The
Brazilian
Journal
of
INFECTIOUS
DISEASES
w w w . e l s e v i e r . c o m / l o c a t e / b j i d
Original
article
Therapy
with
radio-attenuated
vaccine
in
experimental
murine
visceral
leishmaniasis
showed
enhanced
T
cell
and
inducible
nitric
oxide
synthase
levels,
suppressed
tumor
growth
factor-beta
production
with
higher
expression
of
some
signaling
molecules
Sanchita
Datta
a,
Syamal
Roy
b,
Madhumita
Manna
a,∗ aPostGraduateDepartmentofZoology,BarasatGovernmentCollege,Kolkata,India bInfectiousDiseasesandImmunology,IndianInstituteofChemicalBiology,Kolkata,Indiaa
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received7May2014
Accepted6October2014
Availableonline19December2014
Keywords: Radio-attenuatedLeishmania parasites PDK1 PI3K p38MAPK
a
b
s
t
r
a
c
t
Background:Visceralleishmaniasis(VL)orKala-Azar(KA)isoneofthemostdeadlyforms
ofdiseaseamongallneglectedtropicaldiseases.Therearenosatisfactorydrugsor
vac-cinecandidatesavailableforthisdreadeddisease.Ourpreviousstudiesshowedpromising
therapeuticandprophylacticefficacyofthelive,radio-attenuatedparasitesthrough
intra-muscular(I.M.)andintraperitoneal(I.P.)routeinBALB/cmicemodel.
Methods:TheT-cellproliferationlevel,themRNAexpressionlevelofinduciblenitricoxide
synthase(iNOS)andtumorgrowthfactor-beta(TGF-)genesandfinallythephosphorylation
levelsofphosphoinositidedependentkinase1(PDK1),phosphoinositide3kinase(PI3K)and
p38mitogenactivatedproteinkinase(p38MAPK)moleculeswerecheckedinBALB/cmice
modelimmunizedwithradio-attenuatedLeishmaniadonovaniparasitesthroughI.M.route.
Results:HigherT-cellproliferation,increasediNOSlevel,andsuppressedTGF-levelwere
foundintreatedinfectedanimalgroups(100and150Gy)inrelationtountreatedinfected
animals.Likewise,phosphorylationlevelsofPDK1,PI3Kandp38MAPKofthesetwogroups
wereincreasedwhencomparedtountreatedinfectedcontrols.
Conclusion:Theclearanceofthe parasitesfromtreated infectedgroupsofanimalsmay
bemediatedbytherestorationofT-cellduetotherapywithradio-attenuatedL.donovani
parasites.Thekillingofparasiteswasmediatedbyincreaseinnitricoxidereleasethrough
PDK1,PI3Kandp38MAPKsignalingpathways.AlowerTGF-expressionhasaugmented
therestoredTh1ambienceinthe100and150Gytreatedanimalgroupsprovingfurtherthe
efficacyofthecandidatevaccine.
©2015PublishedbyElsevierEditoraLtda.
∗ Correspondingauthorat:PostGraduateDepartmentofZoology,BarasatGovernmentCollege,10K.N.C.Road,Kolkata700124,India.
E-mailaddress:madhumita.manna09@gmail.com(M.Manna).
