www.jped.com.br
ORIGINAL
ARTICLE
Placental
and
colostral
transfer
of
antibodies
reactive
with
enteropathogenic
Escherichia
coli
intimins
␣
,

,
or
␥
夽
,
夽夽
Silvia
P.N.
Altman
a,b,
Milene
Tino-De-Franco
a,
Cristiane
B.
Carbonare
a,
Patricia
Palmeira
c,
Solange
B.
Carbonare
a,∗aInstitutoButantan,LaboratóriodeImunogenética,SãoPaulo,SP,Brazil
bHospitalIsraelitaAlbertEinstein,SãoPaulo,SP,Brazil
cHospitaldasClínicas,InstitutodaCrianc¸a,LaboratóriodeInvestigac¸ãoMédica(LIM-36),SãoPaulo,SP,Brazil
Received21September2016;accepted29December2016 Availableonline19March2017
KEYWORDS
Enteropathogenic
Escherichiacoli; Diarrheagenic
Escherichiacoli; Intimin;
Maternallyacquired immunity;
ImmunoglobulinG; Colostrum
Abstract
Objective: Intiminsareproteinadhesins ofenteropathogenicEscherichia coliand enterohe-morrhagic E. coli capable of inducing attachment and effacement lesions in enterocytes. Anti-intimin antibodiesareimportantfor the protection fromenteropathogenic E. coliand enterohemorrhagicE.coliinfectionsbecausetheseantibodiesinhibitbacterialadhesionand impairtheinitialstepofthepathogenesis.Westudiedthetransferofmaternalanti-intimin anti-bodiesfromhealthyBrazilianmotherstotheirnewbornsthroughtheplacentaandcolostrum. Methods: SerumimmunoglobulinGand secretoryimmunoglobulinA antibodiesagainst con-served and variable regions of intimins ␣, , and ␥ were analyzed using an enzyme linked-immunosorbentassay inthebloodandcolostrum from45 healthywomenas wellas cordbloodserumsamplesfromtheirnewborns.
Results: Theconcentrationsofantibodiesreactivewith␣intiminweresignificantlylowerthan those ofanti-␥ andanti-conserved intimin antibodies inthe colostrum samples.IgG serum antibodiesreactivewithallthesubtypesofintiminsweretransferredtothenewborns,butthe concentrationsofanti-conservedintiminserumantibodiesweresignificantlyhigherinmothers andnewbornsthanconcentrationsofantibodiesagainstvariableregions.ThepatternsofIgG transferfrommotherstonewbornsweresimilarforallanti-intiminantibodies.Thesevalues aresimilartothepercentagetransferenceoftotalIgG.
夽
Pleasecitethisarticleas:AltmanSP,Tino-De-FrancoM,CarbonareCB,PalmeiraP,CarbonareSB.Placentalandcolostraltransferof antibodiesreactivewithenteropathogenicEscherichiacoliintimins␣,,or␥.JPediatr(RioJ).2017;93:568---75.
夽夽
StudyconductedatInstitutoButantan,SãoPaulo,SP,Brazil. ∗Correspondingauthor.
E-mail:[email protected](S.B.Carbonare). http://dx.doi.org/10.1016/j.jped.2016.12.005
Conclusions: Anti-intimin antibodiesaretransferred frommothers tonewborns throughthe placenta,andreinforcetheprotectionprovidedbybreastfeedingagainstdiarrheagenicE.coli infections.
©2017SociedadeBrasileiradePediatria.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/ 4.0/).
PALAVRAS-CHAVE Escherichiacoli
enteropatogênica;
Escherichiacoli
diarreiogênica; Intimina;
Imunidadematerna adquirida;
ImunoglobulinaG; Colostro
TransferênciaplacentáriaecolostraldeanticorposreativosaEscherichiacoli
enteropatogênicacomexpressãodasintiminas␣,ou␥
Resumo
Objetivo: AsIntiminassãoadesinasproteicasdeEscherichiacolienteropatogênicase entero-hemorrágicascapazesdeinduziraslesões‘‘attachingandeffacing’’nosenterócitos.Anticorpos anti-intiminassãoimportantesparaaprotec¸ãocontrainfecc¸õesporE.colienteropatogênica eE. colienterohemorrágicaporqueestes anticorposinibemaadesão bacterianaimpedindo opassoinicial domecanismopatogênicodestasbactérias.Nósestudamosatransferênciade anticorposmaternosanti-intiminasdemãesbrasileirassaudáveisparaosseusrecém-nascidos atravésdaplacentaedocolostro.
