ORIGINAL ARTICLE INTRODUCTION

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1 Allergology Interntionl. ;6:8-9 DOI:. llergolint.-oa-6 ORIGINAL ARTICLE Elevte Serum IgE ginst MGL_ in Ptients with Atopi Dermtitis n Cholinergi Urtiri Mkiko Hirgun, Tkki Hirgun,KoriIshii, Hienori Suzuki,, Akio Tnk, Yuhki Ynse, Shoji Mihr, Yoshinori Hrut, Nouoki Kohno n Mihihiro Hie ABSTRACT Bkgroun: MGL_ serete y Mlssezi gloos is ontine in humn swet n inues histmine relese from sophils in ptients with topi ermtitis (AD) t high positive rte. The ims of this stuy were to estlish the enzyme-linke immunosorent ssy (ELISA) mesuring speifi immunogloulins ginst MGL_ n to investigte the levels of these immunogloulins in ser of ptients with vrious llergi iseses. Methos: Purifie MGL_ from humn swet (QRX) n reominnt MGL_ (rmgl_) were prepre for ELISA. To quntify the mount of MGL_-speifi immunogloulins, the stnr serum ws rete y pooling ser of ptients with AD whose sophils relese histmine in response to QRX. A monolonl ntioy whih exhiite the highest neutrlizing ility ginst QRX ws estlishe s Smith-, n use s pture ntioy for the ssy of. A totl of 56 sujets [norml ontrols (n = ), AD (n = 6), holinergi urtiri (CU) (n = ), ronhil sthm (n = ), n llergi rhinitis (n = )] were enrolle in this stuy. Results: ELISA methos to quntify the speifi IgE, IgG n IgG ginst MGL_ in ser were suessfully estlishe. Levels of in ser of ptients with AD n CU were signifintly higher thn those of norml ontrols. Moreover, the levels of n rmgl_-speifi IgE in ptients with AD were signifintly orrelte with their isese severities. Conlusions: These ELISA methos to quntify the speifi immunogloulins ginst MGL_ re esy n useful mens to ssess llergy to MGL_. MGL_ ontine in swet is n importnt ntigen for ptients with AD n CU. KEY WORDS topi ermtitis, holinergi urtiri, ELISA, Mlssezi gloos, MGL_ INTRODUCTION Atopi ermtitis (AD) is n inflmmtory pruriti, hroni or hronilly relpsing skin isese ourring often in fmilies with other topi iseses. - The involvement of IgE hs een suggeste s n importnt ftor in the pthogenesis of AD, s in other topi iseses, suh s sthm or llergi rhinitis. Moreover, high levels of totl IgE in ser of ptients with AD, n severl reports presenting the effetiveness of omlizum for AD,5 enfore the importne of IgE in AD. Reently, filggrin she light on the evelopment of AD, 6 ut muttions of filggrin re lso etete in ptients with ihthyosis vulgris without AD. Moreover, only 5% of mil-to-moerte AD n 5% of moerte-to-severe AD ses hve revele filggrin muttions. 7 Thus, preise role of IgE in the pthogenesis of AD remins to e investigte. Numerous triggering ftors, suh s irritnts, erollergens, foo, miroil orgnisms, n sweting, re lso known to e involve in the evelopment n or the ggrvtion of AD. We n other uthors Deprtment of Dermtology, Deprtment of Moleulr n Internl Meiine, Grute Shool of Biomeil n Helth Sienes, Hiroshim University n Fulty of Humn Siene, Hiroshim Bunkyo Women s University, Hiroshim, Jpn. Conflit of interest: No potentil onflit of interest ws islose. Corresponene: Mihihiro Hie, MD,PhD, Deprtment of Dermtology, Grute Shool of Biomeil n Helth Sienes, Hiroshim University, Ksumi, Minmi-ku, Hiroshim 7 855, Jpn. Emil: eh we ro@hiroshim u..jp Reeive August. Aepte for pulition Otoer. Jpnese Soiety of Allergology Allergology Interntionl Vol 6, No, 8