http://dx.doi.org/10.1016/j.bjid.2014.10.009
Introduction
Visceralleishmaniasis(VL)orKala-Azar(KA)hasbeenranked
second to malaria in terms of mortality and fourth for
morbidity.1 Globalestimate is50,000newcasesofVL each
yearand90%ofVLexistinBangladesh,Brazil,India,Nepal
andSudan.2,3TheinabilityofKApatientstomountan
anti-leishmanialcell-mediatedimmuneresponseisthehallmark
ofthedisease.4Thetargetpathogensarerapidlydeveloping
resistanceagainsttheavailabledrugsincludingpentavalent
antimonials.5Absenceofanylicensedvaccineforcombating
thediseaseisanotherseriousprobleminthecontrol
man-agementprogramforVLworldwide.Ontheotherhand,the
observationsthat peoplecuredfrom VL developprotection
mediatedbyThelper1(Th1)typecellularresponsesagainst
newinfections6giveimpetusforthesearchofeffective
vac-cinecandidates.InvadingLeishmaniaparasitesareeliminated
uponactivationofmacrophagesbyinterferon-gamma(IFN-␥)
secretedbyTh1cellsandnaturalkiller(NK)cellsthatempower
themtodestroytheintracellularLeishmania amastigotesby
reactive nitric oxide(NO) mediatedpathway.7 Inhibition of
nitricoxideproductionfrominduciblenitricoxidesynthase
(iNOS)bytheparasitesmakesmacrophagesinactivetofight
againsttheinfection.8Anyvaccineforleishmaniasisshould
triggertheTh1pathwayand suppressthe Thelper2(Th2)
cytokinesthatexacerbatethedisease.Sofar,manyresearch
groups have undertaken serious attempts in developing a
successfulvaccineagainstthediseasewhichincludeuseof
liveparasites,9 killedparasites,10attenuatedorganisms,11–13
recombinant protein vaccine,14 DNA vaccines,15,16 etc. Use
of live non-attenuated parasites as vaccine candidate has
notbeen recommended by WHO foramatter ofsafety as
reversion of virulent forms would result in adverse side
effectsincludingimmunosuppression.17Leishmaniaparasites
withoutvirulencefactor,yetmaintaining theimmunogenic
attributes,isofgreatinterestasattenuatedparasitesclosely
mimicthenaturalinfectionthatmayleadtosimilarimmune
responses without the fear of infection with live virulent
parasites.18 Ionizingradiationhaslong beenusedto
atten-uateparasitesforthe purposeofvaccinedevelopment.19–24
Irradiation oftencauses lossor reductionof virulenceand
reproductive ability of the pathogens but retains their
metabolicactivities,morphologyandantigenicprofile.Thus,
radio-attenuatedparasitesareabletotriggerspecificimmune
responseswithoutposingriskofprogressiveinfection.25More
interestingly,insomecases,theradio-attenuatedpathogens
are more immunogenic than the normal counterparts.26
Use ofradio-attenuated Leishmania parasites is in practice
forquite a long timenow.22,27,28 Previously our group had
checkedtheprophylacticandtherapeuticefficacyofthe
irra-diatedLeishmaniadonovaniparasitesinexperimentalmurine
model and got encouraging results.29–31 The present study
isanextensionofearlierworkstoseewhetherthereisany
restorationfrom Tcell anergy,inductionofinducible iNOS
andsuppressionofTh2armofimmuneresponsebystudying
Tcellproliferation,iNOSgene, andTGF-geneexpression,
respectively.It isalsoaninitialattemptto understandthe
underlyingmechanismofparasiteclearancebymacrophages
followingtheexpressionsofsomesignaling moleculeslike
phosphoinositide 3 kinase (PI3K),
phosphoinositide-dependent kinase 1 (PDK1) and p38 mitogen-activated
proteinkinase(p38MAPK),respectively,inthegroupsof
ani-malsreceivingtherapywithattenuatedhomologousvaccine
throughI.M.route.
Materials
and
methods
Reagents
HEPES, penicillin, streptomycin, sodium bicarbonate,
2-mercaptoethanol (2-ME), bovine serum albumin (BSA),
histopaque, fetal calf serum (FCS), RPMI 1640 medium, M
199mediumwerepurchasedfromSigma–Aldrich.Anti-PI3K
andanti-phospho-PI3K(p85)wereobtainedfromSantaCruz
Biotech, Inc. All other antibodies
[anti-phosphoinositide-dependent kinase 1 (PDK1) and anti-phospho-PDK1,
anti-p38 mitogen-activated protein kinase (p38MAPK) and
anti-phospho-P38MAPK] and LumiGlo reagents for
chemi-luminescence were obtained from Cell Signaling (Beverly,
MA). Tritiated thymidine [3H]-thymidine (6.7Ci/mmol) was
obtained from PerkinElmer®, deoxynucleoside
triphos-phatesobtainedfromPromega,TaqpolymeraseandMoloney
leukemia virus reverse transcriptase were purchased from
InvitrogenandRNeasyminikitwaspurchasedfromQiagen.
