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Two- and three-dimensional human cell cultures: an in vitro tecnique for gliadin's cytotoxicity
Small Bowel
99
97 PREVENTION OF EARLY POST-TIPS HEFAX ENCEFALOPATHY: COMPARATION BKWEEN RIFAXIMIN, LACTlTOL AND NO TREATMENT m, Efrati C, Nicolao F, Mm-ii M, Attili AF and Riggio 0. II Gastmenterologia, Universita “La Sapienza” Roma The incidence of Fast TIPS heptic encepWapathy (HE) in cinhatic patients ranges from 29 to 54% at one year, with a m&mum during the Rrst month. Risk factors are considered: age>65yrs. previous episodes of HE, advanced Child class and a low oorto-heotic oressure uadient 3fter TIPS. There is IK) consensus about &e oppdrtunic to treat &tients submitted to TlPS for the prevention of HE and about which Watment L to prefere. Aim of this tidy was to test the eificacy of Wee diiferent treatment skedules inthe prevention ofeaiiy post-TIPS HE: l&l [L) 60 g/day, ,+&mine CR) ‘200 mgiday and co hafmant (NT). Xrtytiree c:nsecutive cirrhotic patient :25M, SF, age 57~11, Child-Pugh T‘i2ss3A, : as, X) w?n :‘-d ?rn :G T!PS for ,dar;ceal bleeding (24 pts) or refractory zscI!es (9 pts) ‘we??enrclled. 3oiccy c,i .IV~? :ichosis was alcoholic in 10 and viral in 19. Eight ::i &se patients -&e,w:ra.zt T;?S n emergency and 6 had previous episodes af HE. All the ptients asmmed a non prorein-testicted diet tirosghout Be penzd of saoy ar.d :,ere rarccmtzed :o -eceIve one oirhe above tsealments. Patiew were evaluated beiore 7 ?S and T, i4,21 and 30 days afbr TIPS by examining and grading mental status. asterixis. plasma ammonia and Bail making test Tne PSE index was calculated from aese paramatars. TWO patients died during the first month iollowinc TIPS. The 3 groups studied Were comparable for age, sex, Child-Pugh class. etiology, par@-Cad pressure gradient after TIPS and previous episodes oi YE. Ten patients aeveloped HE in the study pericd (W3 %); 4 in -he ‘a&tot gnuo (grade WIV=3, l-11=1)4 in tie r!faxintne group (c:ade :ll-IV=3. I-!!=I) and 2 in tie io sesment group (grade WIV=3, l-11=2).ice incidence oi iiE ivas no si@candy different in the three groups studied flc? rank tas: p=:?.f7). “-a--?5 ~vencus blood ammonia l&S and bail ma!zng test pericrr?anca were ;crrq;s~e ‘n ze :bree groups studied. MOUS blood ammonia &Jfs In tie patients WM did not deveicped +E (L-124152 YS ~=ll~z:6.5 IS ?“;=‘?7=:+,dl; ?=0.76), Sad making test performance (L-E.:30 ‘is ~.=&I=7 JS \;=:&?3 set: o=O,68) and PSE index (L=0.16 YS ?=O.lC ‘is LsT=O ! 5; 3= 3 ti) ;,~re ilmilar one month ai?er ms, independengy on tie ueatmenr skeaute received. Our data show mat the testcent wt! la&o or afaxirrin ;o nettier ytetd a lower incidence of HE, rw improve piesma z-fzoql 2 3r xx;cmetm :cz: wing the first month a&r rips placement
CELIAC DISEASE DIAGNOSIS IN CHILDREN USEFULNESSOF IN VITRO GLIADIN CHALLENGE A. Picarelli, L. Sabbatella,M. Di Tola, P. Mariam*, A. La Vecchia’, M. Bonamico*