2 Hirgun M et l. reporte tht skin test with utologous swet ws positive in the mjority of ptients with AD, 8- n linil symptoms of hilren with AD signifintly improve uring the summer if they took showers t shools., Moreover, we previously revele tht semi-purifie swet ntigen inue histmine relese from the sophils of 77% ptients of AD n 66% ptients of holinergi urtiri (CU), n the histmine relese y swet ntigen ws meite y speifi IgE. 9, Finlly, we extensively purifie histmine relese tivity of the semi-purifie swet ntigen y omintion of vrious hromtogrphies n ientifie puttive protein, MGL_ of Mlssezi gloos (M. gloos), s mjor llergen in humn swet. 5 MGL_ inue histmine relese of sophils otine from the mjority of ptients with AD. 5 In Jpn, ommeril histmine relese test (HRT) ginst swet ontining MGL_ hs een ville sine. However, HRT nees fresh loo ells of ptients n its results re qulittive rther thn quntittive. In the present stuy, to overome the isvntges of HRT, we estlishe enzyme-linke immunosorent ssys (ELISAs) to mesure speifi IgE ginst ntive MGL_ ollete from humn swet (QRX) n reominnt MGL_ (rmgl_) y using ser of ptients with AD n other llergi iseses. METHODS PATIENTS AND ETHICS A totl of 56 sujets were enrolle in this stuy. Sixty-three ptients with AD (6 men n 7 women; -65 yers of ge, men ± SD: 9.9 ±.5), ptients with holinergi urtiri (CU) n no history of AD ( men n women; 7-65 yers of ge, men ± SD:. ±.7) n ptients with ronhil sthm (BA) n without AD (5 men n 7 women; -8 yers of ge, men ± SD: 5.6 ± 6.) who visite Hiroshim University Hospitl were inlue in this stuy. Fourteen ptients with llergi rhinitis (AR) without other llergi iseses ( men n women; -75 yers of ge, men ± SD:. ± 5.5) n norml ontrol sujets (NC) without llergi symptoms (5 men n 8 women; - yers of ge, men ± SD: 6. ± 6.5) were reruite s volunteers n inlue in this stuy. The severity of AD ws evlute using severity inex of Jpnese guieline for AD. This stuy ws rrie out in orne with the guielines stte in the Delrtion of Helsinki n ws pprove y the Ethis Committee of Hiroshim University Institute of Biomeil & Helth Sienes (Approve No 556, 6). Written informe onsent ws otine from every sujet. PREPARATION OF PURIFIED MGL_ (QRX) AND RECOMBINANT MGL_ The semi-purifie swet ntigen purifie from humn swet of helthy volunteers esrie in the previous report, QR (semi-purifie swet ntigen), ws use for histmine relese test (HRT). QR ws further frtionte y gel hromtogrphy olumn, n the frtions with histmine relesing tivity were ollete s purifie MGL_ (QRX) s esrie previously. 5 QRX ws use for immunolot nlysis n ELISA. By using Esherihi oli (JM9) n pcol TF vetors oing MGL_, the poly histiine-tgge n trigger ftor (TF)-fuse rmgl_ protein (TF-rMGL_) ws prepre s esrie previously. 5 MONOCLONAL ANTIBODY AGAINST PURIFIED MGL_ (QRX) A mouse monolonl ntioy, Smith-, 5 whih hs neutrliztion ility ginst histmine relese tivity from sophils of ptients with AD in response to QRX, ws use for ELISA mesuring QRX-speifi IgE. WESTERN BLOT ANALYSIS Smples were loe into SDS-PAGE gel n trnsferre to polyvinyliene fluorie (PVDF) memrne s reporte elsewhere. 6 The memrnes were inute with AD stnr serum or nti-pent-his ntioy (Qigen, Hilen, Germny) t overnight. The memrne oun primry ntioies were visulize with horserish peroxise-onjugte seonry ntioies n hemiluminesene. HISTAMINE RELEASE TEST HRT with sophils otine from peripherl loo were performe s esrie previously. 7 Cells were stimulte with μg ml of got nti-humn IgE ntioy (Bethyl Lortories, Montgomery, TX, USA) n ng ml of QR. ESTABLISHMENT OF ELISA SYSTEMS FOR MEASUREMENT OF MGL_-SPECIFIC IMMU- NOGLOBULINS To quntify the mount of MGL_-speifi immunogloulins, the AD stnr serum ws prepre y pooling ser of ptients with AD whose sophils relese histmine in response to QR. Ninety-six well ELISA-pltes (High-Bining, Greiner io-one, Frikenhusen, Germny) were use for ll ELISA. For the mesurement of, 96 well pltes were iretly ote with μg ml QRX for iret ELISA or μg ml Smith- ntioy for snwih ELISA in phosphte uffere sline (PBS) without ny rrier proteins n inute over night t. The remining non-speifi ining sites were loke with % ovine serum lumin PBS (BSA- PBS) for hour t room temperture. The pltes ote with Smith- ntioy were sequentilly inute with μg ml QRX in.% BSA-PBS for 9 minutes t room temperture. The wells were inute 8 Allergology Interntionl Vol 6, No,