Animals,parasitesandinfection
BALB/cmice4–6weeksoldwithalmostequalweightandsame
sex,rearedinInstitutefacilities,wereusedforexperimental
purposeswithpriorapprovaloftheAnimalEthics
Commit-teeoftheIndianInstituteofChemicalBiology,Kolkata and
allanimalexperimentationswereperformedatIndian
Insti-tuteofChemicalBiology,Kolkata,IndiafollowingtheNational
RegulatoryGuidelinesissuedbyCommitteeforthePurposeof
ControlandSupervisionofExperimentsonAnimals(CPCSEA),
MinistryofEnvironmentand Forest,Govt.ofIndia.L.
dono-vaniisolate,AG83(MHOM/IN/1983/AG83),originallyobtained
fromconfirmedanIndianKala-Azarpatientwasusedforthe
preparationofthelive-attenuatedvaccinecandidate.This
iso-lateismaintainedinGoldenhamstersandthepromastigotes
obtained aftertransforming the amastigotes from infected
animalspleenwereculturedinmediumM199supplemented
with10% FCSalong with100U/mlpenicillin and100g/ml
streptomycin,maintainedat22◦C.32
Therapywithattenuatedparasites
L.donovaniisolate,AG83,was attenuatedbygamma
irradi-ation froma60Co gammachamberwitharateofradiation
of10Gy/60satIndianInstituteofChemicalBiology,Kolkata,
India. AG83 parasites (5×106/BALB/c mice) were irradiated
and washed three times insterile phosphate buffer saline
(PBS)andirradiatedinthegammachamberatthree
differ-ent doses:50,100, and 150Gyrespectively. Thedescription
oftheanimalgroupsforthestudywasasfollows:Group1,
normalcontrolanimalswithoutanyinfectionand
therapeu-ticimmunization;Group2,infectedcontrolanimalswithout
animals that had been infected and received therapeutic
immunizationswithL.donovaniisolate,AG83 (5×106
para-sites/animal)attenuatedat50,100and150Gydosesofgamma
radiation,respectively,at75 dayspost-infection.The
treat-ment was repeated for three immunizedgroups at15-day
interval.Themiceweresacrificed15daysafterthelast
treat-ment,forexperimentalpurposes.
T-cellproliferationassay
T-cell proliferation assay was performed as described
previously.33 Briefly, singlecell suspensions ofsplenocytes
fromdifferentexperimentalgroupsofBALB/cmicewere
pre-paredafter Ficoll density gradient centrifugationand then
suspendedincompleteRPMI1640.Cellswereplatedin
trip-licate at105 cell/well concentrations in96-well plates and
allowed to proliferate for 72h at 37◦C in 5% CO2
incuba-torinthepresenceofSLAconcentration(5g/ml).Eighteen
hours beforethey were harvested in GFC membrane, cells
werepulsedwith1Ci[3H]-thymidine/well.[3H]-Thymidine
incorporationwas measured byliquid scintillation counter
(Tri-Carb2100TR;PackardInstrument).
RNAisolationandsemi-quantitativeRT-PCRanalysesof TGF-ˇandiNOSgenes
RNAisolationfromthesplenocyteswasperformedusingthe
RNeasy minikit procedure (Qiagen).15 Forward and reverse
primerswereusedtoamplifythetranscripts.Samples(1g)
ofRNAfromdifferentexperimentalgroupsofmicewerefirst
utilizedforcDNAsynthesiswithreverseprimers(IDT;Sigma)
usingMouseMoloneyLeukemiaVirusreversetranscriptase
(Invitrogen)at37◦C for90min.A commonmaster mixture
containingdeoxynucleosidetriphosphates(Promega)andTaq
polymerase(Invitrogen),aswellasgenespecificprimersand
0.25 volume of cDNA, was used foramplification with an
AppliedBiosystemsthermocycler.Thecyclingconditionsfor
genesofinterestwere5minat95◦C,followedby40cyclesof
denaturationat95◦Cfor30s,annealingat56◦Cfor40s,and
extensionat72◦Cfor40s.TheidentitiesofthePCRamplified
geneproductswere verifiedbyagarose gelelectrophoresis.
Identicalaliquotswereprocessedinparallelwithoutreverse
transcriptasetoruleoutthepresenceofresidualgenomicDNA
contamination in PCR amplification preparations.
Densito-metricanalysesweredoneusingtheQuantityOnesoftware
(Bio-Rad),ethidiumbromidestaining,andvisualizationunder
a UV transilluminator. For densitometric calculations, the
sameband areawasusedtodetermineband intensityand
normalizedforHGPRT.
