Rev. Bras. Hematol. Hemoter. vol.39 número2

w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Original

article

Telomere

length

correlates

with

disease

severity

and

inflammation

in

sickle

cell

disease

Marina

Pereira

Colella

_,

_Barbara

_A.

_Santana

,

Nicola

Conran

,

Vinicius

Tomazini

,

Fernando

F.

Costa

_,

_Rodrigo

_T.

_Calado

,

Sara

T.

Olalla

Saad

a_{UniversidadeEstadualdeCampinas/Hemocentro(UNICAMP),Campinas,SP,Brazil}

b_{UniversidadedeSãoPaulo(USP),RibeirãoPreto,SP,Brazil}

c_{Fundac¸ãodeAmparoàPesquisadoEstadodeSãoPaulo(FAPESP),RibeirãoPreto,SãoPaulo,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17January2017 Accepted15February2017 Availableonline11March2017

Keywords:

Sicklecelldisease Telomerelength Inflammation

a

b

s

t

r

a

c

t

Background:Telomeres,theendsoflinearchromosomes,shortenduringmitoticcelldivision

anderosionmaybeaggravatedbyinflammationorproliferativeandoxidativestress.Asthe bonemarrowisunderhyperproliferativepressureinsicklecelldiseaseandseveraltissues aresubmittedtochronicinflammation,thisstudysoughttodeterminethetelomerelength ofpatientswithsicklecelldisease.

Methods:Themeantelomerelengthwasmeasuredinperipheralbloodleukocytesby

quan-titativepolymerasechainreaction.Theage-adjustedtelomeretosinglecopygeneratiowas comparedbetween91adultsicklecelldiseasepatientsand188controls.

Results:Sicklecelldiseasepatientshadsignificantlyshortertelomeresthanthecontrols

(p-value<0.0001).Moreover,amongsicklecelldiseasegenotypes,HbSSpatientshad sig-nificantlyshorter telomeres compared to Hb SC and Hb S␤ patients (p-value<0.0001). Patientsonhydroxyureaalsohadshortertelomeresincomparisontothoseoffthedrug(p -value=0.02).Apositivecorrelationwasobservedbetweentelomerelengthandhemoglobin level(r=0.3;p-value=0.004),whereasnegativecorrelationsweredetectedbetween telo-merelengthandlymphocytecount(r=−0.3;p-value=0.005)andinterleukin-8serumlevels

(r=−0.4;p-value=0.02).

Conclusions:Thefindingsofthisstudyindicatethattelomeresareshortinsicklecelldisease

patientsandthattelomereerosiondirectlycorrelateswithdiseasegenotype,inflammation markers,andtheuseofhydroxyurea.

©2017Associac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthorat:HematologyandHemotherapyCenter,UniversidadeEstadualdeCampinas(UNICAMP),RuaCarlosChagas,}

480,13083-878Campinas,SP,Brazil.

E-mailaddress:sara@unicamp.br(S.T.Saad).

http://dx.doi.org/10.1016/j.bjhh.2017.02.007

Introduction

Sicklecelldisease(SCD)ischaracterizedbythepresenceof apathologicalhemoglobin,denominatedhemoglobin(Hb)S. TheformationofHbSiscausedbyamutationinthe␤-globin gene atthe 17th nucleotide,in which thymine is changed toadenine, resultinginthesubstitutionofthesixth amino acidofthe␤-globinchainfromglutamicacidtovaline.The termSCDincludesseveralgenotypeswiththepresenceof␤S allele,including thehomozygousform(HbSSor sicklecell anemia)andheterozygousformswithco-inheritanceofother mutationssuchas ␤C allele (Hb SC or Hb SC disease) and the␤-thalassemiaallele(HbS␤orS␤Thalassemia).The clini-calhallmarksofSCDarechronicintravascularhemolysisand acutevaso-occlusiveevents,whichinvolveendothelial dys-function,increasedcellularadhesion,chronicinflammation, leukocytosis, coagulation activation and oxidative stress.1

Hemolysis plays a central role in the disease mechanism due to the constant release of free hemoglobin and free heme from red blood cells (RBC) into plasma, leading to nitric oxide (NO) depletion causing potent immediate sys-temic and vascular inflammation.2 _{NOdepletion may lead}

toahighly adhesiveendothelium,and platelet and coagu-lation activation.3 _{Unbound extracellular heme causes the}

generation of reactive oxygen species (ROS) and activa-tionofinnateimmunitypathwaysthroughToll-likereceptor 4(TLR4).4,5