3 ELISA of Serum IgE ginst MGL_ with μl of ptient ser ilute : with % BSA- PBS t room temperture for 9 minutes n susequently with peroxise-lele ntioy to humn IgE (ε-hin speifi) (KPL, Githersurg, MD, USA) ilute : with % BSA-PBS, t room temperture for hour. For the mesurement of rmgl_-speifi IgE, 96 well pltes were iretly ote with μg ml TFrMGL_ or the sme onentrtion of TF lone ilute in PBS without rrier protein, n inute over night t. The remining non-speifi ining sites were loke with % BSA-PBS for hour t room temperture. The wells were inute with μl of ptient ser ilute : with % BSA-PBS t room temperture for hours n susequently with peroxise-lele ntioy to humn IgE esrie ove n ilute : with % BSA-PBS, t room temperture for hour. For the mesurement of rmgl_-speifi IgG, 96 well pltes were iretly ote with μg ml TFrMGL_ or TF lone in PBS without rrier protein, t over night. The remining non-speifi ining sites were loke with 5% skim-milk in PBS (skim-milk-pbs) for hour t room temperture. The wells were inute with μl of ptient ser ilute : in % skim-milk-pbs t room temperture for hours n susequently with peroxiselele ntioy ginst humn IgG (F speifi) (KPL) ilute : in % skim-milk-pbs t room tempertureforhour. For the mesurement of rmgl_-speifi IgG, 96 well pltes were iretly ote with μg ml TFrMGL_ or TF lone in PBS without rrier protein, n inute over night t. The remining non-speifi ining sites were loke with 5% skimmilk-pbs for hour t room temperture. The wells were inute with μl of ptient ser ilute : with % skim-milk-pbs t room temperture for hours n susequently with peroxise-lele ntioy to humn IgG (γ hin speifi) (Southern Bioteh) ilute : 5 with % skim-milk-pbs, t room temperture for hour. All inutions were followe y three wshes with wsh uffer (PBS with.5% Tween-), n the ntioy ining ws visulize y inution with TMB Mirowell Peroxise Sustrte n TMB Stop Solution (KPL). The optil ensity t 5 nm (OD5) wsthenrewithnutomtipltereer(benhmrkplus,bio-r,herules,ca,usa).whenwe use TF-rMGL_ s n ntigen, we lulte ifferenes etween OD5 otine with TF-rMGL_ n tht with TF lone. Smples were teste in uplite, n the onentrtions of speifi ntioies (Units ml) were lulte y using stnr urve otine from seril ilution of the AD stnr serum stte ove. The units of eh speifi ntioy in one milliliter of the AD stnr serum were efine s Units. MEASUREMENT OF MALASSEZIA-SPECIFIC IgE AND TARC Serum levels of Mlssezi-speifi IgE (UA ml) n TARC (pg ml) were mesure y using the Immuno- CAP instrument (Phi AB, Portge, MI, USA) n ELISA in SRL In. (Tokyo, Jpn), respetively. STATISTICAL ANALYSIS Anlyses were performe y the use of GRAPHPAD PRISM version 5. (GrphP Softwre, Sn Diego, CA, USA). All t re presente s men ± SEM. RESULTS ESTABLISHMENT OF THE ELISA SYSTEM FOR IgE, IgG, AND IgG AGAINST MGL_ The semi-purifie swet ntigen, QRX (7 kd), TF (6 kd) n TF-rMGL_ (77 kd) were etete with immunolots y using AD stnr serum n or nti-polyhistiine-tg ntioy (Supplementry Fig. ). However, with iret ELISA using QRX s ote ntigen, we oul hrly etet QRX-speifi IgE (Fig. ), IgG n IgG (t not shown). The minimum etetion limit of the snwih ELISA for y using Smith-ntioy s ote (pture) ntioy ws gretly improve s ompre with the iret ELISA (Fig. ). In ontrst to IgE ginst QRX, IgG n IgG ginst QRX oul not e etete y the snwih ELISA using Smith- ntioy ue to ross tivity of Smith-ntioy for the seonry ntioies ginst humn IgG n IgG (t not shown). However, speifi inings to rmgl_ y IgE, IgG n IgG were ll suessfully etete with the iret ELISA (Fig. -). rmgl_-specific IgE VS QRX-SPECIFIC IgE The onentrtions (Units ml) of QRX-speifi-IgE n rmgl_-speifi-ige in ser of ptients with AD were highly orrelte (Fig., R =.95, P <., N = 6). The levels of QRX- or rmgl_- speifi IgE in ser n histmine relese inue y QR from sophils of the ptients with AD were lso orrelte (Fig., R =.778, P <., N = 5) (Fig., R =.78, P <., N = 5). Aoring to the ROC urves of or rmgl_- speifi IgE n HRT (Fig., e) mong ptients n NC, we efine the ut off vlues s 5 Units ml for QRX speifi-ige (sensitivity 8.9%, speifiity 85.7%, P <., N = ), n.6 Units ml for rmgl_ -IgE speifi-ige (sensitivity 8.8%, speifiity 8.