comparison of iga-class and endomysium antibodies

Gut, 1989, 30, 1225-1232

Comparison of IgA-class reticulin and endomysiumantibodies in coeliac disease and dermatitisherpetiformisOLAVI HALLSTROM

From the Department of Clinical Microbiology and Immunology, University Central Hospital of Tampere,Tampere, Finland

SUMMARY The occurrence of IgA class reticulin and endomysium antibodies was examined with thestandard immunofluorescence method in coeliac disease and dermatitis herpetiformis. Similar highantibody frequencies were detected in 32 untreated adults (91%) and 18 children (100%) with coeliacdisease and in 14 dermatitis herpetiformis patients with subtotal villous atrophy (reticulin antibodies93% and endomysium antibodies 100%). The specificity of IgA class reticulin antibodies andendomysium antibodies was high because all 45 adult patients with ulcerative colitis or Crohn'sdisease, 24 non-coeliac children with abdominal symptoms and 99/100 healthy blood donors were

negative for these antibodies. The only positive blood donor had both IgA class reticulin antibodiesand endomysium antibodies but also she was found to have coeliac disease. IgA class reticulinantibodies and endomysium antibodies declined in parallel during treatment with a gluten free dietand increased on gluten challenge. This suggests that these antibodies can be used to screen forgluten sensitive enteropathy and to monitor dietary treatment. To characterise the tissue specificityof reticulin antibodies and endomysium antibodies four positive sera were absorbed with human andseveral rodent liver homogenates. Absorption with rat or other rodent livers removed the rodent-specific reticulin antibodies but not the reticulin antibodies detectable with human tissues or theendomysium antibodies detectable with monkey oeosophagus. These results show that reticulinantibodies can be divided into the rat and human subtypes. The human subtype could not beseparated from endomysium antibodies in the present absorption experiments.

Reticulin antibodies frequently occur in the sera ofpatients with coeliac disease and dermatitis herpeti-formis and the RI-type of reticulin antibodies havebeen proposed as a screening test for gluten sensitiveenteropathy.' In coeliac disease, reported low fre-quencies (18%-46%) and low specificity (75%-85%)of reticulin antibodies seem to mainly concern IgGclass.`'2 In contrast, IgA class reticulin antibodiesseem to be more sensitive and specific." 12 We pre-

Address for correspondence: Dr 0 Hallstrom, Department of ClinicalMicrobiology and Immunology, University Central Hospital of Tampere,SF-3352()Tampere, Finland.

Accepted for publication 19 January 1989.

viously found a very high (97%) sensitivity andspecificity (98%) of the IgA class reticulin antibodiesin children with coeliac disease,'3 and a similar highsensitivity in dermatitis herpetiformis patients withsubtotal villous atrophy.'4 1'A new group of connective tissue antibodies, the

endomysium antibodies, also determined withimmunofluorescence, and recently described byChorzelski et al,'6 seem to be closely related toreticulin antibodies. Both reticulin and endomysiumantibodies are directed against 'reticulin like', silverstain positive fibres in connective tissues.6 17 Reticulinantibodies are known to react both with human andvarious rodent tissues.'"'8 Endomysium antibodies,

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in contrast, have been claimed to be more speciesspecific, and react only with the connective tissue (theendomysium) around smooth muscle fibres in theintestinal tract of primates.'""" An almost 100%sensitivity and specificity has been reported for IgAclass endomysium antibodies in coeliac disease and indermatitis herpetiformis patients with damagedjejunal mucosa"' and endomysium antibodies seemthus to be a new valuable screening test for glutensensitive enteropathy.The purpose of this study was to compare the

frequencies of IgA class reticulin antibodies andendomysium antibodies in adults and children withcoeliac disease, and in dermatitis herpetiformispatients with normal and damaged jejunal mucosa.Moreover, the specificity of reticulin and endo-mysium antibodies was examined by using differenthuman and animal tissues as substrates and byperforming absorption studies with rodent andhuman liver homogenates.

Methods

PATI ENNS

COELIAC DISEASESera from 32 untreated adult patients with biopsyproven coeliac disease, mean age 36 years (range 18-63), were examined. A second serum sample wasobtained from four patients who after four to 32months diet treatment had a normal jejunal mucosa.