UnivetsitkLa Sapenza Beckground Recent works showedthe possibility to induceantiendomysialantibodies@MA) productionin-vitm by small bowel mucosaof celiec diseasepatientsin clinical remission cultured in the presenceof gliedin peptides.Our aim was to usethis in vitm system to verify if it may predict theclinical at&or thebistologiceI relapseto glutenchallengein CD children on a gluten-freediet (GFD). Materials andMethcds Enrolledin the study were 30 children one glubxwxttaining diet (GCD) showing total or subtotalvillotts atrophy (gmttp I); 17 CD children on GFD who tmdenvent anin-viva glutenchallenge(group 2A) and 7 CD ad&scents who requiredin vitro gliedin challenge(gmup 2B). Fmthemmre, 78 patientswere studiedas diseasecontmls &up 3). Biopsy cultures with andwithout gliadiq mmphometric analysis aswell as IgA andIgGI EMA detection both in seta andculture supematmts were @wmed. Results EMA were detectedin supemetants of all gmttp 1patients.Ten out of 17children belonging to group 2A were IgA EMA positive in culture supemetmts, other 5 children were IgGI EMA positive. All thesechildren but one showed climcsl andfor histologicalrelapseatlet in viva glutenchallenge.All culture su*ematantsfrom group2B were IgA EMA positive, whetws those from diseasecontmls were both IgA andIgGl EMA negativeirrespective of gliedin challenge.Discussion Gut results show that the organ culture with in-vitro gliadin challengeis able to reproducethe results of in-viva challenge,thereforethis system could consistently reduce the necessity of performing the glutenchallengein celiac children. In addition,in-vitro gliadin challengecould be useful to clarify diagnosiswhen it is uncertainor difficult
98 Two- and three-dimensionalhumancell cultures: an in vitm temique for gliedin’s cytotoxicity Elli L (I), Dolfini E (Xl), Desdia T (Z), BuferdeeiB (2), Flmimi I (3), Fslini m (2), C~lleoni MP (2), Ferrem S (2) endBatdella MT (2) l)Gsstroentemlogy, Uniwsty of Udin 2)University of Milan 3)Mario Negri Institute UniversitA di Udine Celiac disease(CD) is a frequentchronic et&empathy. ‘Ibe pathogenesisof CD is not wmpletely understoodbut an alteredimmune regp0n.w8ccms to play a maim mle even if the initial stm of the whole process is still unclear.F’mxde& studies bweshg&d the biological propertiesof g&in obsenringits cytotoxic effects but liudxr studiesare still needed. Aim oftbis work is to developa new in vitro temimte, to study. the cvtotoxicitv._.. of eliadin. mseonsiblefor CD. Dwinn the experimentsLoVo cell line (human colon sdenwsrcitmma), 8mwing in two- endth&imensionel cultures (Multice1hd.wSpheroids,MCS ), hesbeenexposedto peptic&yptic digest& breedwheat gliedin et different concentmtions (62,125,250,500,750 micmgr/ml). We eveltteted:1) cell growth after 48 hours of treatment;2)oxidedve balance:content of tiuced glutethione(GSH); pemxidsse,transferwe and nductasc activity; 3)dietnetem,volumes studviability (colony-forming ability) of treatedMCS. Common food proteins (albmnio,ovwdbumin, cssein, betelectoglobulin) havebeenused as mntmls. Dtmnett test hes bcm performed for statistical analysis. Gut data show a stetistically simifiwnt inhibition of cellular mowtb. in omeortion to &din cmcetttmtion (iiom 26,7%, at 62 ttti~~gr/mI, to 100% at 750 micr&/mlj. O&&e be&e showeda decreasein GSH content (-38% at SO0miemgrhl), ande reduction of enzymatic activities (-30% et 500 micmpr/mI). A&r 8 days of gliedin exposure(50 micm~/ml) the MCS have shown: a volume end diametas decreaset?om 37% to 70%, a reducedcell viability end e different histologicalpsttem with amodified cell-cell interactionand diffemntistion. Controls didn’t show any noxius effect both on two- and three-dimmsianalcell cultures. Our dataconfirm that LoVo cells are usefulin evaluatinggliadin cytotoxicity both in two&ensionel cell cnltmes, andin the new tbxedimensionalMCS cultmes and could be usedto test modified gliadittsor investigatethe biological pmperties of gliadin.
