Role of the low af fi nity IgE re cep tor (CD23) on the IgE response against Ascaris lumbricoides in Wa rao Ame rin dian chil dren from Venezuela.

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1 Invest Clin 47(3): , 2006 Role of the low af fi nity IgE re cep tor (CD23) on the IgE response against Ascaris lumbricoides in Wa rao Ame rin dian chil dren from Venezuela. Isa bel Ha gel 1, Mai ra Ca bre ra 1, Pe dro Sán chez 2, Pa tri cia Ro drí guez 2 and Jean José Lat touf 3. 1 Insti tu te of Bio me di ci ne, Fa culty of Me di ci ne, Cen tral Uni ver sity of Ve ne zue la, 2 Mu ni ci pal Blood Bank, Ca ra cas and 3 Mi nis tery of Health, Del ta Ama cu ro. Ve ne zue la. iha gel@tel cel.net.ve; Isa bel ha gel@yahoo.com Key words: Warao Am er in dian chil dren, A. lumbricoides, IgE, CD20+CD23+ cells. Abstract. The ob jec tive of this study was to in ves ti gate the im mune mech a nisms against gas tro in tes ti nal helminths in volved in the pro duc tion of IgE, par tic u larly the role of the low af fin ity IgE re cep tor (CD23) in Am er in - dian Warao chil dren from the Orinoco Delta, Ven e zuela. We stud ied a group of un se lected (n = 159) Warao school chil dren with high prev a lence and in - ten sity of helminthic in fec tion and a sim i lar non- par a sit ized (n = 70) con trol group of a creole com mu nity also lo cated at the Delta of the Orinoco. The lev els of to tal and spe cific anti-ascaris IgE, de ter mined by ELISA, were ex - tremely higher in the Warao chil dren (p < 0.001) com pared to the con trol group. How ever, only a re duced group of these chil dren were able to de velop a clin i cally sig nif i cant (> 0.7 PRU/mL) IgE re sponse against A lumbricoides an - ti gens. Cir cu lat ing T helper cells (CD3+CD4+), ac ti vated B (CD20+, CD20+CD21+) lym pho cytes and the ex pres sion of the low af fin ity re cep tor for IgE (CD23), de ter mined by flow cytometry, were sig nif i cantly (p < 0.001) el e vated in the Warao chil dren, par tic u larly in those with an en hanced ca pac - ity to mount ef fi cient spe cific anti Ascaris IgE re sponses. Strong cor re la tions (p < 0.001) were found be tween the num ber of cir cu lat ing CD20+CD23+ cells and the level of to tal and anti-a lumbricoides IgE. Also, a sig nif i cant (p < 0.005) cor re la tion be tween CD20+CD23+ and CD4+ cir cu lat ing cells was ob served. We con clude that A lumbricoides an ti gens may stim u late the pro duc tion of to - tal and spe cific IgE through a CD23 de pend ent path way. How ever the dif fer - ent en vi ron men tal and ge net ics fac tors in volved in the ca pac ity to de velop ef - fi cient spe cific re sponses among the Warao pop u la tion still need to be elu ci - dated. Co rres pon ding aut hor: Isa bel Ha gel. Instituto de Biomedicina, Faculty of Medicine, Central University of Venezuela, Apartado Caracas 1010A, Venezuela. ihagel@telcel.net.ve; Isabelhagel@yahoo.com

2 242 Hagel y col. Pa pel del re cep tor de baja afi ni dad para la IgE (CD23) en la res pues ta IgE fren te a Ascaris lumbricoides en ni ños in dí ge nas Wa rao de Ve nezuela. Inves Clín 2006; 47(3): Palabras clave: Niños indígenas Warao, Ascaris lumbricoides, IgE, células B CD20+CD23+. Resumen. El ob je ti vo de este es tu dio fue in ves ti gar los me ca nis mos in - mu ni ta rios fren te a hel min tos gas troin tes ti na les in vo lu cra dos en la pro duc - ción de IgE, par ti cu lar men te el pa pel del re cep tor de baja afi ni dad para la IgE (CD23) en ni ños in dí ge nas de la et nia Wa rao del Del ta del Ori no co en Ve ne - zue la. Estu dia mos un gru po no se lec cio na do (159) de ni ños Wa rao es co la res con una