%, P <., N = ). LEVELS OF QRX- AND rmgl_-specific IgE IN PATIENTS WITH EACH DISEASE, AND COR- RELATIONS WITH SEVERITY OF AD The levels of in ser of ptients with AD n those of ptients with CU were signifintly higher thn those of NC (P <., P <.5) n p- Allergology Interntionl Vol 6, No, 85

4 Hirgun M et l. Bining of speifi IgE (OD5) QRX (Snwih) QRX (Diret orting)..... Dilution of AD stnr serum Bining of speifi IgE (OD5) TF-rMGL_ TF.... Dilution of AD stnr serum Bining of speifi IgG (OD5) TF-rMGL_ TF..... Dilution of AD stnr serum Bining of speifi IgG (OD5) TF-rMGL_ TF.... Dilution of AD stnr serum Fig. Stnr urves of MGL_-speifi immunogloulins plotte y using AD stnr serum in ELISA. in the seril ilute AD stnr serum ws mesure y iret n snwih ELISA (). rmgl_-speifi IgE (), rmgl_-speifi IgG (), n rmgl_- speifi IgG () in the seril ilute AD stnr serum were mesure y iret ELISA. Eh pnel showe representtive t expresse s mens of OD5 vlues one in uplite tients with BA (P <., P <.) (Fig. ). Likewise, the level of rmgl_-speifi IgE in ser of ptients with AD ws signifintly higher thn those of NC (P <.5) n ptients with BA (P <.) (Fig. ). The level of rmgl_ speifi-ige in ser of ptients with CU ws signifintly higher thn tht with BA (P <.), ut not tht of NC. Aoring to the ut off vlues efine y ROC urves, the rte of -positive ptients with AD ws signifintly higher thn those of NC (P <.). However, there were no signifint ifferenes etween NC n CU in the rte of ptients with positive n rmgl_-speifi IgE in ser, respetively (Tle ). Moreover, the rte of rmgl_-speifi IgE-positive ptients with BA ws signifintly lower thn tht in NC (P <.). Disese severities n the levels of n rmgl_-speifi IgE in ptients with AD were signifintly orrelte (Fig., R =.568, P <.) (Fig., R =.8, P <.). LEVELS OF rmgl_-specific IgG AND IgG IN PATIENTS WITH EACH DISEASE, AND COR- RELATIONS WITH SEVERITIES OF AD The levels of rmgl_-speifi IgG in ptients with AD ws signifintly higher thn tht of BA (P <.), ut the ifferene in the levels of rmgl_- speifi IgG in ptients with AD n those of the other iseses ws not pprent (Fig., ). The isese severities n the levels of rmgl_-speifi IgG n IgG in ptients with AD were wekly ut signifintly orrelte (Fig., R =.9, P =.) (Fig., R =.8, P =.). MGL_-SPECIFIC IgE IS CORRELATED WITH rmgl_-specific IgG AND IgG IN PA- TIENTS WITH AD BUT NOT IN PATIENTS WITH CU The levels of rmgl_-speifi IgE n rmgl_ -speifi IgG or IgG in ptients with AD were signifintly orrelte (Fig. 5, R =.69, P =., N = 6) (Fig. 5, R =.5, P <., N = 6). The levels of totl serum IgE n tht of rmgl_- speifi IgE or in ptients with AD were even more signifintly orrelte (Fig. 5, R =.7, P <., N = 9) (Fig. 5, R =.697, P <., N = 9). Moreover, the serum levels of QRXspeifi IgE n rmgl_-speifi IgE in ptients with AD were wekly ut signifintly orrelte with serum TARC (Supplementry Fig., R =.66, P =.67, N = 6) (Supplementry Fig., R =., P =.8, N = 6). However, no orreltions were o- 86 Allergology Interntionl Vol 6, No,