ULTRASONOGRAPHIC SIGNS IN ANTICIPATING CELLK DISEASE. A PROSPECTIVE STUDY Fmquelli M., Tmvato C., PmnettaR., BardellaM.T., IColli A., Colucci A. md D. Conte. Csitedm di Gss’uoentemlogia, IRCCS GspedeleMaggiom, Milsno, md IDivisione di Me&ins, Ospedale di Lecco, Italy IRCCS Osp Matim Backgm& an&tn. Different uhmsonogmphic(US) tindingshave beenrepottedin association with overt celiec disease(CD) (1,2). This prcspective study wes aimedto evaluatetheir eccumw in anticipatingCD. Patientsandmethods. 162c&emtive patients(67 M and 95 F, meanage46. +SD I8 years) investigetedfor chmnic diarrhea, dyspepsia or imn-deficimcy enemia (# 105, 25 end 32 cases, mspectively) underwentanti-endomysium(EmA) IgA antibodiesdetezminstion, distal duodens.lbiopsiesendto e blind US sssegwent of the presenceof : 1) enlarged gallbladder, 2) diffisely dilated andebnmmeIly fluid tilled smell bowel Iwps, 3) diflitsc thickening of smell bowl we& 4) increasedpetistelsis, 5) ftee fluid in the sbdominsl cavity and6) enlargedmesentmiclymph n&e. Definite diagnosisof CD was basedon positive EmA and consistenthistologic findings.The Sensitivity (Sn), spaifieity (Sp), positive ettd negative likelihood ratio (LH+ md LB). positive and neeetivemedictive v8hte-s(PPV end NPVl were calculatedfor eech US parameter.Giving apr&t probability of CD oi about 10%(B;iderived from a comparableour own study proup), we ceIcuIetedthe post-test onewhich atmmximetesthe positive andnegetivepredictive value. Results CD wes dieg&wd in 12c=s (7.6%) with chmnic die&es (# 7), imn-deficiency anemia(# 4) or dyspepsia(# 1).The tablemmm&es the diagnosticperformancesof the different us tindings. As also given, the presmce of at leastone or of all the six US signs together,allows to obtaina low negativeLH (0.10) anda hi& positive LH (33.0) respectively, thus seppottinge scmeningor a confitmatoty diqnostic strategy. US CD NoCDSnSp LH PPV NPVsiy g2) (#150) (“h) (%) (+) (-) (%) I%) I 92 8* 1% 57 7 i5’ 77 6” 4.0 0.10 24 99 II. I nx 9-;; ,”9x 75 il’ 91 8.3 0.27 39 97 4 IO 19 83 87 6.3 0.15 34 98 5 6 6 50 96 12.5 0.52 50 96 6 5 4 42 97 14.0 0.59 55 99 At leastone II 35 92 77 4.0 0.10 24 99Al16 4 1 33 99 33.0 0.67 80 95 * S/l1 (1 cholecystectomized); “6/143 (7 cholecystectomized)Comments Presentresults obtainedin e populationwith a pm-testpmbebility of CD of less than IO%, indicate that different US signs can scwmtely detectpatientswith celisc disease.The value obtainedfor LH+ andLH - respecttvely support the possibility to trmsfer these datealso to B populationwith different pretest probability of CD 1) Radialogy 1999;21I: 389.394 2) Am J Gsstmenteml 1999;94: 1866-1870.
A63
99
97 PREVENTION OF EARLY POST-TIPS HEFAX ENCEFALOPATHY: COMPARATION BKWEEN RIFAXIMIN, LACTlTOL AND NO TREATMENT m, Efrati C, Nicolao F, Mm-ii M, Attili AF and Riggio 0. II Gastmenterologia, Universita “La Sapienza” Roma The incidence of Fast TIPS heptic encepWapathy (HE) in cinhatic patients ranges from 29 to 54% at one year, with a m&mum during the Rrst month. Risk factors are considered: age>65yrs. previous episodes of HE, advanced Child class and a low oorto-heotic oressure uadient 3fter TIPS. There is IK) consensus about &e oppdrtunic to treat &tients submitted to TlPS for the prevention of HE and about which Watment L to prefere. Aim of this tidy was to test the eificacy of Wee diiferent treatment skedules inthe prevention ofeaiiy post-TIPS HE: l&l [L) 60 g/day, ,+&mine CR) ‘200 mgiday and co hafmant (NT). Xrtytiree c:nsecutive cirrhotic patient :25M, SF, age 57~11, Child-Pugh T‘i2ss3A, : as, X) w?n :‘-d ?rn :G T!PS for ,dar;ceal bleeding (24 pts) or refractory zscI!es (9 pts) ‘we??enrclled. 3oiccy c,i .IV~? :ichosis was alcoholic in 10 and viral in 19. Eight ::i &se patients -&e,w:ra.zt T;?S n emergency and 6 had previous episodes af HE. All the ptients asmmed a non prorein-testicted diet tirosghout Be penzd of saoy ar.d :,ere rarccmtzed :o -eceIve one oirhe above tsealments. Patiew were evaluated beiore 7 ?S and T, i4,21 and 30 days afbr TIPS by examining and grading mental status. asterixis. plasma ammonia and Bail making test Tne PSE index was calculated from aese paramatars. TWO patients died during the first month iollowinc TIPS. The 3 groups studied Were comparable for age, sex, Child-Pugh class. etiology, par@-Cad pressure gradient after TIPS and previous episodes oi YE. Ten patients aeveloped HE in the study pericd (W3 %); 4 in -he ‘a&tot gnuo (grade WIV=3, l-11=1)4 in tie r!faxintne group (c:ade :ll-IV=3. I-!!=I) and 2 in tie io sesment group (grade WIV=3, l-11=2).ice incidence oi iiE ivas no si@candy different in the three groups studied flc? rank tas: p=:?.f7). “-a--?5 ~vencus blood ammonia l&S and bail ma!zng test pericrr?anca were ;crrq;s~e ‘n ze :bree groups studied. MOUS blood ammonia &Jfs In tie patients WM did not deveicped +E (L-124152 YS ~=ll~z:6.5 IS ?“;=‘?7=:+,dl; ?=0.76), Sad making test performance (L-E.:30 ‘is ~.=&I=7 JS \;=:&?3 set: o=O,68) and PSE index (L=0.16 YS ?=O.lC ‘is LsT=O ! 5; 3= 3 ti) ;,~re ilmilar one month ai?er ms, independengy on tie ueatmenr skeaute received. Our data show mat the testcent wt! la&o or afaxirrin ;o nettier ytetd a lower incidence of HE, rw improve piesma z-fzoql 2 3r xx;cmetm :cz: wing the first month a&r rips placement