Telomeres are hexameric T2AG3 tandem repeats coated by specialized proteins covering the ends of lin-ear chromosomes.6 _{Telomeres confer protection against}

chromosomeinstabilityand activation ofthe DNA-damage response(DDR)machinery.Telomereshorteningisa chrono-logicalmarkerofaging, andits acceleratedshorteningrate hasbeeninvolvedinthedevelopmentofseveraldiseases,the telomeropathies.7 _{In addition, cumulative events resulting}

from replicative stress, such as exposure to biochemical stressors,excessiveoxidation,andinflammation,maycause excessive telomere erosion.8,9 _{Maintenance of telomere}

integrityrequirestelomerasereversetranscriptase(TERT),its RNAtemplate(TERC),andotherproteins,whichcomposethe telomerasecomplex.

Inviewoftheinflammatoryandoxidativestressfeatures observedinSCD,thetelomerelengthwasdeterminedinSCD patientsandhealthycontrolsinordertocorrelatethiswith diseasegenotype,severity,andinflammationmarkers.

Methods

Patientsandcontrols

ThisstudywasperformedinacohortofadultSCDpatients seen at the Outpatient Clinic of the Hematology and Hemotherapy Center, Universidade Estadual de Campinas (UNICAMP).PatientsthatwerehomozygousforHbS(HbSS), heterozygous with Hb C and S (Hb SC), and heterozygous with␤-thalassemiaand S(HbS␤)were included.All ofthe patientswereinsteadystatethereforenonehadhadpainful crises,hospitalizationsorbloodtransfusionsduringthethree

months precedingblood sample collection. Agroup of188 age-matchedhealthysubjects(HbAA)wereanalyzedas con-trols for telomere lengthmeasurement.10 _{Anadditional 70}

age-andgender-matchedhealthysubjects(HbAA)were eval-uated forlymphocytecountsandinterleukin-8(IL-8)levels. Venousbloodsamplesforallstudyanalyses,including periph-eralbloodcountsandhemolysismarkers,wereobtained dur-ingclinicvisits.TheUniversity’sEthicsCommitteeapproved thestudyandallpatientsgavetheirwritteninformedconsent.

DNAextraction

GenomicDNAwasextractedfromthebuffycoatofhealthy controls’peripheralbloodleukocytesupto48hafter collec-tion, usingthe GentraPurege Blood Kit(Qiagen, Maryland, USA).DNAsampleswerequantified,dilutedto50ng/␮Land storedat−20◦C.GenomicDNAfromSCDpatientswas

iso-lated from frozen white blood cells (WBC). Briefly, whole peripheralbloodsampleswerewashedinphosphatebuffered saline(PBS)with0.1%bovineserumalbumin(BSA)upto16h after collection and incubated on ice coldammonia chlo-ride (NH4Cl) for osmotic red blood cell lyses. WBCs were

counted,aliquoted,andfrozenat−80◦_{Cin10%dimethyl}

sulf-oxide(DMSO)untildefrostingandDNAextraction,performed usingtheGentraPuregeBloodKit(Qiagen,Maryland,USA). Genomic DNA (50ng/␮L) was checked forintegrity in0.8% agarose gelat80Vfor40min. For quantitativepolymerase chainreaction(qPCR),DNAdilutionsof5ng/␮Lwereprepared andkeptat4◦_{Cuptosevendays,whenthesubsequent}

dilu-tionsof0.2ng/␮Lwereusedforeveryrunpreparedjustbefore experiments.