alta pre va len cia e in ten si dad de in fec ción por pa rá si tos hel min tos, y un gru po con trol de 70 ni ños no pa ra si ta dos de una co mu ni dad ru ral crio lla del mis mo Esta do. Los ni ve les to ta les y es pe cí fi cos de IgE anti-a lum bri coi des, de ter mi na dos por ELISA, fue ron ex tre ma da men te ele va dos en los ni ños Wa rao (p < 0,001). Sin em bar go, solo un re du ci do gru po de esos ni ños fue ca paz de de sa rro llar una res pues ta de IgE es pe cí fi ca clí ni ca men te sig ni fi ca ti va (> 0,7UI/mL) fren te a an tí ge nos de A lum bri coi des. El nú me ro de cé lu las cir - cu lan tes, T CD4+, B CD20+ CD21+ y la ex pre sión del re cep tor de baja afi ni - dad para IgE (CD23) de ter mi na dos por ci to me tría de flu jo, fue ron sig ni fi ca ti - va men te (p < 0,001) más ele va dos en los ni ños Wa rao, par ti cu lar men te en aque llos con ade cua dos ni ve les de IgE es pe cí fi ca fren te al pa rá si to. Se en con - tró una alta co rre la ción (p < 0,001) en tre los ni ve les to ta les y es pe cí fi cos de IgE es pe cí fi ca y la ex pre sión del re cep tor para la IgE (CD23) y una co rre la ción sig ni fi ca ti va (p < 0,005) en tre el nú me ro de cé lu las cir cu lan tes CD20+CD23+ y CD4+. Con clui mos en ton ces que los an tí ge nos de A. lum bri - coi des po drían es ti mu lar la pro duc ción de IgE to tal y es pe cí fi ca a tra vés de una vía de pen dien te del re cep tor CD23. Sin em bar go, los fac to res am bien ta - les y ge né ti cos que po drían de ter mi nar la ca pa ci dad de de sa rro llar res pues tas IgE efi cien tes en la po bla ción Wa rao, re quie re de ma yor es tu dio. Re ceived: Ac cepted: INTRODUCTION The Am er in dian Warao eth nic group con sti tutes a pop u la tion of around peo ple dis persed into 250 iso lated fish ing and hunt ing com mu ni ties along the delta of the Orinoco River, in east ern Ven e zuela. They live in ex treme pov erty with very poor san i tary con di tions and lim ited ac cess to health as sis tance, which place the chil dren at high bio med i cal risk (1). Our pre vi ous stud ies in these com mu ni ties have shown a high prev a lence of helminthic in fec tion (75%), par tic u larly Ascaris lumbricoides (62%), as well as el e vated to tal (8500 IU/mL) and spe cific anti-a lumbricoides (3.5 PRU/mL) IgE se rum lev els (2). It has been ex ten sively re ported that the stim u la - tion of spe cific IgE an ti bod ies by helminth an ti gens con sti tutes an im por tant com po - Investigación Clínica 47(3): 2006

3 IgE re sponses in Warao Am er in dian chil dren 243 nent of the pro tec tive re sponse against these par a sites (3, 4). Our pre vi ous in ves ti - ga tions in chil dren from slum ar eas, with a high prev a lence of helminthic in fec tion, have shown that chil dren with the high est spe cific IgE lev els against adult worm an ti - gens are less likely to be re-in fected with A. lumbricoides af ter antihelminthic treat - ment (5). In ad di tion, chil dren with an atopic back ground that have dem on strated an en hanced ca pac ity to pro duce high lev - els of spe cific IgE an ti bod ies against these par a sites, had sig nif i cantly lower in ten si ties of in fec tion than their non atopic coun ter - parts (6). IgE switch ing is de pend ent on two sig - nals pro vided by spe cif i cally stim u lated T cells: IL-4 in duces the ex pres sion of the ster ile tran script, and the trig ger ing of CD40 with its ligand in duces the ex pres sion of the ma ture tran script cod ing for IgE (7). It has been pro posed that ad di tional sig nals are also re quired for IgE pro duc tion; these would be pro vided by