Westernblotanalysis
Splenocytes from different groups of animals were
har-vested,resuspendedinchilledlysisbuffer(containing20mM
Tris–HCl (pH 7.5),0.5mM EGTA, 1mM EDTA, 0.1%(vol/vol)
2-mercaptoethanol)34 and rapidly freeze-thawedthrice and
passedthrougha 26-gaugeneedle (10times) forlysis.The
lysates were centrifuged (800×g for 10min at 4◦C), the
supernatants were collected, and the proteins were
esti-matedusingBradfordreagent.Thesupernatantsweremixed
with Laemmli buffer, heated in a boiling water bath for
five min and cooled to room temperature. About 20g
of protein was loaded in lanes of Sodium Dodecyl
Sul-fate Poly Acrylamide Gels (SDS-PAGE) for electrophoresis.
After the run, the gel was transferred to polyvinylidene
difluoride membrane.Immunoblottingwas performedwith
appropriately diluted specific primary antibodies for PI3K
(anti-phosphoinositide3kinaseMab)andphosphoPDK1
(anti-phosphoinositide-dependent Kinase 1 Mab) and p38MAPK
(anti-p38mitogen-activatedproteinkinaseMab)respectively.
Finally, chemiluminescence was performed with LumiGlo
reagent as described by the manufacturer.35 Densitometry
analysesweredoneusingQuantity1(Bio-Rad)software.15
Statisticalanalysis
Eachexperimentwasperformedthreetimesandresultsare
expressedasmeans±SD.Student’st-testwasperformedfor
significanceandap-valueof<0.05wasconsideredsignificant.
WesternblotsandRT-PCRshowrepresentativedatafromthree
independentexperiments.*p<0.05,**p<0.01,***p<0.001.
Results
ReversalofTcellresponsivenessaftertherapywithradio attenuatedparasites
Cell mediatedimmunityisimpairedinLeishmania infection
characterizedbymarkedTcellanergytospecificLeishmania
antigens.15Afterobservingthepotentialtherapeuticeffectsof
100and150Gyattenuatedparasitesintermsofreductionin
organparasiteburden,31webecameinterestedtoseewhether
theTcellanergyobservedduringprogressiveinfectioncould
bereversedbythetreatment.Splenocytesofinfectedanimals
(Group2)failedtomountanti-leishmanialTcellproliferation
toleishmanialantigenssuggestingglobalimmune
unrespon-sivenessattheactivestageofthedisease.Though Group3
animalsweretreatedwith50Gyradio-attenuatedparasites,
theyalsofailedtodoso.Ontheotherhand,thesplenocytes
from treatedgroups(Group4andGroup5)stimulatedwith
SLAshowedasignificantly(p<0.001)higherlevel(3.5–4folds)
ofTcellproliferation(Fig.1).
ExpressionsofiNOSandTGF-ˇgenes
ThemRNAexpressionlevelsofiNOSandTGF-genes(Fig.2)
werecheckedindifferentanimalgroupsbyRT-PCRmethod.
Asignificantlyhigherlevelofexpression(six-fold,p<0.001)of
iNOSmRNAwasseeninanimalsofgroups4and5,whereas
expression of TGF- mRNA has been lowered by two-fold
(p<0.001)inthesamegroupsofanimalscomparedtothatof
infectedgroupofanimals(Group2).TreatedanimalsofGroup
3didnotshowsuchincrementordecrement.