Leukocytetelomerelengthmeasurementbyquantitative polymerasechainreaction

The mean leukocyte telomere length was determined by qPCR as hasbeen described previously.11–13 _{All qPCR}

reac-tions were prepared on a QIAgility automated pipettor (Qiagen, California, USA), amplification was conducted in triplicate inthe Rotor-Gene Q5plex HRMInstrument (Qia-gen), and analysis was completed using Rotor-Gene Q Software Version 2.2.3. The 24␮L final volume reactions included: 8␮L of genomic DNA (0.2ng/␮L), 2× Rotor-Gene

SYBR Green PCR kit (Qiagen, Hilden, Germany), RNase-free water (Qiagen), and the primers Telomere Fw (CGGT-TTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT – 300nM) andRv(GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT –300nM)orsinglegene(36B4)Fw (CAGCAAGTGGGAAGGTG-TAATCC–300nM)andRv(CCCATTCTATCATCAACGGGTACAA – 500nM). Telomere reactions were performed as follows: denaturationat95◦_{Cfor5minfollowedby25cyclesof7sat}

98◦_{Cand 10s at60}◦_{C,whereas singlegenereactionswere}

denaturedat95◦_{Cfor5minfollowedby35 cycles of7sat}

98◦_Cand10sat58◦_{C.Thetelomerelengthforeachsample}

wasdeterminedusingthetelomeretosinglecopygeneratio (T/Sratio)withthecalculationofCt[Ct(telomere)/_Ct(singlegene)_].

TheT/Sratioforeachsample(x)wasnormalizedtothemean T/Sratioofthereferencesample[2−(Ctx−Ctr₎₌₂−Ct_],which

Table1–Clinicalcharacteristicsofsicklecelldiseasepatients.

HbSS HbSC/HbS␤+

Numberofpatients–n 51 40

Meanage–years(range) 37(18–58) 41(19–66)

Gender:male/female–n(%) 18/33(35/65) 14/26(35/65)

␣-Thalassemiatrait*_{–n(%) 11(23) 11(32)}

Useofhydroxyurea–n(%) 37(73) 3(8)

Meanage-adjustedT/S–ratio(range) −0.34(−0.61to0.21) −0.21(−0.51to0.68)

T/S:telomeretosinglecopygeneratio. ∗ _{␣-Thalassemiatrait:−3.7kbdeletion(−␣3.7).}

Theresultswereavailablefor47patientsintheHbSSgroupand34patientsintheHbSC/HbS␤group.

variations(CV)oftriplicatemeasurementswere2%and1%or less,fortelomereandsinglegenereactions,respectively.The consideredinter-assayCVwasupto5%.

LeukocytetelomerelengthbySouthernblotanalysis

To validate the qPCR terminal restriction fragment (TRF), analysiswasperformedbySouthernblotof17samples accord-ing tothe manufacturer’sinstructions withminorchanges (TeloTAGGGTelomereLengthAssay–RocheAppliedScience, Mannheim,Germany). Briefly, 800ngof genomic DNA was digestedbyanoptimized mixtureofHinfI andRsaI FastDi-gestrestriction enzymes (ThermoScientific,Waltham, MA, USA)at37◦_{Cfor2h.FollowingDNAdigestion,DNAfragments}

were separated byelectrophoresisin 0.8%agarosegel dur-ing four hoursat80V. Gel wasdenaturedand neutralized, samplesweretransferredtoanylonmembranebySouthern blottingandprobed,andthe terminalrestrictionfragments weredetectedbychemiluminescence.MeanTRFlengthwas determinedaccordingtotheformulaTRF=(ODi)/(ODi/Li), whereODiisthechemiluminescentsignalandLiisthelength ofthefragmentatagivenposition.

Inflammationmarkers

Tumornecrosisfactor-alpha(TNF-␣)andIL-8wereassessedas pro-inflammatorymarkers.TNF-␣andIL-8levelswere mea-sured,in duplicate,inserum samplesusing ultra-sensitive enzyme-linkedimmunosorbentassay(ELISA)kitsaccording tothemanufacturer’sinstructions(TNF-␣USandIL-8US, Invi-trogen,Camarillo,CA,USA).

Statisticalanalyses

The relationship between telomere length and age was estimated using linear regression. Estimated regression coefficients were used to calculate the observed minus expected(O-E),orage-adjustedtelomerelengthforeach sub-ject. Age-adjusted T/S ratios were used for all the study analysesexceptforthecorrelationwithage.Spearman’srank correlationcoefficientwasusedtoanalyzebivariate associa-tionsbetweentelomerelengths,hemolysisandinflammation markers.T/Sratioswerecomparedaccordingtothediagnosis, useofhydroxyureaandgenderusingWilcoxonranksumtest. Linearregressionwasusedtoanalyzethecorrelationbetween

telomerelengthmeasuredbyqPCRandTRFbySouthernblot. Allp-values≤0.5wereconsideredsignificant.Statistical

anal-yses wereperformedusingtheRstatisticsprogramversion 3.1.3.

