+ cells from patients (119899 = 37) infected with dengue virus during the acute phaseof the disease (a) represents the gate strategy a gate was drawn on SSC times FL1CD14+ cells (b) represents anti-TLR4-PE mean fluorescenceintensity (MFI) of CD14+ cells from dengue fever (DF 119899 = 26) dengue hemorrhagic fever (DHF 119899 = 11) and healthy controls (HC 119899 = 20)(c) represents anti-TLR4-PE MFI of CD14+ cells from dengue infected patients with low (NS1 lt 100 times 103 BRUmL) and high (NS1 ge 100 times103 BRUmL) dengue virus nonstructural protein 1 (NS1) serum levels Results on (b) and (c) graphs were expressed as median using a boxplot Equivalent symbols (lowast ) represent statistically significant differences (119875 lt 005) between groups

Ethical Approval

This work was approved by local ethics research commit-teeinstitutional review board of Universidade Federal doTriangulo Mineiro (CEP-UFTM) Brazil All patients wereinformed about the study and signed a free consent term(including healthy donors) prior to blood draw When theparticipant was a minor the informed consent was signed byat least one parent andor guardians

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Denise Maciel Carvalho participated in all experimentsFernanda Goncalves Garcia participated in all experiments

Ana Paula Sarreta Terra participated in selection of patientsAna Cristina Lopes Tosta participated in cytometry analysisLuciana de Almeida Silva participated in selection of patientsand design of the study Lucio Roberto Castellano partici-pated in design of the study and drafted the paper and DavidNascimento Silva Teixeira conceived the study participatedin its design and coordinated and drafted the paper DeniseMaciel Carvalho and FernandaGoncalvesGarcia contributedequally to this work

Acknowledgments

This work was supported by the Coordenacao de Aperfei-coamento de Pessoal de Nıvel Superior (CAPESPNPD)Fundacao de Amparo a Pesquisa do Estado de MinasGerais (FAPEMIG) Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq) and Fundacao de Ensino ePesquisa de Uberaba (FUNEPU)


References

[1] M G Guzman S B Halstead H Artsob et al ldquoDengue acontinuing global threatrdquo Nature Reviews Microbiology vol 8no 12 pp S7ndashS16 2010

[2] O J Brady P W Gething S Bhatt et al ldquoRefining the globalspatial limits of dengue virus transmission by evidence-basedconsensusrdquo PLoS Neglected Tropical Diseases vol 6 no 8Article ID e1760 2012

[3] N E A Murray M B Quam and A Wilder-Smith ldquoEpidemi-ology of dengue past present and future prospectsrdquo ClinicalEpidemiology vol 5 no 1 pp 299ndash309 2013

[4] J G Rigau-Perez G G Clark D J Gubler P Reiter E JSanders and A V Vorndam ldquoDengue and dengue haemor-rhagic feverrdquoThe Lancet vol 352 no 9132 pp 971ndash977 1998

[5] I Kurane and F E Ennis ldquoImmunity and immunopathology indengue virus infectionsrdquo Seminars in Immunology vol 4 no 2pp 121ndash127 1992

[6] M G Guzman and S Vazquez ldquoThe complexity of antibody-dependent enhancement of dengue virus infectionrdquo Virusesvol 2 no 12 pp 2649ndash2662 2010

[7] I Kurane B L Innis A Nisalak et al ldquoHuman T cell responseto dengue virus antigens Proliferative responses and interferongamma productionrdquo The Journal of Clinical Investigation vol83 no 2 pp 506ndash513 1989

[8] A L Rothman ldquoImmunity to dengue virus a tale of originalantigenic sin and tropical cytokine stormsrdquo Nature ReviewsImmunology vol 11 no 8 pp 532ndash543 2011

[9] A L Rothman and F A Ennis ldquoImmunopathogenesis ofdengue hemorrhagic feverrdquoVirology vol 257 no 1 pp 1ndash6 1999

[10] U C Chaturvedi R Agarwal E A Elbishbishi and A SMustafa ldquoCytokine cascade in dengue hemorrhagic feverimplications for pathogenesisrdquo FEMS Immunology and MedicalMicrobiology vol 28 no 3 pp 183ndash188 2000