dif fer ent cytokines, sol u ble CD23 and the pair ing of B cell sur face mol e cules CD21 and CD23 (8). An ti bod ies to CD23 have been shown to in hibit IL-4 in duced hu man IgE pro duc - tion in vi tro (9) and to in hibit an ti gen -spe - cific IgE re sponses in rat mod els in an isotype-se lec tive man ner (10). Stud ies per - formed in hu man pop u la tions have re ported an in crease in the pro por tion of CD23+ cir - cu lat ing B cells in asth matic chil dren al ler - gic to Dermatophagoides pteronyssinus, with high lev els of spe cific IgE against this al ler gen (11). The aim of this study was to eval u ate the pos si ble as so ci a tions be tween se rum lev els of to tal and spe cific IgE against Ascaris lumbricoides and the low af fin ity IgE re cep tor (CD23) ex pres sion on B lym - pho cytes cir cu lat ing cells in a group of Am - er in dian Warao school-chil dren chron i cally ex posed to gas tro in tes ti nal helminths. SUB JECTS AND METH ODS Study group We per formed a cross sec tional study in a group of 159 school chil dren from a Warao Amerindian community, Arawuaymujo, lo - cated at the delta of the Orinoco River. The pop u la tion stud ied was sex bal anced and the age range of the group (6-11 years old; mean age: years) was se lected ac - cord ing to pre vi ous stud ies in which the prev a lence and in ten sity of helminthic in - fec tion, have shown to be com pa ra ble at this age range (12). A con trol group of 70 healthy non parasitized school-children (mean age years) was also eval u - ated. These chil dren be long to the com mu - nity of El Caigual, also lo cated near the Orinoco river at Delta Amacuro state. Eth ni - cally, this com mu nity be longs to the creole pop u la tion. How ever, most of these chil dren may have a Warao an ces tor. So cio - eco nomic as pects that may in flu ence the prev a lence and in ten sity of helminthic in fec - tion, including sanitary conditions, mother s level of ed u ca tion and the de gree of pov erty were de ter mined in both groups of chil dren (Ta ble I). The de gree of pov erty of the fam i - lies was de ter mined by ap ply ing the cri te ria in of fi cial use in Ven e zuela, as de fined by the Cen tral Bank of Ven e zuela and the Cen tral Of fice of the Co-or di na tion of the Pres i dent of the Bolivarian Re pub lic of Ven e zuela (CORDIPLAN) (12). Ac cord ing to this clas si - fi ca tion, the to tal fam ily in comes are com - pared to the cost of a shoping bas ket of ba sic food items, and the cost of ba sic food and ser vices for a stan dard fam ily. Ac cess ing to al i men tary prac tices like fish ing or hunt - ing was also con sid ered in the study of these communities. Those families whose incomes did not cover the bas ket of ba sic food items and had lim ited ac cess to other al i men tary prac tices were con sid ered as be ing in ex - treme pov erty. Vol. 47(3): , 2006

4 244 Hagel y col. TABLA I SOCIOECONOMIC CONDITIONS OF THE DIFFERENT GROUPS OF CHILDREN EVALUATED % of children Araguaymujo (159) El Caigual (70) Statistical significance Without access to tap water P < 0,0001 Odds ratio: 101,24 Lack of toilet or latrine P < 0,0001 Odds ratio:21,287 Illiterate mother P = 0,5239 Odds ratio: 0,7279 Extreme poverty (according to their family income) P = 0,3630 Odds ratio: 1,359 95% confidence interval 38,788 to 264,24 10,039 to 45,137 0,3262 to 1,624 0,7368 to 2,507 Eth i cal con sid er ations Informed parental consent was obtained to par tic i pate in the study. This pro - ject was ap proved by the eth i cal com mit - tees of the Re gional De part ment of the Min is try of Health at Delta Amacuro State, and of