Results

Patients’characteristics

Ninety-one adultSCDpatients were includedin thestudy: 51 Hb SS, 38 Hb SC,and 2 Hb S␤+. Forty were on hydrox-yureawithamediandoseof1g/day(range:500–1750mg/day). The dose was titrated according to clinical improvement or to the maximum tolerated dose. The clinical indica-tions for hydroxyurea were frequent painful crises (n=14; 35%), acute chest syndrome (n=5;13%), stroke (n=6; 16%), and severehemolyticanemia(n=15;37%).Patients’ clinical characteristics are described in Table 1. Thecontrol group consisted of188healthy individuals (Hb AA),witha mean ageof38years(range:18–88years),including97womenand 91men.

Telomeresareshortinsicklecelldiseasepatients

Telomeres were significantly shorterin SCD patients com-pared toage-matched healthy controls[T/S ratio: −0.28 in

SCD vs.−0.01incontrols;standard deviation(SD)=0.20 vs.

0.23; p-value<0.0001 – Figure 1A]. When genotypes were compared, the telomeres were significantly shorter in Hb SS patients than in the Hb SC and Hb S␤ genotypes (T/S ratio: −0.34 vs. −0.21, respectively; SD=0.17 vs. 0.21; p

-value<0.0001 – Figure 1B). Telomereswere also shorterin patientsonhydroxyurea(T/Sratio:−0.34vs.−0.25;SD=0.20

vs.0.20;p-value=0.02)(Figure1C).Althoughtelomeres short-enedwithageinhealthycontrols(r=−0.5;_p-value<0.0001),

age did not affect telomere length in SCD patients (r=−0.02;p-value=0.8).Telomerelengthwasnotinfluenced

by genderin either SCD patients (p-value=0.2) or controls (p-value=0.9).

Inordertovalidatethesefindings,telomerelengthswere also measured by Southern blot for 17 patients showing thatT/SratiosandTRFswerehighlycorrelated(R2_=0.86;p

A

B

C

0.5

0.0

–0.5

–1.0

1.0

0.5

0.0

–0.5

–1.0

1.0

0.5

0.0

–0.5

–1.0 Controls

Age-adjusted T/S

ratio

Age-adjusted T/S

ratio

Age-adjusted T/S

ratio

SCD HbSS HbSC/HbSβ HbSS/HbSC HU HbSS/HbSC/HbSβ

P<0.0001

P<0.0001

P<0.02

Figure1–Age-adjustedT/Sratioincontrolsandsicklecelldisease(SCD)patients.(A)SCDpatientspresentedshortened telomerelengthcomparedtonormalcontrols.(B)HbSSpatientspresentedshortenedtelomerelengthcomparedtoHbSC andHbS␤patients.(C)HbSSandHbSCpatientstreatedwithhydroxyurea(HbSS/HbSCHU)presentedshortenedtelomere lengthswhencomparedtopatientsnottreatedwithhydroxyurea(HbSS/HbSC/HbS␤+).T/Sratioswerecompared

accordingtothediagnosisanduseofhydroxyureausingWilcoxonranksumtest.Meantelomerelengthwasmeasuredin peripheralbloodleukocytesbyquantitativepolymerasechainreaction(qPCR).

Telomerelengthcorrelateswithhemolysisand inflammationinsicklecelldisease

Theassociationbetweentelomerelengthandmarkerlevels for hemolysis (hemoglobin, hematocrit, lactate dehydroge-nase,indirectbilirubin,reticulocytecounts)andinflammation (IL-8, TNF-␣, total leukocyte, neutrophil, lymphocyte and monocytecounts)wereanalyzed(Table2).Therewasaweak positivecorrelationbetweentelomerelengthandhemoglobin concentration(r=0.3;p-value=0.004),butnocorrelationwith other hemolysis markers. Among inflammatory markers, there was a weak negative correlation of telomere length withlymphocytecounts(r=−0.3;p-value=0.005)andwith

IL-8serumlevels(r=−0.4;p-value=0.02;Table2).Ofnote,overall,

SCDpatientshadhigherlymphocytecounts(SCD:2.9×109/L

15.0

12.5

10.0

7.5

5.0

2.5

0.0

0.0 0.2 0.4 0.6

T/S ratio (qPCR)