Métodos: AnticorposséricosdaclasseIgGesecretóriosdaclasseIgAreativoscomasporc¸ões conservadaevariáveisdasintiminas␣,e␥foramanalisadospelotesteimunoenzimáticono sangueenocolostrode45parturientessaudáveis enosanguedecordãoumbilicaldosseus respectivosrecém-nascidos.
Resultados: Asconcentrac¸õesdeanticorposreativoscomintiminav␣foramsignificativamente maisbaixasqueasdosanticorposanti-␥eanti-intiminaconservadanasamostrasdecolostro. Anticorpos IgG séricos reativos com todas as intiminas foram transferidos para os recém-nascidos,masasconcentrac¸õesdeanti-intiminaconservadaforamsignificativamentemaisaltas tantonasmãescomonosrecém-nascidosdoqueosanticorposreativoscomasregiõesvariáveis dasintiminas.OpadrãodetransferênciadeIgGdasmães paraosrecém-nascidosfoimuito semelhanteparatodososanticorposanti-intiminas.Osvaloresdeporcentagemdetransferência foramsemelhantesàtransferênciadeIgGtotal.
Conclusões: Anticorposanti-intiminasãotransferidosdasmãesparaosrecém-nascidospela pla-centaecorroboramaprotec¸ãocontrainfecc¸õesporEscherichiacolidiarreiogênicasconferida peloaleitamentomaterno.
©2017SociedadeBrasileiradePediatria.PublicadoporElsevierEditoraLtda.Este ´eumartigo OpenAccesssobumalicenc¸aCCBY-NC-ND(http://creativecommons.org/licenses/by-nc-nd/4. 0/).
Introduction
Inthedevelopingworld,themorbidityandmortalityrelated topediatricbacterialdiarrheaarelargelyattributedtothe diarrheagenicformofEscherichiacoli(DEC).Newbornsand infants up to1 year of age areparticularly vulnerableto diarrheacausedbyDECwhentheyhavenotbeenbreastfed.1
Epidemiological studies among Brazilian children have revealed the presence of DEC strains in feces not only of children with diarrhea but also in those of healthy children. Typical (tEPEC) and atypical (aEPEC) forms of enteropathogenicE.colihavebeendetectedamongthese isolates from children in both rural and metropolitan areas.2,3 Enterohemorrhagic E. coli (EHEC) is an enteric
pathogenthatisgenetically andphenotypicallyrelatedto EPEC,butdistinguishablebyitstoxinproduction.Bothkinds ofbacterialstrainsproduceattachingandeffacinglesions (A/E) in the gut mucosa, resulting in intimate contactof thebacteriawiththehost cell.This phenomenonleadsto protein phosphorylation, cell membrane destruction, and expressionofbacterialgenes,clusteredinagenomicisland
called locusof enterocyte effacement (LEE).1 Oneof the
mostimportantproteinsencodedbyLEEisintimin,a94-kDa outer-membraneproteininvolvedinintimatebacterial-cell attachment.Theintiminmoleculeiscomposedof939amino acidresidues (aa) and hastwo functional regions: the N-terminalportion,whichishighlyconservedandisinserted intothebacterialexternal membrane,andtheC-terminal 280aaportion,whichisvariableandwhosepolymorphisms determinethevariousintiminsubtypes.4,5Atleast27
sub-typesofintiminhavebeendescribed,butonlyafewhave been implicated in a disease in humans. The prevalence ofintiminsubtypesvariesamongtEPEC,aEPEC,andEHEC, in different geographic regions and periods, as demon-stratedbyepidemiologicalstudies.Intimins␣,,and␥were amongtheprevalentintiminsubtypesinBrazilintheearly 2000.1,6---8
Some studies have identified anti-intimin antibodies in the serum of children and adults infected with EPEC or EHEC.9,10 Theauthorshaveidentifiedanti-intimin
antibod-iesintheserumofhealthyadultsandchildren,11---13aswell
findings have been described for American and Mexican women.19 Nonetheless,the transfer of maternal antibody
subtypes reactive with intimin to newborns through the placentahasnotyetbeendescribed.