WesternblotanalysesforPI3K,PDK1andp38MAPK
Inordertoscorethechanges,ifany,inthesignaling
mecha-nismsofsplenocytesoftheimmunizedanimals,immunoblot
N I 50Gy 100Gy 150Gy 0 2000 4000 6000 8000 10000 12000 14000 16000 CPM
N, Normal; I, Infected; Gy, Gray; CPM, Count per minute; *, P<.05, **, P<.01,
***, P<.001.
Fig.1–TcellproliferationassayinBALB/cmicegroups. NumberofproliferatedTcellsisexpressedasCPM(count perminute).ProliferationsofTcellswerecheckedand comparedbetweeninfectedcontrolgroupandtreated animalgroups(50,100and150Gygroups),respectively. HigherT-cellproliferationscomparedtoinfectedanimals werefoundinthetreatedanimalgroups,100and150Gy, respectively.Lefttoright,barsrepresentN,healthycontrol; I,Infected:micereceivedinfectiononly;50,100,150Gy representthreegroupsofmicetreatedwithattenuated
Leishmaniadonovaniparasites,dosesofattenuationbeing
50,100and150Gyabsorbeddosesof␥-radiation,
respectively.Infected(5×106parasites/animal)andtreated
(twice@5×106parasitesperanimalin15-dayinterval)
miceweresacrificedat120dpi.Splenocyteswereisolated andculturedinmediumRPMI-1640in24-wellplates@ 2×106splenocytes/well.After72hthesplenocyteswere
pulsedwithH3thymidineandafter18hofpulsingthe
countwastakeninliquidscintillationcounter.Data representmean±SDofsixanimalspergroupandare representativeofthreeindependentexperiments;paired two-tailedStudent’st-testwasperformed.p<0.05was consideredsignificant.
molecules (Fig. 3). These are some ofthe most important
moleculesintriggeringtheproductionoffreeradicalslikeNO
andROS.Inourpreviousintramusculartherapeuticstudies,
weidentifiedasignificant increaseofnitric oxide(NO)and
reactiveoxygen species(ROS)levelsinanimals ofdifferent
immunized/treatedgroups.30,31 Wemeasuredthe
phosphor-ylationlevelofthesethreemoleculesinsplenocytesofthe
animalsandobservedfrom 3.5- to4-fold increasein
phos-phorylationin 100 and 150Gy animal groups compared to
theinfectedgroups(Fig.3).Thisindicatesthe participation
ofthesemoleculesinactivatingtheeffectormoleculessothat
protectioncouldbeachieved.AnimalsofGroup3thatreceived
immunizationwithparasitesattenuatedwith50Gy doseof
radiationdidnotshowanyhigherexpressionintermsof
phos-phorylationofanyofthethreesignalingproteinsstudiedhere.
Thiscorroboratesourpreviousobservationswherewenoticed
thatthisgroupofanimalshadnoprotectionandsuccumbed
tothedisease.30,31
Discussion
Wesawthatattenuatedparasitesactivatedthemicrobicidal
mechanisms of macrophages in the model host body and
thentheactivatedmacrophagesefficientlycleared
intracellu-larparasitesfromspleenandlivercellsoftheprotectedanimal
groups. Inourtherapeuticstudy,31the increasedreleaseof
IFN-␥(three-foldhigherthantheinfectedgroupofanimals)
from theinfectedandthentreatedgroups(100and 150Gy)
supportedourhypothesisthattheTh2ambiencealready
cre-ated in infected conditions has been overrodeby the Th1
atmospheregeneratedbytherapeuticuseofradio-attenuated
L. donovani. While in case of our prophylactic study,30 the
radio-attenuatedL.donovaniparasitesgeneratedanactiveTh1
ambienceinsidetheimmunizedhostbodybeforethehostwas
exposedtovirulentL.donovaniparasiteswhichdidnotallow
thediseaseprogress.Theprimedandthenchallengedgroups
ofmiceshowedIFN-␥release15-foldhigherthanInterleukin4
(IL-4)andaboutfive-foldhigherthanIL-10.30Mechanistically
thusbothapproachesaredependenteitheronthepresenceor
ontheestablishmentofTh1milieu.Attainmentoftheright
doses ofattenuationis stilla difficulttaskasit is directly
relatedtotheefficacyofthevaccineinonehandandtosafety
issuesinanother.Ifthedoseofattenuationbygamma
radia-tionistoolow,thereisachanceofreversiontovirulentformof
thepathogenscausingdiseaseprogressionunderminingthe
purpose.Ifthedoseofattenuationbygammaradiationistoo
high,itwillimmediatelykilltheparasites;thus,hostimmune
responsewillnotbeevoked aslivepromastigoteshavethe
potentialtoinducevariouscellstoproduceIFN-␥,aproperty
whichdeadpromastigoteslacked.36 Mannaetal.37reported
thatthelimitingdoseofattenuationbygammaradiationfor
L.donovaniparasitesis20Krad(200Gy)uptowhichcellscan
employhomeoviscousadaptationtosurviveandcompensate
thealteredconditions.Inourownexperiencesfrom
prophy-lactic and therapeuticstudies, we haveseen that 100 and
150Gyweretherightdosesofattenuationtoevokeimmune
responseinexperimentalhostswhile50Gymaycause
rever-sionasevidencedfromsurvivalkineticsandparasiteburden
analysesofspleenand livercells of50Gy treatedanimals.