[11] S Yacoub J Mongkolsapaya and G Screaton ldquoThe pathogen-esis of denguerdquo Current Opinion in Infectious Diseases vol 26no 3 pp 284ndash289 2013

[12] R M Scott A Nisalak U Cheamudon S Seridhoranakul andS Nimmannitya ldquoIsolation of dengue viruses from peripheralblood leukocytes of patients with hemorrhagic feverrdquo Journal ofInfectious Diseases vol 141 no 1 pp 1ndash6 1980

[13] W C Hall T P Crowell D M Watts et al ldquoDemonstrationof yellow fever and dengue antigens in formalin-fixed paraffin-embedded human liver by immunohistochemical analysisrdquoTheAmerican Journal of Tropical Medicine and Hygiene vol 45 no4 pp 408ndash417 1991

[14] K Jessie M Y Fong S Devi S K Lam and K T WongldquoLocalization of dengue virus in naturally infected humantissues by immunohistochemistry and in situ hybridizationrdquoJournal of Infectious Diseases vol 189 no 8 pp 1411ndash1418 2004

[15] E L A Braga P Moura L M O Pinto et al ldquoDetection ofcirculant tumor necrosis factorndash120572 soluble tumor necrosis factorp75 and interferon-120574 in Brazilian patients with dengue feverand dengue hemorrhagic feverrdquoMemorias do Instituto OswaldoCruz vol 96 no 2 pp 229ndash232 2001

[16] M T Fernandez-Mestre K Gendzekhadze P Rivas-Veten-court and Z Layrisse ldquoTNF-alpha-308A allele a possibleseverity risk factor of hemorrhagic manifestation in denguefever patientsrdquoTissue Antigens vol 64 no 4 pp 469ndash472 2004

[17] A Chuansumrit N Anantasit W Sasanakul et al ldquoTumournecrosis factor gene polymorphism in dengue infection associ-ation with risk of bleedingrdquo Paediatrics and International ChildHealth vol 33 no 2 pp 97ndash101 2013

[18] Y-T Yen H-C Chen Y-D Lin C-C Shieh and B A Wu-Hsieh ldquoEnhancement by tumor necrosis factor alpha of denguevirus-induced endothelial cell production of reactive nitrogenand oxygen species is key to hemorrhage developmentrdquo Journalof Virology vol 82 no 24 pp 12312ndash12324 2008

[19] P Marianneau A-M Steffan C Royer et al ldquoInfection ofprimary cultures of human Kupffer cells by dengue virus noviral progeny synthesis but cytokine production is evidentrdquoJournal of Virology vol 73 no 6 pp 5201ndash5206 1999

[20] N Valero L M Espina G Anez E Torres and J A MosqueraldquoShort report Increased level of serum nitric oxide in patientswith denguerdquo The American Journal of Tropical Medicine andHygiene vol 66 no 6 pp 762ndash764 2002

[21] P C F Neves-Souza E L Azeredo S M O Zagne et alldquoInducible nitric oxide synthase (iNOS) expression in mono-cytes during acute Dengue Fever in patients and during in vitroinfectionrdquo BMC Infectious Diseases vol 5 article 64 2005

[22] T R Kreil and M M Eibl ldquoNitric oxide and viral infection Noantiviral activity against a flavivirus in vitro and evidence forcontribution to pathogenesis in experimental infection in vivordquoVirology vol 219 no 1 pp 304ndash306 1996

[23] P Nicotera B Brune and G Bagetta ldquoNitric oxide inducer orsuppressor of apoptosisrdquo Trends in Pharmacological Sciencesvol 18 no 6 pp 189ndash190 1997

[24] G Karupiah Q W Xie R M L Buller C Nathan C Duarteand J D MacMicking ldquoInhibition of viral replication byinterferon-120574-induced nitric oxide synthaserdquo Science vol 261no 5127 pp 1445ndash1448 1993

