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Interleukin (IL)-12 receptor β2 subunit expression is up-regulated in helicobacter pylori infected gastric mucosa
ORALPRESENTATIONS
3
1 HELICOBACTER PVLOBI MFECTION Cad-POSITIVE THE OXIDATIVE DNA DAMAGE OF GASTBIC MUCOSA.
INCREASES
Introduction/aim: Reactiw oxygea metabolitescan interacr directly wkb genomic DNA, producing several bax mcdificariarr including the fonnarion of the muragenic adduo 8-bydmxydwxy8uanosme (8OHdG). Gxidauve DNA addud formation has been sbovm to be imporrmd in the prccas of’ carcinogeneris. We have previously demonstrated that, in gastric rnucosa, oxrdstive DNA damage, quantified trough the mucoral cmceatratim of 80HdG. associateswith I-I. pylori infectloo, gland atrophy, and intesrinsl meraplasia 0.M.). H.pylori srrains harboring CagA 8enehave beenassociatedwith the highest risk of developmentof atmphy, LM and &ric cancer. The aim of tba rmdy war to ascertain wbaher H.pylori CagA+ve infection condates with the prcsencc of praancerous conditions more frequently than in CasA-ve H.pvIori infectioo and with hi&r concentrations 8OHdG in 8artric
m”&sa. Pstieots sed Methods: Gris hundredtwo patimrs were studied@UF=62l40, mean me 61+14 m). Fourteen sstients were H ~vlori nesstive (-vel. Anions H. wlori p&rive (+A) patients, t6.wei.e CagA pai& (+ve: and 38 Ca8A negative’(&). In 24 patienrs H. pylon mfer&n bad been eradicatedgi.pylori-Ersd) more thim 6 months before 8xrnc samplisg. H.pylori infsrion was deemiinrd wab modified Oiemsa staining. In ardmm, corpus and cardias. gastritis, d&are acritiy, atrcpbyrl.M. were semiqsantirsdvely scored In asrml biopsies, 8OHdG cnncentmricm was asserscd by HPLC and elecrnxbemical d&&xi and expressed ss number of adduds 8-OHdG/IO’ dG residues. CagA startis ws determinedby PCR Resuk Degree of gastritis was nd associared with CagA+ve infectico in all gasmc sites In corpus, CagA genaype was si8irificantly correlated with disease activity @=0.05). Owd, no si&icant difference was found m avopbyll.M., however, ammg patients younger tlnm 50, gastric avophy significantly associated with Cag+ve status @=0.02). Atrophy and I.M. were lixnd in 55% of CegA+ve and 16% of CagA-ve patients (KR. 6.5 95% C.L.l.240.2). CagA+ve patients had 8OHdG levels significantly higher than all the dher subgroups @=O01) and in particular rban C&Aye patmds @=0.05), also in parients younger than 50 @=O05). No QfFerencein c&dative DNA damagewar deterred betweenH.pylonErad. and H.pylori-vs paients. Higherr levels of 8OHdG were detected in pauenrs who were CagA+ve and hadab-cphy/l.M. Cooelurions: CagA+ve patients were characterizedby I, tugher diseaseacnwty at du corpus, 2. higher RR for atrqhy when younger than 50, 3. l@ber DNA oxnfatm damage,overall andis particular at younger age. Highest DNA oridative damage levels were found in H.pylori+ve CagA+ve patients wnh &c&c metaplsria. This sating may representP cancer-pronebiological ccmtext
NONSTEROIDAL ANTI-INFLAMMATORY DRUGS COUNTERACT H. pylon’ VncA TOXIN-INDUCED VACUOLATION OF GASTRIC EPITHELIAL CELLS IN VITRO. V. Ricci,’ B.A. Manzo,*t P. Sommi,** C. Tuccillo,t M. SsritarigeIo,~ R. Zarrilli,t. M. Romanot ‘Istituh di Fisiologia Umsna e “Patologia Generale, Universit& DI Pavia; tGsr.troenterologia, Seconda Univenitii. di Napoli; **Diwrtimento di Bioloda e Patolonia Cellulare e Molecolare, Univ&sitB Fed&co II, Nipoli BACKGROUND: VacA toxin from H. p$ori induces cytoplasmic vacuolation in gastric epithelial cells in v&o and seems to play a major role in gastric damage. The relationship between nansteroidal antiinflammatory drugs (NSAIDs) and H. pylori-induced