Antibodies from colostrum inhibit bacterial adhesion to HEp-2 cells in vitro; the same anti-EPEC antibod-ies were found in the feces of breastfeed newborns as presentin theirmother’scolostrum,15 pointing toa
possi-blemechanism bywhich breastfeeding confersprotection onto newborns against infections caused by EPEC and EHEC.14,16,17 In addition, secretory and serumanti-intimin
antibodies have been shown to inhibit bacterial adhesion tocultured cells and have been implicated in protection againstinfection15,17,20---22;therefore, the transfer of
anti-intimin antibodies could protect the fetus and newborn againstinfection.Inthepresentstudy,theauthorsassessed the presence of anti-intimin antibodies in the serumand colostrumfromhealthymothersandintheumbilicalcordof theirnewbornbabiesusingpurifiedproteinsobtainedfrom recombinantbacteriaexpressingtheconserved(cons)and variableregionsofintimins␣(v␣),(v),and␥(v␥).4By
simultaneouslymeasuringconcentrationsofthese antibod-iesinserumsamplesobtainedfromthemothersandbabies, theirpossibleplacentaltransferwasalsoassessed.
Materials
and
methods
Biologicalsamplesfromhumansubjects
Thisstudy’sprotocolwasapprovedbytheethicscommittee of theHospital Israelita Albert Einstein(HIAE) andof the Universidadede São Paulo, Brazil (CEP number: 06/434); allprocedures werein accordancewiththe 1964 Helsinki declaration,andawritteninformedconsentwasobtained fromalltheparticipatingmothers.
The samples were obtained at HIAE, a private Hospi-tal serving families of a medium to high socioeconomic level, fromJuly to October 2006. Serum, colostrum,and cord blood serum were obtained from 45 healthy moth-ersandtheirhealthynewbornbabies,correspondingto37 cesareandeliveries.Theinclusioncriteriawereasfollows: healthymothers,without anypathologiesrecordedduring pregnancyorlabor,andwithnegativeresultsonserological testsfor HIV,HTLV I/II,hepatitisBandC,Chagasdisease, syphilis,toxoplasmosis,andcytomegalovirus.Maternaland cordblood were obtained immediately afterdelivery and colostrumsampleswerecollectedupto72hafterdelivery. Allmotherswereagedbetween22and36years(meanage 31.2),andthetotalserumimmunoglobulinG(IgG)ranged from508.8to1389.1mg/dL(mean800.3mg/dL).The moth-ers were all well-nourished, predominantly primiparous, withmediumtohigheducationalandsocioeconomicstatus, livingin goodsanitaryconditionsinthe cityof SãoPaulo. The newborn babieswere healthy, with adequate weight forthegestationalage,37---41gestationalweeks(mean38), weightingbetween2630and3945g(mean3298g).
Acontrol colostrumpool waspreparedwithequal vol-umes of 20 samples collected from healthy women, and acontrolserumpoolwaspreparedwithequal volumesof 100bloodsamplescollectedfromhealthyadultdonors.The total secretory immunoglobulin A (SIgA) concentration of
the colostrumpool was determinedby a capture enzyme linked-immunosorbent assay(ELISA) using a commercially availablestandard(Sigma,USA).Aserumpoolwithaknown immunoglobulinG(IgG)concentrationservedasastandard. Sampleswithlowandhighconcentrationsofantibodieswere included in each ELISAplate asan internal controlof the assay.
Antigens
Purifiedintimins wereobtainedfromculturesof recombi-nantbacteriaexpressingeachoftheconservedandvariable regions ofintimins␣,,and ␥,aspreviouslydescribed.18
Briefly, therecombinant E. coliM15bacteria transformed withplasmidspFLv␣,pFLv,pFLv␥,orpFLconswere ana-lyzed by PCR toconfirm the presence of the insert after cloningof theplasmidvector.Fragmentsof theeaegene, whichencodesintimin,weresequencedforcomparisonwith standard strainsregistered inGenBank.Proteinexpression was induced by culturingthe bacteria withisopropyl--d -thiogalactopyranoside,followed by extraction ina French pressandcentrifugation.Theproteinswerefinallypurified by immobilizedmetal (Ni) ion adsorptionchromatography (Qiagen, USA) andthe products were analyzed bysodium dodecylsulphatepolyacrylamidegelelectrophoresis.