Sera from 14 children with flat small intestinalmucosa on their first biopsy were studied compara-tively for reticulin antibodies and endomysium anti-bodies. In all children jejunal mucosa recovered on agluten free diet but later they will undergo glutenchallenge. The mean age was 11 years (range 2-16).Moreover, serum samples were obtained from fourchildren before and after gluten challenge. Thesechildren had been on a gluten free diet for five years,had a normal jejunal mucosa, and underwent a two tonine months gluten challenge during which all fourdeveloped a flat jejunal mucosa.

DERMATITIS HERPETIFORMISSera from 23 adult dermatitis herpetiformis patients,mean age 38 years (range 18-64), on a normal glutencontaining diet were examined. All patients hadgranular IgA deposits in the skin, and jejunal biopsyspecimens were graded as previously described.'4Fourteen patients had subtotal villous atrophy, fivehad partial villous atrophy and four slight changesor normal mucosa. A second serum sample wasobtained from 10 patients after four to 19 months on agluten free diet and from eight patients after three to14 months on a normal diet.

CONTI ROLSControl sera were obtained from 24 non-coeliacchildren, mean age 7 5 years (range 1-15), biopsiedfor various abdominal symptoms and having anormal jejunal mucosa. Control sera were alsoobtained from 31 adults with biopsy proven ulcera-tive colitis, mean age 41 years (range 28-82), from 14adults with biopsy proven Crohn's disease (four ofthese had also lesions in proximal small bowel), meanage 39 years (range 20-81), and from 100 healthyblood donors, mean age 41 years (range 20-65). Atthe time of scrum sampling 20 patients with ulcera-tive colitis and nine with Crohn's disease had signsand symptoms of active disease.

RETICULIN AND ENDOMYSIUM ANTIBODY TES[SReticulin antibodies and endomysium antibodieswere determined with the standard immunofluores-cence method using unfixed cryostat (4 ptm) sectionsof rat kidney, liver, and stomach for reticulin'' andcommercial slides of monkey oesophagus sections(SciMedx, Denville, NJ) for endomysium anti-bodies'`'4 as antigens. Fluoresceinisothiocyanate(FITC) conjugated antisera against human immuno-globulins were from Kallestad Laboratories (Austin,Texas, USA).

Patient sera were stored from one week to fouryears at -20°C, and screened for reticulin antibodiesand endomysium antibodies at dilutions of 1:5 and1:10 with a polyspecific burro antihuman antiserum(F/P molar ratio 2 60, total protein concentration24-0 mg/mI) and a monospecific goat antihuman IgAantiserum (F/P ratio 2-73, antibody concentration1 85 mg/mI). Positive sera were titrated further (1:50,1:100, 1:200, 1:5()0, 1:1000, 1:2000, 1:4000(, and1:8000) with the goat anti-lgA and anti-lgG anti-serum (F/P ratio 2-27, antibody concentration 2 48mg/mI). All dilutions of sera were made in phosphatebuffered saline (PBS) pH 7-2-7-4. Optimal workingdilutions of the FITC-labelled antisera (1:80-1:120)were determined by block titration both withreticulin and antinuclear antibody positive sera.Positive and negative controls were included inevery batch of tests. Sections were examined on aLeitz Laborlux D epi-illumination fluorescencemicroscope equipped with an Osram HBO 100 Wlight source and interference filterblock 12 for bluelight.

All serum samples were examined for IgA and IgGclass reticulin antibodies and IgA class endomysiumantibodies. Moreover IgG class endomysium anti-bodies were examined in 20 dermatitis herpetiformissera, in three coeliac disease sera negative for IgAclass reticulin antibodies and endomysium antibodiesand in one coeliac disease serum from a patient withIgA deficiency.