[25] M Saura C Zaragoza A McMillan et al ldquoAn antiviral mech-anism of nitric oxide inhibition of a viral proteaserdquo Immunityvol 10 no 1 pp 21ndash28 1999

[26] L G Guidotti and F V Chisari ldquoNoncytolytic control ofviral infections by the innate and adaptive immune responserdquoAnnual Review of Immunology vol 19 pp 65ndash91 2001

[27] AG Bowie and I RHaga ldquoThe role of Toll-like receptors in thehost response to virusesrdquoMolecular Immunology vol 42 no 8pp 859ndash867 2005

[28] S-Y Zhang E Jouanguy V Sancho-Shimizu et al ldquoHumanToll-like receptor-dependent induction of interferons in protec-tive immunity to virusesrdquo Immunological Reviews vol 220 no1 pp 225ndash236 2007

[29] M D de Kruif T E Setiati A T AMairuhu et al ldquoDifferentialgene expression changes in children with severe dengue virusinfectionsrdquo PLoSNeglected Tropical Diseases vol 2 no 4 articlee215 2008

[30] O Takeuchi and S Akira ldquoInnate immunity to virus infectionrdquoImmunological Reviews vol 227 no 1 pp 75ndash86 2009

[31] M Essakalli O Atouf N Bennani N Benseffaj S Ouadghiriand C Brick ldquoToll-like receptorsrdquo Pathologie Biologie vol 57no 5 pp 430ndash438 2009

[32] Y-C Chen S-Y Wang and C-C King ldquoBacterial lipopolysac-charide inhibits dengue virus infection of primary humanmonocytesmacrophages by blockade of virus entry via a CD14-dependent mechanismrdquo Journal of Virology vol 73 no 4 pp2650ndash2657 1999

[33] A C Shurtleff D W C Beasley J J Y Chen et al ldquoGeneticvariation in the 31015840 non-coding region of dengue virusesrdquoVirology vol 281 no 1 pp 75ndash87 2001


[34] E C Holmes and S S Twiddy ldquoThe origin emergence andevolutionary genetics of dengue virusrdquo Infection Genetics andEvolution vol 3 no 1 pp 19ndash28 2003

[35] D A Muller and P R Young ldquoThe flavivirus NS1 proteinmolecular and structural biology immunology role in patho-genesis and application as a diagnostic biomarkerrdquo AntiviralResearch vol 98 no 2 pp 192ndash208 2013

[36] WHO-Dengue Haemorrhagic Fever Diagnosis Treatment Pre-vention and Control World Health Organization GenevaSwitzerland 2nd edition 1997 httpwwwwhointcsrresou-rcespublicationsdengueDenguepublicationen

[37] G A Macedo M L C Gonin S M Pone O G Cruz F FNobre and P Brasil ldquoSensitivity and specificity of the WorldHealth Organization dengue classification schemes for severedengue assessment in children in Rio de Janeirordquo PLoS ONEvol 9 no 4 Article ID e96314 2014

[38] P AvirutnanN Punyadee S Noisakran et al ldquoVascular leakagein severe dengue virus infections a potential role for thenonstructural viral protein NS1 and complementrdquo Journal ofInfectious Diseases vol 193 no 8 pp 1078ndash1088 2006

[39] C-F Lin H-Y Lei A-L Shiau et al ldquoEndothelial cell apoptosisinduced by antibodies against dengue virus nonstructural pro-tein 1 via production of nitric oxiderdquoThe Journal of Immunologyvol 169 no 2 pp 657ndash664 2002

[40] P Vallance and S Moncada ldquoNitric oxide-from mediator tomedicinesrdquo Journal of the Royal College of Physicians of Londonvol 28 no 3 pp 209ndash219 1994

[41] W C Sessa ldquoThe nitric oxide synthase family of proteinsrdquoJournal of Vascular Research vol 31 no 3 pp 131ndash143 1994

[42] A Sanchez M Lukwiya D Bausch et al ldquoAnalysis of humanperipheral blood samples from fatal and nonfatal cases of Ebola(Sudan) hemorrhagic fever cellular responses virus load andnitric oxide levelsrdquo Journal of Virology vol 78 no 19 pp 10370ndash10377 2004