Anti-intiminantibodies
Theconcentrationsofanti-intiminserumIgGandsecretory IgA(SIgA)weredeterminedbyELISAbasedontheFomsgaard technique.23Concentrationsofspecificanti-intimin
antibod-iesweredeterminedin comparisonwithtotalIgGandIgA concentrations,asdescribedelsewhere.18 For
standardiza-tion,halfofamicrotiter96-wellELISAplatewascoatedwith anti-IgG asa capture antibody (2.5g/mL)and theother halfwascoatedwitheachofthepurifiedintiminsobtained fromrecombinantbacteriaexpressingcons,v␣,v,andv␥4
at thesameconcentration(2.5g/mL).Appropriateserial dilutions ofthe control serumpool(with known totalIgG concentrationdeterminedbynephelometry)wereaddedon both sidesofthe plate.The assaywasdeveloped withan anti-IgGconjugate.Theopticaldensityvalueswere estab-lishedandtheconcentrationofanti-intiminantibodieswas determinedbycomparisonwiththestandardcurveoftotal IgG.ThisstandardprocedurewasalsoperformedforSIgAin acolostrumpool.Then,theindividualserumandcolostrum samplesinserialdilutionsstartingfrom1:10wereanalyzed inELISAplatescoatedwiththerecombinantintimins,with theserumorcolostrumpoolusedtoconstructthestandard curve. Samples withhigh and low concentrations of anti-bodieswereincludedin eachplateasaninternal control. The totalIgG concentrations in maternal and cordserum samples weremeasured by an immunoturbidimetric assay (AbbottDiagnostics,USA).Theplacentaltransfer percent-agesof the totalandintimin-specific IgGantibodies were definedineachassayasfollows:
%T =
concentrationofantibodiesincordserum concentrationofantibodiesinthematernalserum
Statisticalanalysis
All resultswere subjected tostatistical analysisincluding descriptiveanalyses withbox plot graphs,non-parametric ANOVAwithFriedman’smultiple comparisontest,and the Spearmancorrelationtest,ata95%confidencelevel,using the GraphPad Prism software (Graph Pad Software Inc., USA).
Results
Fig.1showstheconcentrationsofantibodiesreactivewith thefourpurifiedrecombinant antigens (cons,v␣, v, and v␥) present in 45 colostrum samples, 45 maternal serum samples,and45cordserumsamplesfromtheirrespective newborns. The box plots show the maximal and minimal values,25%and75%percentiles,medians,andmeans.
TheanalysesofcolostrumsamplesbytheFriedmantest formultiplecomparisonsshowedthattheconcentrationsof anti-v␣IgAantibodiesweresignificantlylowerthanthoseof anti-v␥andanti-cons,buttherewerenostatistically signif-icantdifferenceswithintheotherpairsofvalues(Fig.1A). TheconcentrationsofIgAantibodiesvariedwidely,andthe meansabovethemediansindicatedthatitwasnotanormal distribution.
ThecomparisonofspecificIgGrevealedthatthe concen-trationofanti-conswassignificantlyhigherwhencompared with the other subtypes in the mothers’ and newborns’ serumsamples.Theanti-v␣antibodylevelswerehigherthan thoseof anti-v,buttherewerenosignificant differences betweenthesetwosubtypesandanti-v␥antibodies(Fig.1B andC).Theseresultswereconfirmedbythefrequency dis-tributionanalysis(datanotshown).
Fig. 2 shows that the IgG transfer from mothers to newbornswasverysimilarforalltheantibodysubtypes ana-lyzed. The mean transference percentage of anti-intimin antibodiesrangedfrom142%to149%andthemedians,from 130% to146%.These valuesaresimilartothe percentage oftransferenceoftotalIgG(mean=143%, median=137%). Therewere nosignificant differences amongthe transfer-ence percentages of antibodies reactive with the various intiminsubtypes.
Table1showsresultsoftheSpearmancorrelationtests. Inmaternal serumsamples, therewasasignificant corre-lationbetweenanti-v␣andanti-vantibodies(p<0.0001). Therewasnostatisticallysignificantcorrelationbetweenv␥
andcons.Thesamepatternofcorrelationwasobservedin newborns’serumsamples.Thecorrelationcoefficientswere highinallpairsanalyzedforthecolostrumsamples.