TRF (Kb)

0.8 1.0

Figure2–Correlationbetweentelomerelengthmeasured byquantitativepolymerasechainreaction(qPCR)and terminalrestrictionfragment(TRF)bySouthernBlot. Analysisof17sampleswasperformedtovalidateqPCR TRF.TelomerelengthsmeasuredbyqPCRpresentedan adequatecorrelationwithmeasurementsofTRFby Southernblot,analyzedbylinearregression(R2=0.86;

p-value<0.0001).

vs.controls:1.9×109/L;SD=1.4vs.0.51;p-value<0.0001)and

IL-8levelscomparedtocontrols(SCD:3.3pg/mLvs.controls: 2.3pg/mL;SD=1.9vs. 0.8; p-value=0.009). Telomerelength wasnotassociatedwiththeplasmalevels ofother inflam-matorymarkers.

Discussion

Inthepresentstudy,wefoundthattelomeresofperipheral bloodleukocytesofpatientswithSCDareshortindependent ofageandtelomereattritionwasmorepronouncedinpatients withHb SScomparedtoHb SCorHb S␤+ genotypes. Telo-mere lengthcorrelated withhemoglobinconcentrationand inverselycorrelatedwithIL-8levelandabsolutelymphocyte count.Takentogether,thesefindingssuggest thattelomere lengthisassociatedwithdiseaseseverityandchronic inflam-mationinSCD.

Ourresultsareinsharpcontrastwithonesingleprevious reportbyDrasaret al.,whofoundSCDpatientshad longer telomeres in comparison to age-matched controls.14

How-ever,theirstudypresentedsometechnicalissuesprecluding appropriate interpretation oftheir findings. First, it is not clearintheirwork whethertheyusedfreshorfrozen sam-plesforDNAextraction.Second,telomerelengthwashighly heterogeneousamongtheirpatients,andasignificant propor-tionofolderpatientshadtelomereslongerthanexpectedfor healthycordbloods.Finally,theirqPCRresultswerenot vali-datedwithasecondmethod.Takentogether,theseconcerns mightsuggestthattheDNAusedforanalysiscouldhavebeen degraded. During qPCR,DNAdegradation causes T/S ratios tobehigherandconsequentlytelomeres tobeerroneously interpretedaslonger.10_{Thiseffectisexplainedbythe}

Table2–Correlationsoftelomerelengthswithhemolysisandinflammationmarkersinsicklecelldiseasepatients.

Mean;median(range) Correlationwithtelomerelength

(T/Sratio)r(p-value)a

Hemoglobin(g/dL) 9.8;9.6(6.0–16.0) 0.3(0.004)

Hematocrit(%) 29.2;29.1(16.7–48.4) 0.2(0.2)

Lactatedehydrogenase(U/L) 716;615(257–1680) −0.2(0.08)

Indirectbilirubin(mg/dL) 2.2;1.3(0.6–9.0) −0.2(0.3)

Absolutereticulocytecount(×109_{/L) 274;261(80–667) −0.1(0.3)}

Absoluteleukocytecount(×109_{/L) 8.7;8.6(3.6–16.5) −0.1(0.3)}

Absoluteneutrophilcount(×109_{/L) 4.5;4.6(1.5–9.5) 0.04(0.7)}

Absolutemonocytecount(×109_{/L) 0.5;0.4(0.1–1.4) −0.03(0.7)}

Absolutelymphocytecount(×109_{/L) 3.1;2.9(0.7–7.3) −0.3(0.005)}

Absoluteplateletcount(×109_{/L) 406;407(81–1164) −0.2(0.1)}

TNF-␣(pg/mL) 2.6;2.6(0–6.9) 0.07(0.7)

IL-8(pg/mL) 3.5;3.3(0.8–11.6) −0.4(0.02)

IL-8:interleukin8;TNF-␣:tumornecrosisfactor-alpha.

a _{Spearman’srankcorrelationcoefficient(r)wasusedtoanalyzebivariateassociationsandallp-valuesaretwo-tailed.p-Values<0.05are}

consideredsignificant.