the In sti tute of Biomedicine. The study was also ap proved by the Na tional Coun cil for Sci en tific and Tech no log i cal In - ves ti ga tion of Ven e zuela, and the Post grad - u ate Com mis sion of the Fac ulty of Med i cine of the Cen tral Uni ver sity of Ven e zuela. Dur - ing the study, med i cal as sis tance was pro - vided to all the peo ple liv ing in the com mu - nity and chil dren were treated for helminthic in fec tions and other com mon child hood dis eases. Parasitologic eval u a tion Fae ces sam ples were col lected and di - rect ex am i na tion of the sam ples was per - formed. The helminth in ten si ties were de - ter mined by the method of Kato (14). Strongyloides in ten sity was as sessed by the Baermann con cen tra tion tech nique (14). The num ber of helminth eggs was cal cu - lated per gram of fae ces (epg) in all the sam ples. To tal se rum IgE lev els These were mea sured by a cap ture ELISA de vel oped in our lab o ra tory and stan - dard ized against the com mer cial Phadebas IgE PRIST (Pharmacia Swe den) test. Briefly, flat-bot tom 96-well microtitre plates (Immunolon IV Dynatech Lab o ra to - ries Inc, Vir ginia USA) were coated over - night at 4 C with 1µg/well of murine monoclonal anti-hu man IgE (pro duced in the In sti tute for Child Re search, Perth Aus - tra lia) in car bon ated buffer (ph 9.6) and blocked with 1% bo vine se rum al bu min (BSA). The plates were washed with phos - phate buffer sa line con tain ing 0.1% Tween 20 (PBS-T, ph 7.4) then in cu bated with se - rum sam ples di luted 1:400 for 2 hours at 37 C. Af ter an other wash, biotinylated murine anti-hu man IgE di luted 1:500 (Vec - tor lab o ra to ries, Cal i for nia, USA) was added and in cu bated for 1 hour at 37 C. The plates were washed thor oughly and al lowed to re act with Avidin-Bi o tin-peroxidase com - plex (Vectastain kit, Vec tor lab o ra to ries) for 30 min utes at 37 C. -phenylenediamine (OPD; SIGMA, USA) plus H 2 O 2 were added, and the O.D. was read at 490 nm. A stan - dard ti tra tion curve us ing a pool of sera with high IgE lev els, ac cord ing to the Investigación Clínica 47(3): 2006

5 IgE re sponses in Warao Am er in dian chil dren 245 Phadebas IgE PRIST, was run with each as - say. Re sults were ex pressed as In ter na tional units/ml (IU/mL). Anti-Ascaris lumbricoides IgE lev els These were also mea sured by an ELISA de vel oped in our Lab o ra tory and stan dard - ized against the com mer cial RAST tech - nique. A. lumbricoides adult worm an ti gen (15), 3µg/well, was coated onto 96-well microplates (Immunolon IV, Dynatech Lab - o ra to ries Inc, Vir ginia USA) and in cu bated over night at 4 C. Ex cess an ti gen was washed off with PBS-T and the plates were blocked for two hours at 37 C with 1% BSA. The test un di luted sera, were plated and in - cu bated for 1 hour at 37 C. Af ter fur ther washes with PBS-T, the plates were in cu - bated with 1µg/well of murine monoclonal anti-hu man IgE (pro duced in the In sti tute for Child Re search, Perth, Aus tra lia) for 1 hour and then washed off with PBS-T, then the plates were in cu bated with peroxidase -con ju gated anti-mouse IgG di - luted 1:3000 (Sigma, USA). The wash ing pro cess was re peated and o-phenylene - diamine (OPD) plus H 2 O 2 were added. The op ti cal den si ties were read at 490nm. A stan dard ti tra tion curve us ing a pool of sera with high spe cific anti-ascaris IgE lev els in the com mer cial Phadebas test was run with each as say, and the re sults were ex pressed as Phadebas RAST units (PRU/mL). Lymphocyte surface markers Pe riph eral blood sam ples were ob - tained by venipuncture, and col