Table2 showssignificant correlationcoefficientsfor all intimintypes betweenmaternal and newbornserum sam-ples;therefore,thetransferenceofantibodiesfrommothers to newborns was equally efficient for all the subtypes ofintimin.Low correlationcoefficients betweenmaternal serumIgGandcolostrumSIgAantibodieswereobserved,as the generationof serum IgGand secretory IgA antibodies proceedsviaindependentmechanisms.
Discussion
Inthisstudy,theauthorsprovidedevidenceofplacentaland colostraltransferofanti-EPECandanti-EHECantibodiesto
10 000
a
b
c
SlgA anti-intimin conc.
(µg/mL)
lgG anti-intimin conc.
(µg/mL)
lgG anti-intimin conc.
(µg/mL)
1000
100
10
1
0.1
0.01
v α v β v γ Cons
v α v β v γ Cons
v α v β v γ Cons 100
10
1
0.1
0.01
100
10
1
0.1
0.01
Figure 1 Box-plot distributions of samples by their anti-intiminconcentrations:maximalandminimalvalues(topand bottom lines outside the box, respectively), means (䊉)and
Table1 Correlationcoefficientsoftheconcentrationsofanti-intiminantibodiesinserum(IgG)andcolostrum(SIgA)samples andIgGtransferencepercentagefrommotherstonewborns,inpairsofintimintypes.
Anti-intimin antibodies
Maternalsera Newbornsera Colostrum IgGtransference (%)
IgG IgG SIgA
v␣vs.v 0.8014
(0.0001)
0.8132
(0.0001)
0.8235
(0.0001)
0.3447 (0.0204) v␣vs.v␥ 0.3590
(0.0154)
0.4586 (0.0015)
0.7768
(0.0001)
0.4603 (0.0015) v␣vs.cons 0.3159
(0.0345)
0.5995
(0.0001)
0.7809
(0.0001)
0.3121 (0.0369) vvs.v␥ 0.4433
(0.0023)
0.5034 (0.0004)
0.7232
(0.0001)
0.5306 (0.0002) vvs.cons 0.3495
(0.0186)
0.4934 (0.0006)
0.7111
(0.0001)
0.2462 (0.1031) v␥vs.cons 0.1043
(0.4954)
0.2221 (0.1426)
0.7628
(0.0001)
0.3306 (0.0265)
v␣,variableregionofintimin␣;v,variableregionofintimin;v␥,variableregionofintimin␥;cons,conservedregionofintimin. Thecoefficients were determined bytheSpearman correlation test, ata 95%confidence level. p-Valuesare shown initalics; p -values<0.0001arehighlightedinbold.
newbornbabies;theseantibodiesarelikelytoconfer pro-tectionontotheneonatesagainstdiarrheacausedbythese pathogens.Anti-intiminantibodiescaninhibitthebacterial adhesiontoepithelialcells,andthisabilityhasbeen demon-stratedforSIgAantibodiespresentinhumancolostrumand milk and serum antibodies from humans and immunized animals.13---17,22,24Basedonthisproperty,intiminshavebeen
proposedasapotentialcandidateforavaccineagainstEPEC andEHECinfection.20,21
Antibodiesagainsttheconservedandvariableregionsof intimins␣,,and␥werefoundinallserumandcolostrum samplesanalyzed,probablyduetothewideprevalenceof EPECintheBrazilianpopulation.Thisstudywasconducted
400
300
200
100
0
v α v β v γ Cons
lgG tr
ansf
erence
, %
Total lgG
Figure2 TransferofIgGanti-intiminantibodiesandtotalIgG frommotherstonewborns.Box-plotdistributionofsamplesby theirpercentagesoftransferenceofanti-v␣,anti-v,anti-v␥, andanti-consandtransferoftotalIgG:maximalandminimal values(top andbottom lines outsidethe box, respectively), means(䊉)andmedians(), and75%and 25%quartiles (top andbottomsidesofthebox,respectively).
Table2 Correlation coefficientsofanti-intiminantibody concentrationsbetweenmaternalserum(IgG)andcolostrum (SIgA)samples,andbetweenmaternalandcordserum sam-ples(IgG).