andSouthernblotting.Toavoidthisproblem,allsamplesin ourstudywerecollectedandprocessedwithin16hfromblood drawingforthepurposeoftelomerelengthmeasurementover athree-monthperiodunderrigorousqualitycontrol.To vali-date the qPCR findings ofthis study,telomere lengthwas alsoperformedbySouthernblottingfor17patients,withhigh correlationbetweenbothmethods(R2_{=0.86).Analyzingthe}

distributionofourpatientssimultaneously withonesfrom theEnglishstudy,the agesofourpatientsweredistributed morebetween20and60 yearsold whereasthe patientsof theEnglishstudyweremainlyconcentratedbetween20and 40yearsold.Infact,themedianageofoursampleisalmost ten years higher than the English sample (median age of Campinas vs. England – Hb SS: 39 vs. 32 years; Hb SC/Hb S␤+: 43 vs. 34 years). Thus,we believe thatdivergences in sample number, disease severity, co-morbidities, socioeco-nomicstatus,ethnicityand agedistributionofthepatients and possibly DNA collection might be responsible for the conflictingresultsandencouragefurtherstudiestoclarifythis issue.

Theinvolvementofwhitebloodcellsintheseverityof clin-icalmanifestationsofSCDpatientsiswellknown.15–18_Indeed,

thenumberofbloodcellsincreasesinSCDformanyreasons, includinghighlevelsofcirculatinggranulocytemacrophage colony-stimulatingfactorandincreasedcellsurvival.19–22

Thisstudyalsoobservedthattelomeres wereshorterin patients using hydroxyurea. Hydroxyurea has a cytostatic effectandiscapableofreducingthenumberofhighturnover cellssuchasplatelets,neutrophils,andreticulocytes. Hydrox-yurea may have modulatedblood counts, producing more prominentlymphocytecounts.Inaddition,mostpatientswith moreseverediseasewereonhydroxyurea,whichmayexplain theassociation.

TelomerelengthdidnotcorrelatewithageinSCDpatients. Asinflammationischronicandstartsatayoungage,itmay provokeexcessive telomere shortening early inlife, induc-ingearly“aging”ofthehematopoietictissueinSCDpatients. Thisfindingmaycontributetoearlymultipleorganfailurein SCDandmayexplaintheshorterlifeexpectancyrelatedto

the disease.Thishypothesis shouldbeaddressedinfuture studies.

Our study has some limitations. More significantly, we recruitedasmallnumberofpatientswiththeHbS␤+ geno-type.Inaddition,wedidnotprospectivelyevaluatetheeffects oftelomerelengthondiseasecomplicationevents,suchas acutechestsyndromeandstroke.Finally,hydroxyureamay haveinterferedwithtelomerelengthbymodulatingleukocyte subsets.

In conclusion, our data confirm that telomere lengths aregreatlyinfluencedbyinflammationandreactiveoxygen speciesinSCD,awell-knownphenomenaofthisdisorder.

Authorship

Contribution:M.P.C.participatedintheselectionofpatients, clinicalfollowupofpatients,performedexperimentalwork, analyzedtheresultsandwrotethemanuscript;B.A.S. partic-ipatedintheselectionofcontrols,performedexperimental workandwrotethemanuscript;N.C.andF.F.C.participated intheanalysisoftheresultsandwritingofthemanuscript; R.T.C.conceivedthestudy,designedexperiments,analyzed resultsandwrotethemanuscript;S.T.O.S.conceivedthestudy, participatedintheselectionofpatients,clinicalfollowupof patients,analyzedtheresultsandwrotethemanuscript.All authorsapprovedthefinalversionofthemanuscript.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

r

e

f

e

r

e

n

c

e

s

1. ColellaMP,DePaulaEV,ConranN,Machado-NetoJA, Annicchino-BizzacchiJM,CostaFF,etal.Hydroxyureais associatedwithreductionsinhypercoagulabilitymarkersin sicklecellanemia.JThrombHaemost.2012;10(9):1967–70.

2. AlmeidaCB,SouzaLE,LeonardoFC,CostaFT,WerneckCC, CovasDT,etal.Acutehemolyticvascularinflammatory processesarepreventedbynitricoxidereplacementora singledoseofhydroxyurea.Blood.2015;126(6):711–20.