gastric mucosal cell injury is controversial. AIM: I) To evaluate whether NSAIDs interfere with VacA-induced vacuole formation in gastric epitbelial cells in culture and 2) to assess the mechanism of any such effect. METHODS: Cell Q&&x8: MKh’ 28 cells &urn a human well differentiated gastric adenocarcinoma; Cell Vacuolation: by neutral red dye uptake; QII :by Pmllferatlon: by [‘HI-tbymidine uptake; VacA confocal microscopy. EXPERIMENTAL DESIGN: I) MKN 28 cells were incubated for 16-24 h with VacA+ broth culture filtrate (BCF) without (contra!) or with aspirin (AP 4) (n C-10 mM). indorncrhncin (IND) (0.1-2.5 mM), NS-398 (I-100 pM), and with the DNA synthes.s inhibitor I-b-D-arabinofUranosylcytosine (ABA-C) (0.05-l mg/ml); 2) MKN 28 cells were incubated with VacA+ BCF for I6 h and then IND at different concentrations was added for 8 more h; 3) MKN 28 cells were incubated with 16,16 dimethyl (dm) PGE2 10”-2.5~10.‘M and ASA 1 mM for 30 min, VacA+ BCF was then added and cells incubated for 24 h. RESULTS: I) NSAIDs, but not ABA-C, prevented dose-dependently and significantly (pcO.05) VacA-induced vacuole formation; 2) NSAIDs and ARA-C dose-dependently and significantly (p
2 INTERLEUKIN (IL)-12 RECEPTOR f32 SUBUNIT EXPRESSION IS UP-REGULATED IN HELICOBACTER PYLORI INFECTED GASTRIC MUCOSA T. Parr&o, F. Luua, L. Sebkova, P. Pingitore, G. Mont&one, M. Imeneo, F. Pallone University of Catanzaro;SLBartholomsw’s and Royal London School of Medicine and Dentistry, London, UK, University of Rome Tor Vergata, Italy Bsckgroud. lnterleukin (IL)-12 is up-regulated in H.py/on (HP) infection and contributes to the Thl-type polarisation in this condition. Biological functions of IL-12 are mediated through interaction with a specific receptor foned by two subunlts: 31 and p2. IL-12RS2 seems crucial in maintaining IL-12 responsiveness and controlling Thl lineage commitment. Aim. To investigate whether IL-12R92 expression is up-regulated in Hp gastritis. Methods. Fourteen patients (8 M. aae ranoe 18-69 vrs. median 39) who underwent aastrosooov for byspepiic complaints &re studied. Six patients were ;e-endoszped after eradication therapy. During endoscopy. four antral biopsies were obtained from each patient. Two samples were used for gastric histology (Sydney system score) and to assess Hp status (urease quick test and Giemsa staining). Hp eradication was further assessed by urea Cl3 breath test. Autologous peripheral blood mononuclear cells (PBMC) were, also, obtained from each patient IL-f2RSl and 82, IL12@40. IFN-y, IL4, and IL-5 mRNA were tested by semiquantitetive RT-PCR. Levels of RNA transcripts were compared with S-actin by laser densitomehy and expressed as arbitrary units. Results. IL-1ZRSl and IL-12R92 transcripts were found in all samples tested. However, a more pronounced expression of IL-12RS2, but not IL-12RSl. was detected in gastric mucosa of Hp positive (n=9) than negative (n=5) patients (0.58 r 0.11 vs 0.33 r 0.08, p-zO.05). In Hpinfected gastric mucosa. up-regulation IL-12RS2 was associated with an increased expression of IL-12/p40 and IFN-y. IL-4 was expressed at low level in all samples tested, whereas IL-5 transcripts were not detectable. According to the improvement of gastritis score, levels of IL-12Rp2, IL-12/p40 and IFN-y expression significantly (~~0.05) decreased after HD eradication while levels of IL-4 did not change. Both IL-12RSl anoIL-12RE2 were barely detectable in PBMC. ” Conclusion. IL-12RS2 is up-regulated in Hoinfected gastric mucosa. further supporting the concept that IL-12 is involved in modulating local immune response during gastric Hp colonisation.