Thus,extremecautionmustbetakentoselecttherightdoses
ofattenuation.
OurpreviousstudieshadshownthatBALB/cmicetreated
with100and150Gyradio-attenuatedparasitesthroughI.M.
route(andalsothroughI.P.route)havemuchlowerparasite
burdenandlesshepatoandsplenomegalycomparedto
unim-munizedinfectedanimals.Asignificantincreaseinlevelsof
freeradicals,anincreasedTh1cytokinesprofileanddecreased
Th2levelsintheseanimalscomparedwiththeinfected
ani-malgroupsindicatedthesuccessfulclearanceofintracellular
parasites in these two groups, but not in 50Gy irradiated
group.6BothROSandNOareknowntobeinvolvedin
para-sitekillingintheearlystageofLeishmaniainfectioninmice
andNOaloneisinvolvedinthelatephaseofinfection.38ROS
generationisreportedtoinvolvemoleculeslikePI3K,39PKC,40
ERKandRas.38,41P38MAPKhasbeenreportedtobeinvolved
in thegeneration ofNO42 while activation ofP38MAPK is
dependent upon PI3K activation.43Among other associated
1 2 3 4 5 TGF-B iNOS HGPRT 0 1 2 iNOS/HGPRT 1 2 3 4 5 1 2 3 4 5 0 0.5 1 1.5 2 TGT β / HGPRT
N, Normal; I, Infected; Gy, Gray; TGF–β, Transforming growth factor –β; iNOS, Inducible nitric oxide synthase;
HGPRT, Hypoxanthene-guanine phophoribo syl transferase; *, P<.05, **, P<.01, *** , P<.001.
Fig.2–RT-PCRinBALB/cmicegroupsforcheckingtheexpressionsofiNOSandTGF-genes.LevelsofiNOSandTGF-
genesexpressionwerecomparedbetweeninfectedcontrolgroupandtreatedanimalgroups(50,100and150Gygroups), respectively.Lefttoright,bandsandbarsrepresent1,healthycontrol;2,Infected:micereceivedinfectiononly;3,4,5 representthreegroupsofmicetreatedwithattenuatedLeishmaniadonovaniparasites,dosesofattenuationbeing50,100 and150Gyabsorbeddosesof␥-radiation,respectively.Infected(5×106parasites/animal)andtreated(twice@5×106
parasitesperanimalin15daysinterval)miceweresacrificedat120dpi.SplenocyteswereisolatedfromwhichRNAwas prepared.cDNAwaspreparedfromRNAandPCRexperimentswerecarriedoutforiNOSandTGF-geneexpressionstaking HGPRTgeneasthehousekeepinggeneforinternalcontrol.DensitometryanalysiswasdoneandtheratioofiNOSand TGF-expressedinthebardiagram.Alldatawerecomparedtotheinfectedcontrolgroup.Datarepresentmean±SDofsix animalspergroupandarerepresentativeofthreeindependentexperiments;pairedtwo-tailedStudent’st-testwas performed.p<0.05wasconsideredsignificant.*,p<0.05;**,p<0.01;***,p<0.001.
activationpathway,44andP38MAPKiswellknowntotrigger
theproductionofTNF-␣whichinducesiNOSexpressionand
NOgeneration.45Inourprophylacticstudy,weobserved3.5-to
3.9-foldhigherTNF-␣releasesin100and150Gygroups.30In
thepresentstudy,theexpressionofiNOSwasobservedtobe
six-foldhigherinthesametreatmentgroups.TGF-causes
P-PDK1 PDK1 P-PI3K PI3K P-P38MAPK P-P38MAPK β -Actin N I 50Gy 100Gy 150Gy
N I 50Gv100Gy150Gy 150Gy 100Gy 50Gy I N 1 0 2 1 0 2 P-p38MAPK/ p38MAPK P-PI3K/PI3K P-PDK1/PDK1 0 0.5 1 1.5
N I 50Gy100Gy150Gy
N, Normal; I, Infected; Gy, Gray; PI3K, Phosphoinositide 3 kinase; PDK1, Phosphoinositide dependent kinase 1; p38 MAPK , p38 mitogen activated protein kinase; P, Phospho; *, P<.05, ** , P<.01, *** , P<.001.