lected into EDTA an ti co ag u lant. Rou tine haema to logi - cal stud ies were per formed in the clin i cal laboratory of the nearest Hospital (Tucupita, Delta Amacuro State), and flow cytometry as says were un der taken at the Mu nic i pal Blood Bank of Ca ra cas, us ing a FACScan Flow Cytometer (Becton Dickinson, San Jose, Cal i for nia, USA). Dual col our (FITC-PE) immunofluorescent stain ing of cell sur face antigens in unseparated blood was performed us ing monoclonal an ti bod ies for the de tec - tion of the fol low ing celular phe no types: T helper cells, CD3+CD4+, (FITC/PE), ac ti - vated B cells, CD20+CD21+, (PE/FITC), and B cells ex press ing the low af fin ity re - cep tor for IgE, CD20+CD23+, (FITC/PE). The monoclonal an ti bod ies used were pur - chased by DAKO, Den mark cells were ac quired for each anal y sis per formed. The re sults were ex pressed as ab so lute num ber of lym pho cytes/mm 3. Sta tis ti cal Anal y sis The to tal and spe cific IgE lev els and the eggs per gram of fae ces were logarithmically trans formed. Geo met ric mean and geo met ric mean + 1 stan dard de - vi a tion were cal cu lated for these pa ram e - ters. A para met ric anal y sis was per formed. Stu dents t test and Welch t test were used for com par ing means of the dif fer ent pa - ram e ters be tween the groups. Comparison be tween the pro por tions of chil dren was ac - cessed by the Fisher ex act test. The Pearson rank cor re la tion co ef fi cient was used to de - ter mine the sig nif i cance of the dif fer ent cor re la tions cal cu lated. RE SULTS We found that the de gree of pov erty, ac - cording to family income, was comparable between the two communities evaluated. However, extremely poor sanitary conditions were found among the Warao chil dren from Arawaymujo, with an el e vated prev a lence and intensity of gastrointestinal helminthic infection. In contrast, improved sanitary facilities and ad e quate ac cess to pri mary health care were found among the non-par a sit ized group from El Caigual (Ta ble I). De spite the high to tal se rum IgE lev els ob served in the par a sit - ized group (7630 UI/mL), only 32.7% of these chil dren ex hib ited clin i cally sig nif i - cant ( 0.7 PRU/mL) spe cific anti-ascaris Vol. 47(3): , 2006

6 246 Hagel y col. IgE lev els (geo met ric mean: 4.5 PRU/mL) ac cord ing to the in ter na tional RAST Clas si - fi ca tion (PHARMACIA, Swe den). These were clas si fied as high spe cific anti-ascaris IgE re spond ers. The oth ers had spe cific anti-ige lev els un der 0.7 PRU/mL (geo met - ric mean: 0.36 PRU/mL) and were con sid - ered as low spe cific-anti-ascaris IgE -re - spond ers". Geo met ric mean of Ascaris spe - cific IgE among the non-par a sit ized group was 0.46 PRU/mL. Ta ble II shows there was no sig nif i cant dif fer ence in the prev a lence of helminthic in fec tion be tween high spe cific anti-ascaris IgE re spond ers and their low re sponder coun ter parts. The prev a lence and in ten sity of Strongyloides in fec tion was very low in all the chil dren and was con sid ered as sub-clin - i cal. The intensity of Ascaris and Trichuris in fec tion was sig nif i cantly lower (p = and p < re spec tively) in the high spe cific anti-ascaris IgE re sponder group (Ta ble III). To tal se rum IgE lev els were ex - tremely higher in the Warao chil dren com - pared to the non-par a sit ized con trol group (p < 0.001), and they were also higher (p = ) in the high spe cific anti -IgE re sponder group (Ta ble III). We also found that the ab so lute num - ber of pe riph eral cir cu lat ing T cells (CD3+, CD3+CD4+) was significantly higher (p < ) in the Warao