5 ELISA of Serum IgE ginst MGL_ rmgl_-speifi IgE N = 6 R =.95 P <. rmgl_-speifi IgE N = 5 R =.778 P < Histmine relese test ginst QR/ nti-ige (%) N = 5 R =.78 P < Histmine relese test ginst QR/ nti-ige (%) e Sensitivity (%) 5 HRT positive N = 66 HRT negtive N = 5 P <. 6 8 % - Speifiity (%) 5 Sensitivity (%) HRT positive N = 66 HRT negtive N = 5 P <. 6 8 % - Speifiity (%) Fig. Correltion etween QRX-speifi IgE, rmgl_-speifi IgE, n sophil histmine relese ginst QR. Levels of QRX- n rmgl_-speifi IgE in ser of ptients with AD (). Histmine releses of sophils inue y QR re orrelte with serum levels of QRX-speifi IgE () n rmgl_-speifi IgE () in ptients with AD. Histmine relese ws expresse s (net histmine relese inue y QR) / (net histmine relese inue y nti-ige) (%). ROC urves of () n rmgl_-speifi IgE (e) were rete. tine etween rmgl_-speifi IgE n rmgl_ -speifi IgG or IgG, or etween rmgl_ speifi IgE or n totl serum IgE in ptients with CU (Fig. 6). No orreltion ws oserve etween or rmgl_ speifi IgE n the numer of peripherl eosinophils in ptients with CU (Supplementry Fig. ). MALASSEZIA-SPECIFIC IgE AND MGL_- SPECIFIC IgE WERE CORRELATED, BUT NOT COMPLETELY MATCHED The levels of or rmgl_-speifi IgE n the levels of Mlssezi-speifi IgE were signifintly orrelte (Fig. 7, R =.79, P <., N = 59) (Fig. 7, R =.7, P <., N = 59), ut not ompletely mthe in ptients with AD. The levels of Mlssezi-speifi IgE in ptients with AD were Allergology Interntionl Vol 6, No, 87

6 Hirgun M et l. * *** *** ** NC AD CU BA AR rmgl_-speifi IgE * *** *** NC AD CU BA AR R =.568, P <. R =.8, P <. NC Mil Moerte Severe Most severe AD rmgl_-speifi IgE NC Mil Moerte Severe Most severe AD Fig. Comprisons of serum levels of QRX- n rmgl_-speifi IgE mong iseses n orreltions with severities of AD. Serum levels of () n rmgl_-speifi IgE () in ptients with eh isese were plotte n nlyze y Kruskl-Wllis test. The serum levels of QRX-speifi IgE n rmgl_-speifi IgE in ptients showing vrious severities of AD were plotte n nlyze y Spermn rnk orreltion (, ). NC, norml ontrols; AD, topi ermtitis; CU, holinergi urtiri; BA, ronhil sthm; AR, llergi rhinitis. *P <.5, **P <., ***P <.. Tle Numer of ptients with positive or negtive QRX-speifi IgE n rmgl_-speifi IgE in eh isese QRX-speifi IgE NC AD CU BA AR Positive Negtive Positive rte P-vlue -.** rmgl_-speifi IgE NC AD CU BA AR Positive 9 9 Negtive Positive rte P-vlue **.75 Positive rtes were ompre with NC y Chi-squre test. NC, norml ontrols; AD, topi ermtitis; CU, holinergi urtiri; BA, ronhil sthm; AR, llergi rhinitis. **P <.. lso highly orrelte with totl serum IgE (Supplementry Fig., R =.79, P <., N =.6), n wekly orrelte with eosinophils (%) (Supplementry Fig., R =.8, P =.9, N = 9). DISCUSSION In this stuy, we estlishe ELISA systems to mesure speifi IgE, IgG n IgG ginst ntive n 88 Allergology Interntionl Vol 6, No,

7 ELISA of Serum IgE ginst MGL_ rmgl_-speifi IgG * NC AD CU BA AR rmgl_-speifi IgG NC AD CU BA AR rmgl_-speifi IgG R =.9, P =. NC Mil Moerte Severe Most severe AD rmgl_-speifi-igg R =.8, P =. NC Mil Moerte Severe Most severe AD Fig. Serum levels of rmgl_-speifi IgG n IgG in iseses n their orreltions with severities of AD. Serum levels of rmgl_-speifi IgG () n IgG () in eh isese were plotte n nlyze y Kruskl-Wllis test. The serum levels of rmgl_-speifi IgG () n IgG () in ptients with vrious severities of AD were plotte n nlyze y Spermn rnk orreltion. *P <.5. rmgl_ in ser of ptients with AD n other iseses. MGL_ is seretory protein of M. gloos n mjor ntigen ontine in humn swet n highly inues histmine relese from sophils of mny ptients with AD. By using monolonl ntioy ginst purifie MGL_ (QRX), Smith, s pture ntioy, we oul etet the speifi IgE ginst QRX in ser of ptients with AD with high sensitivity. We hve lrey reporte tht IgE from ptients with AD reognize the steri struture, ut not short mino-i sequene of MGL_. 