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Comparison ofIgA -class retichulin and endomyvsilm antibodies in coeliac disease

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Specimens were considered reticulin antibodypositive, if the characteristic R,-type staining patternwas found both in rat liver and kidney sections."RI-pattern included staining of the fibres around theglomeruli and tubuli in the kidney (Fig. la), stainingof the well defined adventitial fibres around bloodvessels in the kidney and liver, staining of the nodularor lumpy fibres in portal areas of the liver (Fig. lb),and with strong positive sera, staining of discrete,wavy, worm-like strands in the walls of liversinusoids. The other staining patterns of reticulin

Fig. 1 Inunzuntiofluiore.sceit .staininig patternii ofR1-vipe oflgA class reticiilin anitibodies in c}rvostat sectio i.s of rat anidhium#Zanti tissuies. (a) Rat kidnieyt. Stainizng oft thefibres aronntidglotneriuli, tuibutli and a blood vessel. (b) Rat liver. Stainini,g oJthe coarsefibres ini thze portal a-rea around blood-vessels andin the walls of sitnzusoids. (c) Hwmnan liver. Staininig oft/ienetwork offibr.es in the walls ofsinusoids.

antibodies (Rs, R2, Rz,, RkJ' in rat tissues, weredisregarded. Staining of the endomysium around thesmooth muscle fibres in monkey oesophagus wasconsidered as positive for endomysium antibodies(Fig. 2).16 24

TISSUE SPECIFICITY OF RETICULIN ANDENDOMYSIUM ANTIBODIESThe tissue specificity of reticulin antibodies andendomysium antibodies positive sera from fivepatients with coeliac disease and two with dermatitis

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Fig. 2 Immunofluorescent staining pattern ofIgA classendomysium antibodies in a section ofmonkey oesoplbagus.Reticular staining ofthe endomysium ar-ound smooth musclefibres is seen in thle muscularis mucosae.

herpetiformis, was studied with tissue sections ofhuman adult skin, liver, kidney, spleen, jejunum andof fetal skin, lung, spleen, thymus, and jejunum. Thespecificity was also studied in three coeliac diseasesera with human fetal skin fibroblasts cultured on

sterile slides for four to five days at +37°C and fixed10 minutes in acetone at -10OC.21The lgA class reticulin antibodies and endo-

mysium antibodies positive sera from three coeliacchildren and IgG class reticulin antibodies andendomysium antibodies positive serum from one

adult coeliac patient with IgA-deficiency (serum IgA<0-01 g/l) were absorbed with human and severalrodent (rat, mouse, guinea-pig, and rabbit) liverhomogenates, respectively. Pieces of liver, stored at-700C, were minced with knife and scissors andwashed many times with PBS. 0.5 g of the washedhomogenate was then added to 4 5 ml of patientserum (diluted 1:100 with PBS), mixed two hours atroom temperature in a cell mixer, centrifuged 60 min/3000 g at +10°C and absorbed once more with a

further 0-5 g of liver homogenate and left to standovernight at +4°C before centrifugation. Super-natants were stored at -20°C until studied. Torecover the antibodies from the liver homogenates,4*5 ml of 0 1 M citrate buffer (pH 3.0) was added to0.5 g of homogenate, mixed 10 min in the cell mixer,centrifuged 60 min/3000 g at + 10C and the super-natants dialysed overnight against 1 1 of PBS at roomtemperature, centrifuged again as before and storedat -20°C until studied. Reticulin antibodies and

endomysium antibodies concentrations weremeasured in the absorbed and eluted sera withseveral rat and human tissues and with monkeyoesophagus.

GLIADIN ANTIBODIESGliadin antibodies were measured with a micro-ELISA method as previously described.26 IgA-gliadin antibody concentrations over 0-2 EU/mI(arbitary ELISA unit) and IgG-gliadin antibodyconcentrations over 5 EU/mI were consideredpositive.

STATISTICAL ANALYSISThe effect of the diet treatment on the antibody levelswas analysed with Wilcoxon's matched-pairs signed-ranks test.

Results

COELIAC DISEASETwenty nine (91%) of the 32 untreated adult CDpatients had positive IgA-reticulin antibodies andIgA-endomysium antibodies and 14 (44%) had IgG-reticulin antibodies (Table 1, Fig. 3). The threepatients with negative IgA class reticulin antibodiesand endomysium antibodies had IgG class reticulinantibodies (titres 1:100 to 1:8000) and endomysiumantibodies (titres >1:500, the endpoint could not bediscerned). One of these patients had IgA deficiency(IgA <0-01 g/l). All three patients responded togluten free diet and after one to two years all hadnormal jejunal mucosa and no IgG class reticulinantibodies or endomysium antibodies could befound.