CELIAC DISEASE DIAGNOSIS IN CHILDREN USEFULNESSOF IN VITRO GLIADIN CHALLENGE A. Picarelli, L. Sabbatella,M. Di Tola, P. Mariam*, A. La Vecchia’, M. Bonamico*
UnivetsitkLa Sapenza Beckground Recent works showedthe possibility to induceantiendomysialantibodies@MA) productionin-vitm by small bowel mucosaof celiec diseasepatientsin clinical remission cultured in the presenceof gliedin peptides.Our aim was to usethis in vitm system to verify if it may predict theclinical at&or thebistologiceI relapseto glutenchallengein CD children on a gluten-freediet (GFD). Materials andMethcds Enrolledin the study were 30 children one glubxwxttaining diet (GCD) showing total or subtotalvillotts atrophy (gmttp I); 17 CD children on GFD who tmdenvent anin-viva glutenchallenge(group 2A) and 7 CD ad&scents who requiredin vitro gliedin challenge(gmup 2B). Fmthemmre, 78 patientswere studiedas diseasecontmls &up 3). Biopsy cultures with andwithout gliadiq mmphometric analysis aswell as IgA andIgGI EMA detection both in seta andculture supematmts were @wmed. Results EMA were detectedin supemetants of all gmttp 1patients.Ten out of 17children belonging to group 2A were IgA EMA positive in culture supemetmts, other 5 children were IgGI EMA positive. All thesechildren but one showed climcsl andfor histologicalrelapseatlet in viva glutenchallenge.All culture su*ematantsfrom group2B were IgA EMA positive, whetws those from diseasecontmls were both IgA andIgGl EMA negativeirrespective of gliedin challenge.Discussion Gut results show that the organ culture with in-vitro gliadin challengeis able to reproducethe results of in-viva challenge,thereforethis system could consistently reduce the necessity of performing the glutenchallengein celiac children. In addition,in-vitro gliadin challengecould be useful to clarify diagnosiswhen it is uncertainor difficult
98 Two- and three-dimensionalhumancell cultures: an in vitm temique for gliedin’s cytotoxicity Elli L (I), Dolfini E (Xl), Desdia T (Z), BuferdeeiB (2), Flmimi I (3), Fslini m (2), C~lleoni MP (2), Ferrem S (2) endBatdella MT (2) l)Gsstroentemlogy, Uniwsty of Udin 2)University of Milan 3)Mario Negri Institute UniversitA di Udine Celiac disease(CD) is a frequentchronic et&empathy. ‘Ibe pathogenesisof CD is not wmpletely understoodbut an alteredimmune regp0n.w8ccms to play a maim mle even if the initial stm of the whole process is still unclear.F’mxde& studies bweshg&d the biological propertiesof g&in obsenringits cytotoxic effects but liudxr studiesare still needed. Aim oftbis work is to developa new in vitro temimte, to study. the cvtotoxicitv._.. of eliadin. mseonsiblefor CD. Dwinn the experimentsLoVo cell line (human colon sdenwsrcitmma), 8mwing in two- endth&imensionel cultures (Multice1hd.wSpheroids,MCS ), hesbeenexposedto peptic&yptic digest& breedwheat gliedin et different concentmtions (62,125,250,500,750 micmgr/ml). We eveltteted:1) cell growth after 48 hours of treatment;2)oxidedve balance:content of tiuced glutethione(GSH); pemxidsse,transferwe and nductasc activity; 3)dietnetem,volumes studviability (colony-forming ability) of treatedMCS. Common food proteins (albmnio,ovwdbumin, cssein, betelectoglobulin) havebeenused as mntmls. Dtmnett test hes bcm performed for statistical analysis. Gut data show a stetistically simifiwnt inhibition of cellular mowtb. in omeortion to &din cmcetttmtion (iiom 26,7%, at 62 ttti~~gr/mI, to 100% at 750 micr&/mlj. O&&e be&e showeda decreasein GSH content (-38% at SO0miemgrhl), ande reduction of enzymatic activities (-30% et 500 micmpr/mI). A&r 8 days of gliedin exposure(50 micm~/ml) the MCS have shown: a volume end diametas decreaset?om 37% to 70%, a reducedcell viability end e different histologicalpsttem with amodified cell-cell interactionand diffemntistion. Controls didn’t show any noxius effect both on two- and three-dimmsianalcell cultures. Our dataconfirm that LoVo cells are usefulin evaluatinggliadin cytotoxicity both in two&ensionel cell cnltmes, andin the new tbxedimensionalMCS cultmes and could be usedto test modified gliadittsor investigatethe biological pmperties of gliadin.