5 The iffiulty of the iret ELISA using QRX might e ue to its ntigeni property. In ontrst to QRX, TF-rMGL_ oul e pplie to the iret ELISA to mesure rmgl_- speifi IgE, IgG n IgG without the pture ntioy. This vntge of TF-rMGL_ over QRX oul e ue to the tg protein n or TF tht might rise MGL_ on the ELISA plte so s to mke its epitope more esily essile y humn ntioies. Moreover, t of n rmgl_ -speifi IgE in ser of ptients with AD re highly orrelte. However, QRX speifi-ige showe higher sensitivity n speifiity ginst HRT thn rmgl_. Suh ifferenes my e ue to post-trnsltionl moifition of ntive MGL_ in QRX, ut whih is lke y rmgl_ synthesize y Esherihi oli. Sine, QRX is purifie from swet of humn volunteers, it is extremely sre n iffiult to otin in suffiient mounts for ssys in routine linil prties. We re urrently estlishing metho to purify lrge mounts of ntive MGL_ from ulture superntnt of M. gloos. In ptients with AD, the serum levels of speifi IgE ginst MGL_ were signifintly higher thn those of NC. Moreover, the levels of MGL_- speifi IgE in ptients with AD were well orrelte with isese severities of AD n serum levels of totl IgE. These results inite tht the mesurement of MGL_-speifi IgE is useful not only to evlute the llergy to MGL_ in swet ut lso to evlute the isese severity of AD. However, serum levels of MGL_-speifi IgE showe only wek orreltion with serum TARC. It might e ue to more rpi hnges of TARC thn MGL_-speifi IgE in ssoition with isese severities.,8 Therefore, the levels of QRX- n rmgl_-speifi IgE re likely more suitle for the evlution of long-term isese Allergology Interntionl Vol 6, No, 89

8 Hirgun M et l. rmgl_-speifi IgE N = 6 R =.69 P =. rmgl_-speifi IgG rmgl_-speifi IgE N = 6 R =.5 P <. rmgl_-speifi IgG rmgl--speifi IgE N = 9 R =.7 P <. 5 Totl IgE (Log IU/ml) N = 9 R =.697 P <. 5 Totl IgE (Log IU/ml) Fig. 5 Correltions mong rmgl_-speifi immunogloulins in ser of ptients with AD. Signifint orreltions were oserve etween serum levels of rmgl_-speifi IgE n rmgl_-speifi IgG (), rmgl_-speifi IgG (), or totl IgE () in ptients with AD. The levels of serum totl IgE were lso signifi ntly orrelte with QRX-speifi IgE () in ptients with AD. severities rther thn short-term isese tivities of AD. The presene of serum IgG ginst swet ntigens, were suggeste y our previous stuy using histmine relese-neutrliztion ssy. 7 However, there were no signifint ifferenes etween the levels of rmgl_-speifi IgG or IgG in ptients with AD n those of NC. These results suggest tht oth ptients with AD n helthy iniviuls my e sensitize with MGL_, ut IgE lss swithing ginst MGL_ ours muh more strongly in ptients with AD thn in NC, lthough the opportunity of sensitiztion with MGL_ exists even in norml ontrols. Thus, for the ptients with AD, the mesurement of IgE ginst MGL_ is more importnt thn tht of IgG or IgG ginst rmgl_. The influene of Mlssezi speies on the pthogenesis of AD hs ttrte ttention in reent yers. Mny stuies hve shown the effetiveness of ntifungl therpy on AD with Mlssezi llergy, 9 n the ssoition of high levels of Mlssezi-speifi IgE n severe AD., Ishishi et l. ientifie kd protein (MGp) s mjor ntigen of M. gloos for serum IgE of ptients with AD. Moreover, Dri et l. 9 reporte tht the peripherl loo mononuler ells of ptients with AD who hve speifi IgE ginst Mlssezi showe n inrese prolifertion n n inrese proution of inflmmtory ytokines upon the exposure to Mlssezi ompre with ells from helthy ontrol sujets. The ientifition of MGL_ s histmine relesing ntigen in humn swet hs revele the involvement of M. gloos in type llergy ginst swet oserve in ptients with AD n or CU. However, western lotting y using ser of ptients with AD for lystes or ulture superntnt of M. gloos shows mny ns inluing MGp, 5, wheres ptients with AD ret to the frtions of swet elute etween moleulr mrkers of 7 kd n.5 kd y gel hromtogrphy. 9 Moreover, in the present stuy, serum levels of MGL_-speifi IgE n Mlssezi-speifi IgE were not ompletely mthe, suggesting tht the importne of MGL_-speifi IgE rther thn IgE ginst the whole Mlssezi ntigens in ptients whose symptoms re exerte y sweting. The reltionship etween MGL_ n CU is in- 9 Allergology Interntionl Vol 6, No,