chil dren com - pared to the non-par a sit ized con trol group; the cir cu lat ing T helper (CD3+CD4+) cell sub-pop u la tion was sig nif i cantly higher (p < ) in the high spe cific anti-ige re sponder chil dren com pared to the low re sponder group. Sim i larly, B lym pho cyte cir cu lat ing sub-pop u la tions (CD20+, CD20+CD21+ and CD20+CD23+) were sig nif i cantly higher (p < 0.001) in the Warao chil dren (Ta ble IV). The ex pres sion of the low af fin ity IgE re cep tor (CD23) was sig nif i cantly higher (p < ) in the high re sponder group (Table IV). Significant correlations (Pearson rank: ; p < 0.001) be tween CD23 ex - TABLE II HELMINTHIC INFECTION IN A GROUP OF WARAO AMERINDIAN CHILDREN ACCORDING TO THEIR SPECIFIC ANTI-Ascaris IgE LEVELS Prevalence of total helminthes (%) Prevalence of Ascaris lumbricoides Prevalence of Trichuris trichuria Prevalence of Strongyloides stercoralis High specific Anti-Ascaris IgE Responders 0.7IU/mL (52) Low specific anti-ascaris IgE Responders < 0.7IU/mL (107) Statistical significance P = Odds ratio: P = Odds ratio: P = Odds ratio: P = Odds ratio: % confidence interval to to to Non-parasitized control group (70) to Investigación Clínica 47(3): 2006

7 IgE re sponses in Warao Am er in dian chil dren 247 TABLE III T OTAL SERUM IgE LEVELS IN A GROUP OF WARAO AMERINDIAN CHILDREN ACCORDING TO THEIR SPECIFIC ANTI- A scari s IgE LEVEL S G eometric mean of No of A scari s egg s / gram faeces (Geometric mean +1SD ) G eometric mean of N o /gram faeces (Geometric o f T r ichuris egg s mean +1SD) High specific a nti- A scari s Ig E Responders 0.7IU/m L (52) 3214 (8500) 1345 (3450) Low specific a nti- A scari s Ig E Responders < 0.7IU/mL (107) 5550 (12300) 2225 (3850) Statistical significance P = t: P < t: % confidence interval of the difference to to Non-parasitized control group (70) Geometric mean of total serum IgE levels P = to * * (Geometric mean +1 sd) (12650) (16300) t: (1100) * * P < t % confidence interval: to Between Low specific anti- A scari s IgE responders and non-parasitized control group. ABSOLUTE NUMBER OF TABLE IV PERIPHERAL CIRCULATING LYMPHOCYTES IN A GROUP OF T O THEIR SPECIFIC ANTI- A scari s IgE LEVEL S WARAO AMERINDIAN CHILDREN ACCORDING C C C C C Lymphocytes/ Responder s m m 3 0.7PRU/m L (52) D3+ D3+CD4+ D20+ D20+CD21+ D20+CD Non-responders < 0.7PRU/mL (107) Statistical significance P < t: P < t: P = t: P = t: P < t: % confidence interval of the difference to to to to to Non Warao Non Infected control group (70) Statistical significance P < t: P < 0,000 1 t: P < t: P < t: P < t: % confidence interval to to to to to Vol. 47(3): , 2006

8 248 Hagel y col. pres sion and to tal se rum IgE lev els and also with spe cific anti Ascaris IgE lev els (Pearson rank: 0.573; p < 0.05) were ob - served. More over, the num ber of cir cu lat ing CD20+ CD23+ cells cor re lated strongly (Pearson rank: ; p < 0.001) with the num ber of cir cu lat ing T helper cells (CD3+CD4+). DIS CUS SION The Warao peo ple con sti tute the most nu mer ous and eth ni cally con served Am er in - dian pop u la tion in Ven e zuela, de spite the hos tile en vi ron men tal con di tions that sur - round them (1). Prob a bly, the de vel op ment of adap tive de fence mech a nisms against in - fec tious dis eases could play an im por tant role in their sur vival (16). As ex pected, gas tro in tes ti nal helminths were highly prev a lent among chil dren from this Warao com mu nity liv ing in a san i tary de prived en vi ron ment, Ascaris lumbricoides be ing the most fre - quent. In agree ment with this sit u a tion, ex tremely el e vated lev els of to tal se rum IgE were ob served