[43] S Mocellin V Bronte and D Nitti ldquoNitric oxide a doubleedged sword in cancer biology searching for therapeuticopportunitiesrdquo Medicinal Research Reviews vol 27 no 3 pp317ndash352 2007

[44] A Levy N Valero L M Espina G Anez J Arias and JMosquera ldquoIncrement of interleukin 6 tumour necrosis factoralpha nitric oxide C-reactive protein and apoptosis in denguerdquoTransactions of the Royal Society of Tropical Medicine andHygiene vol 104 no 1 pp 16ndash23 2010

[45] J D Teo P A Macary and K S Tan ldquoPleiotropic effects ofBlastocystis spp subtypes 4 and 7 on ligand-specific toll-likereceptor signaling and NF-120581B activation in a human monocytecell linerdquo PLoS ONE vol 9 no 2 Article ID e89036 2014

[46] N Modhiran S Kalayanarooj and S Ubol ldquoSubversion ofinnate defenses by the interplay between DENV and pre-existing enhancing antibodies TLRs signaling collapserdquo PLoSneglected tropical diseases vol 4 no 12 article e924 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


being published around the world [5] Antibody-dependentenhancement [6] inappropriate T cell [7 8] ldquotsunamirdquocytokine response [9 10] and host genetic factors [11] areamongst the postulated causes leading to severe dengue

Monocytes and dendritic and endothelial cells seem to bethe main targets of DV in vivo and in vitro and DV antigenscan be identified inmacrophages of infected patients and alsoon endothelial cells of dead DHF patients [12ndash14] Thus it isapparent that interactions between monocytes and endothe-lial cells leading to a vascular damage play a key role in thepathophysiology of dengue diseaseMonocytesmacrophagescan produce various mediators in response to DV infectionand it is possible that dysregulation of innate and bystanderimmune activation may play a role in aggravating diseaseAmong the mediators produced by activated monocytestumor necrosis factor alpha (TNF-120572) and nitric oxide (NO)might be key molecules A positive association betweenhigh soluble TNF receptor levels and the severity of DHFwas described [15] Single-nucleotide polymorphism analysisidentified TNF-120572 polymorphisms at the TNF-308A allele tobe a possible risk factor for development of hemorrhagicdisease in patients infected with DV [16 17] Using a mousemodel a direct relationship between TNF-120572 and denguehemorrhage was identified because TNF-120572 deficiency greatlydiminished hemorrhage development [18] Moreover pro-duction ofNOcan affect systemic vascular resistance and leadto hypotension shock and death if not corrected NO levelsare increased in many infectious diseases When DVs werecocultured with human Kupffer or spleen cells increasedproduction of NO was reported [19] and elevated levels ofNO were found in DF patients [20] DVs were susceptible toa NO donor treatment and viruses were detected at higherrates in infected cells after iNOS inhibition indicating thatNO might play an important role in controlling monocytesDV infection [21] Thus it seems that TNF-120572 and NO wouldbe involved not only in generating severe symptoms [22 23]but also in the elimination of viruses [24ndash26]

TNF-120572 and NO are produced in response to toll-likereceptor 4 (TLR4) stimulation Toll-like receptors (TLRs) areimportant inmicrobial recognition [27] and they are involvedin the generation of antiviral molecules and proinflammatorycytokines which probably exert immunopathological func-tions [27] Although the implications of TLRs functions inviral infections have been investigated [28] the knowledgeabout dengue is restricted de Kruif et al [29] evaluatedTLR gene-expression profiling of childrenwith severe dengueinfectionsThe authors demonstrated mainly that TLR7 genetranscription was upregulated while TLR2 were downreg-ulated indicating the in vivo role of particular TLRs withdifferent disease-severity parameters

TLR4 is recognized as a LPS receptor [30 31] and a previ-ous study showed an interaction among DV LPS and CD14at themembrane of primary humanmonocytesmacrophages[32] The bacterial lipopolysaccharide (LPS) a ligand of theCD14-TLR4 complex was able to block DV and modulatevirally induced cytokine production by human monocytesand macrophages So based on that we asked if there isa regulatory role for the LPS receptor TLR4 on cytokineproduction during the acute phase of human DV infection