9 ELISA of Serum IgE ginst MGL_ rmgl_-speifi IgE N = R =.87 P =.6856 rmgl--speifi IgG rmgl_-speifi IgE N = R =. P =.55 rmgl_-speifi IgG rmgl_-speifi IgE N = 6 R =.8 P =.8 Totl IgE (Log IU/ml) N = 6 R =.6 P =.9 Totl IgE (Log IU/ml) Fig. 6 Correltions mong rmgl_-speifi immunogloulins in ser of ptients with CU. No signifint orreltions ws oserve etween serum levels of rmgl_-speifi IgE n IgG (), IgE n IgG () or the speifi IgE n totl serum IgE (). Likewise, no pprent orreltion ws oserve etween serum levels of n totl serum IgE in ptients with CU (). N = 59 R =.79 P <. - Mlssezi-speifi IgE (Log UA/ml) rmgl_-speifi IgE N = 59 R =.7 P <. - Mlssezi-speifi IgE (Log UA/ml) Fig. 7 Correltion etween serum levels of MGL_-speifi IgE n Mlssezi-speifi IgE. Serum levels of Mlssezi-speifi IgE n QRX-speifi IgE () or rmgl_-speifi IgE () in ptients with AD were signifi ntly orrelte. triguing. In the present stuy, we revele tht the levels of QRX speifi-ige in ptients with CU were lso higher thn tht of NC n BA. Tkhgi et l. lso reporte tht the levels of sophils histmine relese ginst QR in ptients with CU were signifintly higher thn tht of NC. On the other hn, the levels of rmgl_ speifi-igg or IgG in ptients with CU were not orrelte with their levels of QRX- Allergology Interntionl Vol 6, No, 9

10 Hirgun M et l. or rmgl_-speifi IgE. Tking into ount tht oth IgG n IgG ginst MGL_ likely neutrlize the histmine relese tivity of MGL_, the presene of IgE over IgG n IgG in their ining tivity to MGL_ my e ritil in the pthogenesis of CU. Severl groups hve lrey performe linil trils of hyposensitiztion therpy for CU y the use of utologous swet. -5 Moreover, it is known tht ntigen-speifi immunotherpy for llergi rhinitis inues the inrese of ntigen-speifi IgG. 6,7 Therefore, the methos to evlute levels of serum IgG or IgG ginst MGL_ might e useful to monitor the effetiveness of immunotherpy on ptients with CU. The levels of QRX speifi-ige in ptients with AR tene to e higher thn tht of NC, lthough this ifferene ws not sttistilly signifint. It might e ue to the oloniztion of M. gloos in nsl vity, 8 n suggests tht MGL_ might lso e ustive ntigen for ertin popultion of ptients with AR. In onlusion, MGL_ is n importnt ntigen in swet n the methos to quntify the speifi IgE ginst MGL_ in ser y ELISA were useful mens to ignose llergy to MGL_ ontine in swet n estimte long term isese severities of AD. The mesurement of in ptients with CU ws lso useful for the ignosis n oul e lue to lrify the mehnism of CU. Further stuies on the reltionship etween AD or CU n MGL_ in swet re neee. ACKNOWLEDGEMENTS We thnk for Ms. Kzue Uhi for the expert tehnil ssistnes, n Dr. Fiz Kermni for his ritil review of the mnusript. This work ws supporte y in prt, Helth Siene Reserh Grnts n Helth n Lour Sienes Reserh Grnts from Ministry of Helth, Lour n Welfre of Jpn; A-STEP progrm of Jpn Siene n Tehnology Ageny; Knowlege Cluster Inititive from the Ministry of Eution, Culture, Sports, Siene n Tehnology (MEXT) of Jpn. SUPPLEMENTARY MATERIALS Supplementry Fig. - re ville online. REFERENCES. Ring J, Alomr A, Bieer T et l. Guielines for tretment of topi ezem (topi ermtitis) prt I. JEurA Dermtol Venereol ;6:5-6.. Ring J, Alomr A, Bieer T et l. Guielines for tretment of topi ezem (topi ermtitis) Prt II. JEurA Dermtol Venereol ;6: Ktym I, Kohno Y, Akiym K et l. Jpnese guieline for topi ermtitis. Allergol Int ;6:5-.. Kim DH, Prk KY, Kim BJ, Kim MN, Mun SK. Antiimmunogloulin E in the tretment of refrtory topi ermtitis. Clin Exp Dermtol ;8: Quist SR, Amh A, Goppner D et l. Long-term tretment of severe relitrnt topi ermtitis with