among this group of chil dren. In de pend ently of their ex tremely high to tal IgE lev els, the ma jor ity of the Warao chil dren stud ied were found to pro - duce scarcely de tect able lev els of spe cific anti-ascaris IgE; only a re duced group of chil dren were ca pa ble of mount ing a strong ef fec tive spe cific re sponse against this par a site. The prev a lence of helminthic in fec tion was sim i lar be tween both high and low IgE re sponder groups, in di cat ing a com pa ra ble risk of ex po sure to these par a - sites. How ever the in ten sity of Ascaris in - fec tion was sig nif i cantly lower in the high spe cific anti Ascaris IgE re sponder group. This is in agree ment with our pre vi ous find ings, where atopic chil dren from ru ral ar eas with an en hanced ca pac ity to pro - duce spe cific IgE an ti bod ies against these par a sites, had sig nif i cantly lower in ten si - ties of in fec tion than their non atopic coun ter parts (6), and sug gests that the ca - pac ity to es tab lish ef fi cient spe cific IgE re - sponses may be an ad van tage that con fers better de fence mech a nisms against helminthic in fec tions. In ad di tion, and prob a bly as a re flec - tion of the chronic stim u la tion of the im - mune sys tem by con tin u ous in fec tious dis - eases, par tic u larly those caused by gas tro in - tes ti nal par a sites, a sig nif i cant in crease in the num ber of cir cu lat ing T helper (CD3+ CD4+) and ac ti vated B cell (CD20+, CD20+CD21+) sub-populations was also found among these chil dren com pared to the non-par a sit ized con trol group. Cor re - spon dent to the el e va tion in the lev els of to - tal and spe cific anti Ascaris se rum IgE, the ex pres sion of the low af fin ity IgE re cep tor (CD20+ CD23+) was mark edly aug mented in the Warao chil dren, be ing ex tremely high in the high spe cific anti- Ascaris IgE re sponder group. More over, a strong cor - re la tion be tween the num ber of CD23+ B cells and both to tal and spe cific anti Ascaris se rum IgE lev els was also ob served. The mech a nisms by which helminth par a - sites may en hance CD23 ex pres sion are still not well elu ci dated. Ex per i men tal ev i dence ob tained from in vi tro as says have dem on - strated that ex cre tory-se cre tory com po - nents of filarial par a sites are ca pa ble of stim u lat ing the TH2 de rived, IL-4-de pend - ent ex pres sion of CD23 by hu man splenic B cells (17). Other au thors have pro posed that helminth pro teins with anti-pro te ase ac tiv i ties may af fect the pro cess ing of B cell sur face mol e cules in volved in IgE switch ing, par tic u larly CD23, and con se quently the reg u la tion of IgE syn the sis (18). In this study, we found a sig nif i cant cor re la tion be - tween the num ber of CD23+ B and CD4+T helper cir cu lat ing cells, in di cat ing that an - ti gens de rived from gas tro in tes ti nal Investigación Clínica 47(3): 2006

9 IgE re sponses in Warao Am er in dian chil dren 249 helminths, such as Ascaris lumbricoides, may stim u late the ex pres sion of the low af - fin ity IgE re cep tor (CD23) in a CD4+ de - pendent manner. Stud ies us ing in hib i tory anti CD21 an - ti bod ies have shown that CD23 binds to a sub type of CD21 (8), a mol e cule that has pre vi ously iden ti fied as the com ple ment re - cep tor 2 and as an ac ti va tor B cell fac tor (19). More over, it has been dem on strated that trig ger ing CD21 by re com bi nant scd23 en hances IgE pro duc tion in both T cell de pend ent and T cell in de pend ent sys - tems (20). Thus, it is pos si ble that in chil - dren pop u la tions liv ing in de prived san i tary