Intimin Maternalserum vs.colostrum
Maternalserum vs.newbornserum
v␣ 0.0028(0.9853) 0.8727(0.0001)
v 0.1475(0.3337) 0.9568(0.0001)
v␥ 0.1743(0.2578) 0.9320(0.0001)
cons 0.2265(0.1346) 0.8519(0.0001)
v␣,variableregionofintimin␣;v,variableregionofintimin ; v␥, variableregionofintimin␥; cons,conservedregionof intimin.
CoefficientsweredeterminedbytheSpearmancorrelationtest, at a 95% confidence level. p-Values are shown in italics; p -values<0.0001arehighlightedinbold.
usingsamplescollectedfrommothersfrommediumtohigh socioeconomiclevels.Theauthorshavedemonstrated simi-larfindingswhenanalyzingsamplescollectedfrommothers of lower socioeconomic layers and living in poorly sani-tizedareas,suggestingthatEPECprevalenceisindependent of the socioeconomic statusor hygienicconditions of the patients.11,12,16,18 Similarfindingswere reportedbyAraujo
et al. regarding the detection of DEC in fecal samples of children from poor urban areas and in those of chil-drenreceivinginhigh-socioeconomic-levelprivatemedical services.25
HealthyBrazilianchildrendevelopantibodiesreactiveto EPECvirulence-associatedfactorssuchasintimins.11Bythe
endofthefirstyearoflife,theseinfantsexhibitserumand salivaryanti-EPECantibodyrepertoiresequivalenttothose ofhealthyadultsinthesamepopulation.Intimins␣,,and
that intimin is a common antigen ubiquitously present in theBrazilianpopulation.26
The anti-intimin antibody subtypes found in the sam-plesanalyzedin thisstudymayinhibitbacterialadhesion, becausethisportionoftheintiminmoleculeisresponsible for interactions withthe host cells, pointing toa rolein protectionagainstthebacteria.22
ThestatisticalanalysisofIgGdetectedinbothmaternal andnewborn serumsamples revealed thatconcentrations ofantibodiesagainsttheconservedregionofintiminwere higherincomparisonwithantibodiestothevariableregions ofthesubtypes␣,,and␥.Theconservedregioniscommon toallsubtypes;therefore,itisreasonabletoexpectthatall individualswilldevelop theseantibodies regardlessof the specific intimin subtype present in the contacting bacte-ria. Similar results were observed by Zapata-Quintanilla et al.18 in their analyses of serum samples from healthy
adults (blood donors). Nevertheless, this profile was dif-ferent in colostrum samples, where the concentration of antibodies reactive to v, v␥, and cons were all sim-ilar, but the anti-v␣ antibody concentration was lower than that of anti-v␥ and anti-cons. This pattern was not identifiedby Zapata-Quintanillaetal.,18 whofound lower
anti-v␥ levels. This discrepancy may be attributable to variations in the antibody repertoires among the moth-ers, as the characteristics of the donors in the study by Zapata-Quintanillaweredifferentfromthoseofthepresent study regarding the socio-economic level (public hospital
versus privatehospital, respectively),age (mean 24.6 vs. 31.2years),typeof delivery(24.4% vs.82%cesarean sec-tion), and period of sample collection (September 1997 toFebruary 1998 vs. July toOctober 2006). Epidemiolog-icalstudies have indicatedthat theprevalence of intimin
␥ has increased with the emergence of aEPEC in recent years.1,3,6---8,25
Intiminsarefrequentlyfoundinbacteriaisolatednotonly frompatientswithdiarrhea,butalsofromhealthypeople anddomesticanimalsinBrazil.1,6,7,26Intimin␣wasdetected
in tEPEC and aEPEC strains. Intimin  is associated with tEPEC and aEPEC, as well aswith EHEC, whereas intimin
␥ isfoundin aEPECandEHEC.Isolationof thesebacterial strainsinBrazilcanexplainthepresenceoftheseantibodies in serumandcolostrum samples.In spite of thelow inci-denceofEHECinfectionamongBrazilianadults,anti-intimin
␥ antibodies are present and show a positive correlation withantibodies reactive withother intiminsubtypes. The increasing frequencies of aEPEC strains in the environ-ment maybe responsiblefor the presence ofanti-intimin
␥antibodiesamonghealthyBrazilians.Recentstudieshave implicatedintiminsubtypesand␥intheinductionof diar-rheainhumans,andsubtypewasfoundtobesignificantly morefrequentindiseasecaseswhencomparedwithhealthy carriers.8
Thereisasignificantcorrelationbetweenthe concentra-tionofanti-intimin␣andIgGantibodiesinthematernal serumand newborns’ serum, which may bedue to cross-reactions among IgG antibodies targeting these intimins. Indeed,thegenesequencesofthevariableportionsofthese intiminsare65%homologous,18whichexplainsthepresence
of cross-reacting antibodies. Therefore, this phenomenon impliesthatwhateverthe intiminsubtypecandidatefor a vaccine,thissubtypemayraisespecificantibodiesbutcan
alsoinducecross-reactiveantibodies directedtotheother subtypes.