3. ConranN,Franco-PenteadoCF,CostaFF.Neweraspectsofthe pathophysiologyofsicklecelldiseasevaso-occlusion. Hemoglobin.2009;33(1):1–16.

4. DutraFF,BozzaMT.Hemeoninnateimmunityand inflammation.FrontPharmacol.2014;5:115.

5. FigueiredoRT,FernandezPL,Mourao-SaDS,PortoBN,Dutra FF,AlvesLS,etal.Characterizationofhemeasactivatorof Toll-likereceptor4.JBiolChem.2007;282(28):20221–9.

6. BlackburnEH.Switchingandsignalingatthetelomere.Cell. 2001;106(6):661–73.

7. CaladoRT,YoungNS.Telomerediseases.NEnglJMed. 2009;361(24):2353–65.

8. MasiS,SalpeaKD,LiK,ParkarM,NibaliL,DonosN,etal. Oxidativestress,chronicinflammation,andtelomerelength inpatientswithperiodontitis.FreeRadicBiolMed.

2011;50(6):730–5.

9. WolkowitzOM,MellonSH,EpelES,LinJ,DhabharFS,SuY, etal.Leukocytetelomerelengthinmajordepression: correlationswithchronicity,inflammationandoxidative stress–preliminaryfindings.KiechlS,editor.PLoSOne. 2011;6(3):e17837.

10.Gutierrez-RodriguesF,Santana-LemosBA,ScheucherPS, Alves-PaivaRM,CaladoRT.Directcomparisonofflow-FISH andqPCRasdiagnostictestsfortelomerelength

measurementinhumans.PLoSOne.2014;9(11):e113747.

11.CawthonRM.TelomeremeasurementbyquantitativePCR. NucleicAcidsRes.2002;30(10):e47.

12.BrouiletteSW,MooreJS,McMahonAD,ThompsonJR,FordI, ShepherdJ,etal.Telomerelength,riskofcoronaryheart

disease,andstatintreatmentintheWestofScotlandPrimary PreventionStudy:anestedcase–controlstudy.Lancet. 2007;369(9556):107–14.

13.CaladoRT,CooperJN,Padilla-NashHM,SloandEM,WuCO, ScheinbergP,etal.Shorttelomeresresultinchromosomal instabilityinhematopoieticcellsandprecedemalignant evolutioninhumanaplasticanemia.Leukemia. 2012;26(4):700–7.

14.DraˇsarER,JiangJ,GardnerK,HowardJ,VulliamyT,VasavdaN, etal.Leucocytetelomerelengthinpatientswithsicklecell disease.BrJHaematol.2014;165(5):725–7.

15.OkpalaI.Theintriguingcontributionofwhitebloodcellsto sicklecelldisease–aredcelldisorder.BloodRev.

2004;18(1):65–73.

16.LitosM,SarrisI,BewleyS,SeedP,OkpalaI,Oteng-NtimE. Whitebloodcellcountasapredictoroftheseverityofsickle celldiseaseduringpregnancy.EurJObstetGynecolReprod Biol.2007;133(2):169–72.

17.KinneyTR,SleeperLA,WangWC,ZimmermanRA,Pegelow CH,Ohene-FrempongK,etal.Silentcerebralinfarctsinsickle cellanemia:ariskfactoranalysis.TheCooperativeStudyof SickleCellDisease.Pediatrics.1999;103(3):640–5.

18.PlattOS,BrambillaDJ,RosseWF,MilnerPF,CastroO, SteinbergMH,etal.Mortalityinsicklecelldisease.Life expectancyandriskfactorsforearlydeath.NEnglJMed. 1994;330(23):1639–44.

19.OpdenakkerG,FibbeWE,VanDammeJ.Themolecularbasis ofleukocytosis.ImmunolToday.1998;19(4):182–9.

20.ConranN,SaadST,CostaFF,IkutaT.Leukocytenumbers correlatewithplasmalevelsofgranulocyte-macrophage colony-stimulatingfactorinsicklecelldisease.AnnHematol. 2007;86(4):255–61.

21.AlmeidaCB,FaveroME,Pereira-CunhaFG,Lorand-MetzeI, SaadST,CostaFF,etal.Alterationsincellmaturityandserum survivalfactorsmaymodulateneutrophilnumbersinsickle celldisease.ExpBiolMed(Maywood).2011;236(11):1239–46.