DV genome is a single-stranded positive sense RNAwhich codes for 10 gene products including structuralproteins capsid (C) premembrane (prM) envelope (E) andnonstructural proteins NS1 NS2A NS2B NS3 NS4A NS4Band NS5 [33 34] Due to the fact that intensity of DVreplication might influence clinical outcomes it is importantto investigate the impact of some viral proteins on innateimmune parameters of DV infected patients Among theseproteins the NS1 glycoprotein is a singular glycoproteinsince it does not form part of the virion structure but isexpressed on the membrane of infected cells NS1 circulatesat high levels in the sera of patients during the acute phaseof illness is a well-known early diagnostic marker and maybe involved on the pathophysiology of DV infection [35]Preliminary evidence has shown that NS1 is involved in viralRNA replication [35] but an association between NS1 levelsperturbation of innate immune response and severe diseaseis still unknown In addition toll-like receptor regulation onmonocytes can also theoretically be elicited by proteins suchas viral NS1 Thus the aim of this study was to investigate therelationships between in vivo secreted levels ofNS1 and innateimmune response parameters (TLR4 expression and TNF-120572NO production) in infected dengue patients with differentclinical outcomes

2 Methodology

21 Study Group and Case Classification DV infectedpatients (119899 = 37) 17 female and 20 male subjects wereenrolled in this study Twenty healthy individuals 10 femalesand 10 males were included as healthy controls (HCs) AllHCs tested negative for DV NS1 antigen and DV IgMIgGantibodies and had not been vaccinated against yellow fevervirus Dengue patients were enrolled from February 2010 toApril 2013 in UFTM University Hospital and two healthcarecenters located in the city of Uberaba Brazil Dengue caseswere classified as DF or DHF according to the 1997 WorldHealth Organization (WHO) guidelines [36] We applied theold guidelines since the new WHO guidelines published in2009 are more directly focused on clinical practice and arenot broadly used in research [37]

22 Blood Samples Collection andProcessing After 2nd (acutephase) and 9th day (beginning of convalescence phase)from the commencement of symptoms twenty milliliters ofheparinized peripheral venous blood (PB) and five milliliterswithout anticoagulant (serum) were collected from DVinfected patients and noninfected HCs The heparinizedblood collected was used for isolation of peripheral bloodmononuclear cells (PBMCs) and serum was stored at minus80∘Cuntil further use

23 Serological Diagnosis of Dengue Dengue-specific IgMand IgG were detected using a capture ELISA (PanBio)whereas Dengue NS1 was detected using the immunochro-matographic kit (PanbioQueenslandAustralia) according tomanufacturerrsquos instructions







2






2

minusNO3


2













3 Results





0

100

200

300

400

500


BRUtimes103m

L

lowast

lowast

DF DHF DF DHF




4 Discussion



Acute phase

0

10

20

30

40

50Convalescence

0

10

20

30

40

HC DFDHF HCDF DHF

lowast

lowast

lowast

NO

2minusN

O3minus

(120583M

L)

NO

2minusN

O3minus

(120583M

L)


minus

NO3


0

10

20

30

40

50lowast

lowast


NO

2minusN

O3minus

(120583M

L)


minus

NO3




NS

0

100

200

300

400

500

(pg

mL)

lowast

lowast






NS0

100

200

300

400

500

(pg

mL)

LPS

lowast

lowast










5 Conclusions



1000

800

600

400

200

0

100 101 102 103 104

R1

SSC-

H

SSC

-hei

ght

FL1-H CD14 FITC

(a)

0

1000

2000

3000

4000

MFI

lowast

lowast

lowast

HC DF DHF

(b)

0

1000

2000

3000

4000

MFI


lowast

lowast

(c)