Table 1 Frequency of IgA class reticulin (IgA-RA) andendomysium (IgA-EmA) antibodies in patients witliuntreated gluten sensitive enteropathy and in controls.

Nurmber of Positive Positivepatielit IgA-RA IgA -ErniAs(ra

Patients:Coeliac disease:

adults 32 9 (91%) 29 (91%)childlren 1X* 18 ( 1()'00%) Is (1()1%.)

Dermatitis herpetiformis 14 13 (93%,) 14 ( 10%)Non-coeliac chil(dren ssith

normal mucosa 24 0)Controls:

Ulcerative colitis 31 0 0Crohn's disease 14 (0Blood donors 1(X) 1(1) It 1%)

*Includes four sera obtained dluring gluiteni challenge; t lnclutides onlypatients with subtotal s illous atrophs.

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Titre

1:8000-

1:4000-

1:2000-

1:1000-

1:500-

1:200-

1:100-

1:5071:10-

1:5-

Neg.

0

0

X0ot

AL

IgA-RA

Fig. 3 Titres ofIgA class ret&endomysium (IgA-EMA), andantibodies in the sera of32 aduwith coeliac disease and in 14 apatients with subtotal villous at

All 14 children with untthe four children challengeIgA class reticulin antibodbodies and nine (50%) weiantibodies (Table 1, Fig. 3

DERMATITIS HERPETIFOINineteen (83%) of the 23patients had positive IgA(titres 1:10-1:2000) and(titres 1:5-1:4000). Seventcpositive; one patient had(1:10) and one only endoThirteen (93%) of the 14patients with SVA had IgA14 (100%) had endomysi(13%) had IgG class reticFig. 3). Three of the fivepatients with partial villotreticulin antibodies (titreendomysium antibodies (tiof the four dermatitis heslight changes or normalreticulin antibodies (titresendomysium antibodies (tiof the 20 dermatitis herp4IgG-endomysium antibodi

0

aszot

CONTROLS

No IgA class reticulin antibodies or endomysiumo antibodies or IgG class reticulin antibodies were

found in control patients (Table 1). One (1%) bloodO donor had both IgA-reticulin antibodies (titre 1:200)

and IgA-endomysium antibodies (titre 1:2()0). This38 year old woman had experienced slight abdominalsymptoms for several years. A jejunal biopsy wasperformed and it revealed subtotal villous atrophy.She responded clinically and serologically to glutenfree diet confirming that she had coeliac disease.

EFFECT OF GLUTEN FRE E' DIET ANI) ilU 1TN4

* CHAI.I ENGE ON RETICUL IN ANIIBODII.S ANDENDOMYSIUM ANTIBODIESFour adult patients with coeliac disease and 10 withdermatitis herpetiformis were followed four to 32months on a gluten free diet. The concentrations of

* IgA class reticulin antibodies and endomysium anti-* t_ bodies declined in parallel in both patient groups

000 W (Fig. 4) and the decrease was significant (p<001)both for IgA class reticulin antibodies and endo-

lgA-EMA IgG-RA mysium antibodies. In the coeliac disease group theculin (IgA-RA), IgA class antibodies were negative after four to 12 months on1 IgG class reticulin (IgG-RA) the diet except in one patient who showed low (1: 10)Its (0) and 18 children (0) IgA-endomysium antibodies titre after five months.lermatitis hlerpetiformis In dermatitis herpetiformis group low positive (1: 1(0-trophy (R). 1:50) IgA-reticulin antibodies titres were found in

three patients after six to 12 months and IgA-reated coeliac disease and endomysium antibodies titres (1:5-1:50) in five.d with gluten had positive patients after four to 12 months on a gluten free diet.lies and endomysium anti- Six of eight dermatitis herpetiformis patients whore positive in IgG-reticulin were followed for three to 14 months on a normal diet

had low titres of IgA-reticulin antibodies and five ofthese had lgA-endomysium antibodies (titres 1: 10-