omlizum, n nti-immuno-gloulin E. At Derm Venereol ;9: Plmer CN, Irvine AD, Terron-Kwitkowski A et l. Common loss-of-funtion vrints of the epierml rrier protein filggrin re mjor preisposing ftor for topi ermtitis. Nt Genet 6;8: MAleer MA, Irvine AD. The multifuntionl role of filggrin in llergi skin isese. J Allergy Clin Immunol ; : Ahi J, Aoki T, Ymtoni A. Demonstrtion of swet llergy in holinergi urtiri. JDermtolSi99;7: Hie M, Tnk T, Ymmur Y, Koro O, Ymmoto S. IgE-meite hypersensitivity ginst humn swet ntigen in ptients with topi ermtitis. At Derm Venereol ;8:5-.. Fukung A, Bito T, Tsuru K et l. Responsiveness to utologous swet n serum in holinergi urtiri lssifies its linil sutypes. J Allergy Clin Immunol 5; 6:97-.. Kmeyoshi Y, Tnk T, Mohizuki M et l. [Tking showers t shool is enefiil for hilren with severer topi ermtitis]. Arerugi 8;57:-7 (in Jpnese).. Murot H, Tkhshi A, Nishiok M et l. Showering reues topi ermtitis in elementry shool stuents. Eur J Dermtol ;:-.. Tnk A, Tnk T, Suzuki H, Ishii K, Kmeyoshi Y, Hie M. Semi-purifition of the immunogloulin E-swet ntigen ting on mst ells n sophils in topi ermtitis. Exp Dermtol 6;5:8-9.. Tkhgi S, Tnk T, Ishii K et l. Swet ntigen inues histmine relese from sophils of ptients with holinergi urtiri ssoite with topi ithesis. Br J Dermtol 9;6: Hirgun T, Ishii K, Hirgun M et l. Fungl protein MGL_ in swet is n llergen for topi ermtitis ptients. J Allergy Clin Immunol ;:68-5.e. 6. Towin H, Stehelin T, Goron J. Eletrophoreti trnsfer of proteins from polyrylmie gels to nitroellulose sheets: proeure n some pplitions. Pro Ntl A Si U S A 979;76: Shino H, Ishii K, Ynse Y, Suzuki H, Hie M. Histmine relese-neutrliztion ssy for ser of ptients with topi ermtitis n or holinergi urtiri is useful to sreen type I hypersensitivity ginst swet ntigens. Arh Dermtol Res ;: Kkinum T, Nkmur K, Wkugw M et l. Thymus n tivtion-regulte hemokine in topi ermtitis: Serum thymus n tivtion-regulte hemokine level is losely relte with isese tivity. J Allergy Clin Immunol ;7: Dri K, Hostetler SG, Behtel MA, Zirws M. The role of Mlssezi in topi ermtitis ffeting the he n nek of ults. JAmADermtol9;6:5-6.. Sonesson A, Brtosik J, Christinsen J et l. Sensitiztion to skin-ssoite miroorgnisms in ult ptients with topi ermtitis is of importne for isese severity. At Derm Venereol ;9:-5.. Zhng E, Tnk T, Tjim M et l. Anti-Mlssezispeifi IgE ntioies proution in Jpnese ptients with he n nek topi ermtitis: Reltionship etween the level of speifi IgE ntioy n the oloniz- 9 Allergology Interntionl Vol 6, No,

11 ELISA of Serum IgE ginst MGL_ tion frequeny of utneous Mlssezi speies n linil severity. J Allergy (Ciro) ;: Ishishi Y, Kto H, Ashi Y, Sugit T, Nishikw A. Ientifition of the mjor llergen of Mlssezi gloos relevnt for topi ermtitis. JDermtolSi9;55: Tnk T, Ishii K, Suzuki H, Kmeyoshi Y, Hie M. [Cholinergi urtiri suessfully trete y immunotherpy with prtilly purifie swet ntigen]. Arerugi 7;56:5-7.. Kozru T, Fukung A, Tguhi K et l. Rpi esensitiztion with utologous swet in holinergi urtiri. Allergol Int ;6: Bito T, Sw Y, Tokur Y. Pthogenesis of holinergi urtiri in reltion to sweting. Allergol Int ;6: Sing GW, Shmji MH, Joson MR et l. Sulingul grss pollen immunotherpy is ssoite with inreses in sulingul Foxp-expressing ells n elevte llergen-speifi immunogloulin G, immunogloulin A n serum inhiitory tivity for immunogloulin E- filitte llergen ining to B ells. Clin Exp Allergy ;: Klimek L, Shenzielorz P, Pinol R, Pfr O. Speifi suutneous immunotherpy with reominnt grss pollen llergens: first rnomize ose-rnging sfety stuy. Clin Exp Allergy ;: Zhng E, Tnk T, Tsuoi R, Mkimur K, Nishikw A, Sugit T. Chrteriztion of Mlssezi miroiot in the humn externl uitory nl n on the sole of the foot. Miroiol Immunol ;56:8-. Allergology Interntionl Vol 6, No, 9