Ethical Approval







Acknowledgments



References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















2






2

minusNO3


2













3 Results





0

100

200

300

400

500


BRUtimes103m

L

lowast

lowast

DF DHF DF DHF




4 Discussion



Acute phase

0

10

20

30

40

50Convalescence

0

10

20

30

40

HC DFDHF HCDF DHF

lowast

lowast

lowast

NO

2minusN

O3minus

(120583M

L)

NO

2minusN

O3minus

(120583M

L)


minus

NO3


0

10

20

30

40

50lowast

lowast


NO

2minusN

O3minus

(120583M

L)


minus

NO3




NS

0

100

200

300

400

500

(pg

mL)

lowast

lowast






NS0

100

200

300

400

500

(pg

mL)

LPS

lowast

lowast










5 Conclusions



1000

800

600

400

200

0

100 101 102 103 104

R1

SSC-

H

SSC

-hei

ght

FL1-H CD14 FITC

(a)

0

1000

2000

3000

4000

MFI

lowast

lowast

lowast

HC DF DHF

(b)

0

1000

2000

3000

4000

MFI


lowast

lowast

(c)


Ethical Approval







Acknowledgments



References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results





0

100

200

300

400

500


BRUtimes103m

L

lowast

lowast

DF DHF DF DHF




4 Discussion



Acute phase

0

10

20

30

40

50Convalescence

0

10

20

30

40

HC DFDHF HCDF DHF

lowast

lowast

lowast

NO

2minusN

O3minus

(120583M

L)

NO

2minusN

O3minus

(120583M

L)


minus

NO3


0

10

20

30

40

50lowast

lowast


NO

2minusN

O3minus

(120583M

L)


minus

NO3




NS

0

100

200

300

400

500

(pg

mL)

lowast

lowast






NS0

100

200

300

400

500

(pg

mL)

LPS

lowast

lowast










5 Conclusions



1000

800

600

400

200

0

100 101 102 103 104

R1

SSC-

H

SSC

-hei

ght

FL1-H CD14 FITC

(a)

0

1000

2000

3000

4000

MFI

lowast

lowast

lowast

HC DF DHF

(b)

0

1000

2000

3000

4000

MFI


lowast

lowast

(c)


Ethical Approval







Acknowledgments



References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acute phase

0

10

20

30

40

50Convalescence

0

10

20

30

40

HC DFDHF HCDF DHF

lowast

lowast

lowast

NO

2minusN

O3minus

(120583M

L)

NO

2minusN

O3minus

(120583M

L)


minus

NO3


0

10

20

30

40

50lowast

lowast


NO

2minusN

O3minus

(120583M

L)


minus

NO3




NS

0

100

200

300

400

500

(pg

mL)

lowast

lowast






NS0

100

200

300

400

500

(pg

mL)

LPS

lowast

lowast










5 Conclusions



1000

800

600

400

200

0

100 101 102 103 104

R1

SSC-

H

SSC

-hei

ght

FL1-H CD14 FITC

(a)

0

1000

2000

3000

4000

MFI

lowast

lowast

lowast

HC DF DHF

(b)

0

1000

2000

3000

4000

MFI


lowast

lowast

(c)


Ethical Approval







Acknowledgments



References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NS0

100

200

300

400

500

(pg

mL)

LPS

lowast

lowast










5 Conclusions



1000

800

600

400

200

0

100 101 102 103 104

R1

SSC-

H

SSC

-hei

ght

FL1-H CD14 FITC

(a)

0

1000

2000

3000

4000

MFI

lowast

lowast

lowast

HC DF DHF

(b)

0

1000

2000

3000

4000

MFI


lowast

lowast

(c)


Ethical Approval







Acknowledgments



References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1000

800

600

400

200

0

100 101 102 103 104

R1

SSC-

H

SSC

-hei

ght

FL1-H CD14 FITC

(a)

0

1000

2000

3000

4000

MFI

lowast

lowast

lowast

HC DF DHF

(b)

0

1000

2000

3000

4000

MFI


lowast

lowast

(c)


Ethical Approval







Acknowledgments



References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Elevated Dengue Virus Nonstructural ... · (DF) to the life-threatening syndromes of dengue hemor-rhagic fever (DHF) and dengue shock syndrome (DSS), showing manifestations