RMIS 1:50) at the beginning and at the end of the follow up.dermatitis herpetiformis Two antibody negative dermatitis herpetiformisclass reticulin antibodies patients developed both IgA-reticulin antibodiesendomysium antibodies (1: 10) and IgA-endomysium antibodies (1: 10) during

cen patients had both tests the follow up.only reticulin antibodies All four antibody negative coeliac children becamemysium antibodies (1:5). IgA-reticulin antibodies and IgA-endomysiumdermatitis herpetiformis antibodies positive (titres 1:500-1:4000) whenclass reticulin antibodies, challenged with gluten and one became also IgG-um antibodies and three reticulin antibodies (1:200) positive.-ulin antibodies (Table 1,dermatitis herpetiformis TISSUI SPECIFICITY OF RFTICUI IN AND

is atrophy had both IgA- ENDOMYSIUM ANTIBODIFSs 1:10-1:100) and IgA- All seven sera reacted with reticulin fibres in alltres 1:50-1:200) and three human tissues studied and the IgA and IgG classrpetiformis patients with reticulin antibodies gave identical stainings. Themucosa had IgA class fluorescence was intense in the fetal lung, thymus,1:10) and two IgA class and jejunum; clear in the adult liver, spleen, and

itres 1:10 and 1:50). None jejunum but weak in the adult skin and kidney. Theetiformis sera studied for staining in human liver was most specific on the fibreses were positive. in the walls of the sinusoids (Fig. lc) but not in the

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0)

._

1 :8000O

1:500-

1:2001

1:100-

1:50-

1:10-

1:5-

Neg -

1;80001

1 :5001

1:200 -

1:100-

1:50-

1:10-

1._

'1:

1:5-

Neg -

0 1-3 4-6 7-12 >12 0 1-3 4-6 7-12 >12

Months on gluten free diet

Fig. 4 The effect ofgluten-free diet treatment on IgA class retictulin (RA) atid endom.'5silimn (EnA) amitibodies ini fiour aduiltpatients with coeliac disease (0) and in 10 dermatitis herpetiformis patients wvith slubtotal or partial villous atrophY (R).

portal area as seen in rat liver. A reticular stainingwas also seen in the cytoplasm around the nucleus insome but not in all cultured human fibroblasts.

After absorptions of sera with rodent (rat, mouse,guinea pig, and rabbit) liver homogenates IgA or IgGclass reticulin antibodies could not any more bedetected with rodent tissues but human tissues,fibroblasts and monkey oesophagus gave a clear R,-type reticulin antibodies staining (Table 2). In con-

trast, after absorption with human liver homogenatesboth rat and human tissues, fibroblasts and monkeyoesophagus all gave negative results. These absorp-tions had no effect on IgA or IgG class gliadinantibody concentrations.The IgA and IgG class reticulin antibodies and

endomysium antibodies eluted from rodent liverhomogenates reacted predominantly with rat butonly weakly with human and monkey tissues. In

contrast, the antibodies eluted from human liverhomogenates reacted strongly with all human androdent tissues, fibroblasts, and monkey oesophagus.

Discussion

The present study confirms the importance of IgAclass antibody measurements in coeliac disease anddermatitis herpetiformis. Similar high frequencies(91%-100%) of IgA class reticulin and endomysiumantibodies were found in untreated adults andchildren with coeliac disease and in dermatitisherpetiformis patients with subtotal villous atrophy.The specificities of these two IgA antibody tests werealso high because none of the 69 disease controls andonly one of the 100 blood donors had these anti-bodies. In agreement with the present results, Seah etalt reported IgA class reticulin antibodies in nine of

Tablc 2 Serum titres ofIgA class reticulin (RA) and endomysium (EmA) antibodies before and after absorption wit/l rat andhuman liver homogenates in thlree untreated coeliac children. Reticulin antibodies were measured wit/i rat kidney and hiumanliver and EmA with monkey oesophagus tissue sections by using standard immunofluorescence methods and monospecificantihuman IgA-conjugate

After absorption

Before absorption With rat liver With human liver

RA [mA RA EmA RA FmACase monkey motnikey monkes'nio Rat kidntiey Huiman liver oe.sophagu.s Rat kidney Human liver oesophagus Rat ki(dn1ey Hunmani liver oesophagu.s

1 2(M) 2(KX) 40(X) NEG* 10M() 2M(X) NEG NEG NEG2 10(X)) 10(0 500 NEG 20() 500 NEG NEG NEG3 20(X) 40(X) 1 (X) NEG 10(X) 500 NEG NEG NEG

*Negativc titre <1:1(X).