ULTRASONOGRAPHIC SIGNS IN ANTICIPATING CELLK DISEASE. A PROSPECTIVE STUDY Fmquelli M., Tmvato C., PmnettaR., BardellaM.T., IColli A., Colucci A. md D. Conte. Csitedm di Gss’uoentemlogia, IRCCS GspedeleMaggiom, Milsno, md IDivisione di Me&ins, Ospedale di Lecco, Italy IRCCS Osp Matim Backgm& an&tn. Different uhmsonogmphic(US) tindingshave beenrepottedin association with overt celiec disease(CD) (1,2). This prcspective study wes aimedto evaluatetheir eccumw in anticipatingCD. Patientsandmethods. 162c&emtive patients(67 M and 95 F, meanage46. +SD I8 years) investigetedfor chmnic diarrhea, dyspepsia or imn-deficimcy enemia (# 105, 25 end 32 cases, mspectively) underwentanti-endomysium(EmA) IgA antibodiesdetezminstion, distal duodens.lbiopsiesendto e blind US sssegwent of the presenceof : 1) enlarged gallbladder, 2) diffisely dilated andebnmmeIly fluid tilled smell bowel Iwps, 3) diflitsc thickening of smell bowl we& 4) increasedpetistelsis, 5) ftee fluid in the sbdominsl cavity and6) enlargedmesentmiclymph n&e. Definite diagnosisof CD was basedon positive EmA and consistenthistologic findings.The Sensitivity (Sn), spaifieity (Sp), positive ettd negative likelihood ratio (LH+ md LB). positive and neeetivemedictive v8hte-s(PPV end NPVl were calculatedfor eech US parameter.Giving apr&t probability of CD oi about 10%(B;iderived from a comparableour own study proup), we ceIcuIetedthe post-test onewhich atmmximetesthe positive andnegetivepredictive value. Results CD wes dieg&wd in 12c=s (7.6%) with chmnic die&es (# 7), imn-deficiency anemia(# 4) or dyspepsia(# 1).The tablemmm&es the diagnosticperformancesof the different us tindings. As also given, the presmce of at leastone or of all the six US signs together,allows to obtaina low negativeLH (0.10) anda hi& positive LH (33.0) respectively, thus seppottinge scmeningor a confitmatoty diqnostic strategy. US CD NoCDSnSp LH PPV NPVsiy g2) (#150) (“h) (%) (+) (-) (%) I%) I 92 8* 1% 57 7 i5’ 77 6” 4.0 0.10 24 99 II. I nx 9-;; ,”9x 75 il’ 91 8.3 0.27 39 97 4 IO 19 83 87 6.3 0.15 34 98 5 6 6 50 96 12.5 0.52 50 96 6 5 4 42 97 14.0 0.59 55 99 At leastone II 35 92 77 4.0 0.10 24 99Al16 4 1 33 99 33.0 0.67 80 95 * S/l1 (1 cholecystectomized); “6/143 (7 cholecystectomized)Comments Presentresults obtainedin e populationwith a pm-testpmbebility of CD of less than IO%, indicate that different US signs can scwmtely detectpatientswith celisc disease.The value obtainedfor LH+ andLH - respecttvely support the possibility to trmsfer these datealso to B populationwith different pretest probability of CD 1) Radialogy 1999;21I: 389.394 2) Am J Gsstmenteml 1999;94: 1866-1870.
A63