en vi ron ments, at a sit u a tion of high bio - med i cal risk, early ex pres sion of CD21 re - cep tor may oc cur, fa vour ing the pair ing of CD21/CD23 pos i tive B cells in par a sit ized pop u la tions, there fore, en hanc ing the pro - duc tion of high lev els of to tal and spe cific IgE. How ever, other fac tors may also be in - volved in the gen er a tion of pro tec tive spe - cific anti-ascaris IgE re sponses. The ca pac - ity to rec og nize spe cific epitopes that may con fer pro tec tion against helminths (21) in these Warao chil dren must be iden ti fied. Other as pects, such as mal nu tri tion, that af fect the ca pac ity to de velop ef fi cient mem ory T helper re sponses (22) have been shown to be im por tant for the stim u la tion of high af fin ity spe cific IgE re sponses against these par a sites (23). On the other hand, re cent work per formed by Turner et al, has dem on strated a pos i tive as so ci a tion be tween block ing IgG4 an ti bod ies against Ascaris an ti gens and the in ten sity of Ascaris lumbricoides in fec tion in chron i - cally ex posed chil dren (24). There fore, the com plex in ter ac tions of dif fer ent fac tors that may de ter mine the ca pac ity to es tab - lish pro tec tive IgE re sponses against these par a sites, among these groups of chil dren, need fur ther in ves ti ga tion. ACKNOWLEDGEMENTS This work was sup ported by The Spe - cial Health Care Pro gram for Ven e zue lan Am er in dian Com mu ni ties (Fundación Proyecto País/Guardia Nacional de Ven e - zuela) and FONACIT G We thank Ge ne ral Vic tor Sa lom Ló pez (GN) and the Na tio nal Guard of Ve ne zue la for lo gis tic and tech ni cal sup port in field work. We are gra te ful to Dr. Ma ría Cris ti na Di Pris co and Dr. Neil Lynch for en cou ra - ging us to fo llow our in ves ti ga tions. We also thank MSc Ma ría Eu ge nia Pi nar di for re vie - wing the ma nus cript and Mrs. Loa na Gu tie - rrez for tech ni cal as sis tan ce. REFERENCES 1. Heinen D. Los Wa rao. In: Los Abo rí ge nes de Ve ne zue la. Fun da ción La Sa lle de Cien - cias Na tu ra les, Mon te Avi la Edi to res, Ca ra - cas, Ve ne zue la; pp: Ha gel I, Sal ga do A, Ro drí guez O, Ortiz D, Hur ta do M, Puc cio F, Di Pris co MC, Lat - touf JJ, Pa len que M, Gui llén M, Sa lom V, Lynch NR. Fac to res que in flu yen en la pre - va len cia e in ten si dad de las pa ra si to sis in - tes ti na les en Ve ne zue la. Ga cet Med Ca ra - cas 2001; 109(1): Dunne D, Butterworth A, Fulford A, Kamuki HC, Langley JG, Ouma JH, Capron CA, Pierce RJ, Sturrock RF. Im - mu nity af ter treat ment of hu man schis to - so mi a sis: as so ci a tion be tween IgE an ti bod - ies to adult worm an ti gens and re sis tance to re in fec tion. Europ J Immunol 1992; 22: Dunne DW, Web ster M, Smith P, Langley JG, Rich ard son BA, Fulford AJC, Butterworth AE, Sturrock RF, Kariuki HC, Ouma JH. The iso la tion of a 22Kd band af ter SDS-PAGE of Schistosoma mansoni adult worms and its use to dem - on strate that IgE re sponses against the an - ti gens, it con tains are as so ci ated with hu - man re sis tance to re in fec tion. Par a site Immunol 1997; 19: Vol. 47(3): , 2006

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11 IgE re sponses in Warao Am er in dian chil dren 251 against the in tes ti nal helminth Ascaris lumbricoides in mal nour ished chil dren. J Trop Pediatr 2002; 49: Turner JD, Faulk ner H, Kamgno J, Ken - nedy MW, Behnke J, Boussinesq M, Bradley JE. Al ler gen spe cific IgE and IgG4 are mark ers of re sis tance and sus cep ti bil - ity in a hu man in tes ti nal nematode in fec - tion. Mi crobes and In fec tion 2005; 7: Vol. 47(3): , 2006