1 l 1 91 1 1~~~~~~~~~~~~~~~~~~~

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10 (90%) and Mallas et al" in 13/17 (76%) untreatedchildren with coeliac disease. Moreover, Eade et al''found IgA class reticulin antibodies in 22/28 (79%)children and adults with coeliac disease, andLjunghall et al27 in four of 10 (40%) and Volta et a121 infour of 16 (25%) adult dermatitis herpetiformispatients with subtotal villous atrophy. In all thesestudies the frequencies of IgG class reticulin anti-bodies have been the same or lower than IgA classreticulin antibodies and all sera from healthy anddisease controls, including patients with ulcerativecolitis and Crohn's disease, have been negative forIgA-reticulin antibodies.High prevalence of IgA class endomysium anti-

bodies (65%-100%) have been reported in coeliacdisease and dermatitis herpetiformis recently byChorzelski, Beutner, and coworkers. 17 19 20 22 Thesimultaneous occurrence of IgA class endomysiumantibodies and reticulin antibodies has been pre-viously reported only in dermatitis herpetiformispatients'7 but in contrast with the present study thefrequency of IgA class reticulin antibodies (25%) wasclearly lower than that of endomysium antibodies(65%). One reason for this difference could be thatKumar et al'7 used only mouse kidney sections inreticulin antibodies test and not a composite block ofrat tissues (kidney, liver, and stomach) as was used inthe present and in two recent screening studiesI'IIdetecting a high (93% and 83%) prevalence ofreticulin antibodies in coeliac disease.The present absorption studies showed that the

rodent-specific IgA and IgG class antibodies could beabsorbed with rodent liver homogenates. After theseabsorptions, however, a strong positivity against allhuman tissues and monkey oesophagus persisted.This positivity was regarded as a human (or primates)subtype of reticulin antibodies and interestingly,when this subtype was absorbed away with humanliver homogenates also endomysium antibodies dis-appeared. Therefore, the present absorption experi-ments did not disclose any differences betweenendomysium antibodies and human subtype ofreticulin antibodies. Unfortunately, enough monkeyoesophagus tissue was not available to confirm thatabsorption with this tissue also removes humansubtype of reticulin antibodies. RI-type of reticulinantibodies reacting with human livers 's and spleen2has also been described previously and already Seahet al and Eterman et aP' showed that such antibodiescan react with human jejunum, a finding recentlyconfirmed also by Karpati et al.'2

In conclusion, the present study showed the useful-ness of IgA class reticulin and endomysium antibodytests in detecting gluten sensitive enteropathy andmonitoring dietary treatment in patients with coeliacdisease and dermatitis herpetiformis. Two subtypes

of reticulin antibodies were characterised by absorp-tion studies, the first subtype reacting with rodenttissues and the second also with human tissues. Thehuman subtype of reticulin antibodies seems to bevery similar to endomysium antibodies but moredetailed experiments are needed to confirm theiridentity.

I wish to express my thanks to Drs Timo Reunala,Markku Maki, Olavi Keyrilainen, and Pekka Collinfor patient serum samples, Finnish Red Cross forblood donor sera, and Dr Markku Viander forexamining gliadin antibodies. Mrs Pirjo Saariaho,Riitta Huttunen, and Sairi Koskinen are acknow-ledged for their skilful technical assistance. Thisstudy was supported by a grant from the EmilAaltonen Foundation.

References

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2 Williamson N, Asquith P, Stokes PL, Jowett AW,Cooke WT. Anticonnective tissue and other antitissue,antibodies' in the sera of patients with coeliac diseasecompared with the finding in a mixed hospital popula-tion. J Clin Pathol 1976; 29: 484-94.

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comparison of iga-class and endomysium antibodies

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