Fig.3–WesternblotanalysistoevaluatePDK1,PI3Kandp38MAPKphosphorylationlevelsinBALB/Cmicegroups.Levelsof PDK1,PI3Kandp38MAPKphosphorylationwerecheckedandcomparedbetweeninfectedcontrolgroupandtreatedanimal groups(50,100and150Gygroups),respectively.Splenocytesfromdifferentgroupsofanimalsweretakenandthecell lysateswerepreparedandrunin10%polyacrylamidegelsandthenimmunoblottedwithrespectivemonoclonalantibodies (MAbs)againstnon-phosphorylatedandphosphorylatedformswithactinasaninternalcontrol.Phosphorylationstatus ofeachoftheabovementionedmoleculeswereexpressedasthedensitometricratioofthephosphorylatedform(labeled phosphoorP)versustheinternalcontrol(-actin).Lefttoright,barsrepresentN,healthycontrol;I,infected:micereceived infectiononly;50,100,150GyrepresentthreegroupsofmicetreatedwithattenuatedLeishmaniadonovaniparasites,doses ofattenuationbeing50,100and150Gyabsorbeddosesof␥-radiation,respectively.Infected(5×106parasites/animal)and
treated(twice@5×106parasitesperanimalin15daysinterval)miceweresacrificedat75dpi.Splenocyteswereisolated
andculturedinmediumRPMI-1640in24-wellplates@2×106splenocytes/well.Representativedataofthreesimilar
experimentsarepresentedhere.Datarepresentmean±SDvalueofthreerepeatexperimentswithsixanimalsineach group;pairedtwo-tailedStudent’st-testwasperformed.p<0.05wasconsideredsignificant.*,p<0.05;**,p<0.01;***,
thetranscriptionofmRNAinvolvedinimmunesuppression,
the hall mark offull blown KA state.35 This Th2cytokine
geneexpressionissignificantlyreduced(two-fold,p<0.001)in
thesetwogroupssuggestingthatrestorationofTh1ambience
hasbeen initiatedandestablishedaftersuccessfultherapy
withradio-attenuatedparasites.The100and150Gytreated
animalsalsoshowedasignificantincreaseinPI3K
phosphor-ylation,whichinturnactivatesPDK1phosphorylation.These
areincongruencewithobservationsbyotherworkers.46After
activation,PDK1couldactivatedifferentPKCisotypes,which
areresponsibleforthephosphorylationofERKmoleculesthat
ultimately triggerROSgeneration.ActivatedPI3Kalso
acti-vates P38 MAPK phosphorylation through Akt activation43
leadingtoNOproduction.Inthispreliminarystudy,wehave
alsonoticedhigherlevelofp38MAPKphosphorylation,which
couldincreaseTNF-␣production45andinturninducesNOS2
expressionandsubsequentNOgeneration.45
The results of the present study showed that
radio-attenuatedlivevaccinemayhelptheanimalstorecoverfromT
cellanergy,induceNOS2andsuppressTGF-geneexpression.
TheNOandROSinducedparasitekillingbymacrophages,in
groupsofanimalstreatedwithradio-attenuatedhomologous
vaccine,mayfollowPI3K–p38MAPKmediatedNOgeneration
inonehandandthePI3K–PDK1mediatedROSgenerationon
theother.Furtherstudiesareinprogress.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
Theauthors are thankful to the Council forScientific and
Industrial Research(CSIR), Indiafor the fellowshipof
San-chitaDatta,CSIRSeniorResearchFellow[sanctionno.ACKNo.
112449/2K10/1dt29.03.11].Theauthorsacknowledgethehelp
oftheDirector,PublicInstructions,GovernmentofWest
Ben-gal,IndiaandthePrincipal,BarasatGovt.College,Kolkata.The
kindcooperationoftheDirector,IndianInstituteofChemical
Biology,Kolkata,Indiaisdulyacknowledged.
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