The concentrations ofSIgA antibodiesreactive withall typesofintiminspresentincolostrumsamplesshowed sig-nificantcorrelation coefficients,suggesting thatthereisa certain degree of cross-reaction among these antibodies. Thissituationmaybeexplainedbytheelevated homology betweentheintiminmoleculesandbythelowerspecificity of colostrum SIgA antibodies as compared to serum IgG. Nevertheless,thehigherconcentrationsofanti-intimin anti-bodies in colostrum samples when compared with serum samples supportthe crucialrole of breastfeeding in pro-tectionofinfantsfromDECinfection.Inhumans,milkSIgA antibodiesarenotsignificantlyabsorbedbythemucosaof thebreastfedbaby,butactaslocaldefense.Inaprevious study,theauthorshaveshown,usingimmunoblotting,that thereactivityofanti-EPECSIgAantibodiesofthecolostrum sampleswasthesameasthereactivityofanti-EPEC antibod-iesinthefecesoftherespectivebabies.Inaddition,SIgA antibodiesisolatedfromcolostrumbyaffinity chromatogra-phyinhibitEPECadhesiontoHEp-2cells.15
The present results indicatethat differentanti-intimin antibodiesaretransferredfrommotherstonewborns with equalefficiencythroughtheplacenta.Theseresultssupport thehypothesisthat,inthiscase,thematernal---fetaltransfer isnotinfluencedby antibodyspecificity. The transference percentagesofanti-intiminantibodiesinthepresentstudy werefoundtobeequivalenttothetransferencepercentage oftotalIgG,whichis approximately140%. Similarfindings havebeenreportedbyotherauthors.27,28
Although there is a high frequency of DEC isolates in Brazil, strains of EHEC are not as common as in other countries.Itcanbehypothesizedthat,besides environmen-talfeaturessuchasclimate andalimentaryhabits,people may develop EHEC immunity resulting from contact with otherstrains(e.g.,DEC). Somestudies revealedthe pres-enceofantibodiesreactivewithvariouslipopolysaccharide (LPS)serotypes,suchasO111,O55,O157,andO26,inserum andcolostrum samples fromhealthy Brazilians.12,13 Other
studieshaveshownthematernal---fetaltransferofIgG anti-bodiesreactivewithO111,O16,andO6LPS,aswellasO157, tobeassociatedwithneonatalsepsis.29,30 These LPS
anti-gensarecommonlyfoundindifferentpathotypesofE.coli
that are agents of intestinal or systemic diseases. Taken together,theseobservationsandthepresentworkallowus toconcludethatprotectionfromEHECcanbeconferredby contactwithEPECorotherE.colistrains.
The present results confirm that maternal antibodies reactive with intimins are transferred to the newborns throughtheplacenta, andthat the presenceof high con-centrations of theseantibodies in colostrumsupports the protectiveroleofbreastfeeding.
Funding
ThefinancialsupportwasprovidedbyFAPESP---03/13250-3.
Conflicts
of
interest
Acknowledgments
The authorswould like tothankall themothers for their consenttodonatethesamples.Theywouldalsolikethank theMaternity,theMilkBank,andtheCryopreservation Lab-oratoryofHospital IsraelitaAlbertEinstein,for theirhelp withthesamplecollection;theBrazilian DivisionofAbbot Diagnosis,forthedonationoftheImmunoturbidimetricKit fortheanalysisof IgG;Dr.Lucy B.Zapata-Quintanilla, for the help with experiments, Rosana Prisco, for the statis-ticalanalysis; and Dr.Yanira Riffo Vasquez, for review of the manuscript and her valuable suggestions (Institute of Pharmaceutical Science --- King’s College, London, United Kingdom).
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