ANTI-VACA AM) CAGA
ANTIBODIBS
CBOSS-REACT
WITH
VASCULAR
ANTIGENS. F Fmncescbi,
AR Sepulveda, A Gasbarrioi, EC Nista, G Gasbanini, DY Gmham,RMGcDta. Baylor College of Medicine, VA Medical Center, Houston, TX and Catholic University
of Rome, Italy.
Objective sad aims: H. pykwi-CagA+ strains have been proposed to affect some organic and fimctional cardiovascular diseases. However, the pathogeoesis is still m&ennined. Aim was to dctcrmine whether speciiic anti-CagA and anti-VacA antibodies cross-react with antigens of normal and atherosclerotic wteriea. Mat&ah and Methods: Eight umbilical cord sections, 14 atbemsclemtic attety sections and 10 gastrointestinal tract sections were studied by immunohistcchemishy. Polyclooal anti-C&A and monoclonsl anti-VacA antibodies were used as primary antibodies. Intensity of immunoreactivity was scored on a 4-point scale: 0 (absence), 1 (weak), 2 (moderate) and 3 (intense). Remits: Anti-VacA antibodies strongly reacted with cytoplasmic antigens of smooth muscle cells in all sections of umbilical cord (mean score 2.5M. 19) ami athemsclemtic vessels (mean score 2.6M.2). Anti-G@ reacted with cytoplasm and nuclei of smooth muscle cells in umbilical
cord (mean score
2.37M. 18) sod atbe.rosclerotic
vessel
sections
(mean score 2.4?0.19), cytoplasm of fibroblasts-like cells in intimal atherosclerotic plaques (mean score 2.5io.2), and cell membrane of cndothelial cells (mean score 2.6kO. 18). Interestingly, smooth muscle cells from the gastrointestinal tract did not react with both anti-VacA and CagA antibodies. Conclusions: Anti-CagA and -VacA antibodies cross-react with antigens form both oormal and athemsclemtic blood vessels. Smooth muscle cells, fibmblasts-like cells and endothelial cells represent the target of immttrtorcactivity. tlsnmely, neither antibody reactcd with smooth muscle cells of the gastmhteshal tract. Binding of anti-VacA and-C&A antibodies to those vasdar antigens, if it occurs in viva, could affect vascular physiology and infIue.nce the progression of atherosclerosis in H. pyloriinfected patients.
A65
3
1 HELICOBACTER PVLOBI MFECTION Cad-POSITIVE THE OXIDATIVE DNA DAMAGE OF GASTBIC MUCOSA.
INCREASES
Introduction/aim: Reactiw oxygea metabolitescan interacr directly wkb genomic DNA, producing several bax mcdificariarr including the fonnarion of the muragenic adduo 8-bydmxydwxy8uanosme (8OHdG). Gxidauve DNA addud formation has been sbovm to be imporrmd in the prccas of’ carcinogeneris. We have previously demonstrated that, in gastric rnucosa, oxrdstive DNA damage, quantified trough the mucoral cmceatratim of 80HdG. associateswith I-I. pylori infectloo, gland atrophy, and intesrinsl meraplasia 0.M.). H.pylori srrains harboring CagA 8enehave beenassociatedwith the highest risk of developmentof atmphy, LM and &ric cancer. The aim of tba rmdy war to ascertain wbaher H.pylori CagA+ve infection condates with the prcsencc of praancerous conditions more frequently than in CasA-ve H.pvIori infectioo and with hi&r concentrations 8OHdG in 8artric
m”&sa. Pstieots sed Methods: Gris hundredtwo patimrs were studied@UF=62l40, mean me 61+14 m). Fourteen sstients were H ~vlori nesstive (-vel. Anions H. wlori p&rive (+A) patients, t6.wei.e CagA pai& (+ve: and 38 Ca8A negative’(&). In 24 patienrs H. pylon mfer&n bad been eradicatedgi.pylori-Ersd) more thim 6 months before 8xrnc samplisg. H.pylori infsrion was deemiinrd wab modified Oiemsa staining. In ardmm, corpus and cardias. gastritis, d&are acritiy, atrcpbyrl.M. were semiqsantirsdvely scored In asrml biopsies, 8OHdG cnncentmricm was asserscd by HPLC and elecrnxbemical d&&xi and expressed ss number of adduds 8-OHdG/IO’ dG residues. CagA startis ws determinedby PCR Resuk Degree of gastritis was nd associared with CagA+ve infectico in all gasmc sites In corpus, CagA genaype was si8irificantly correlated with disease activity @=0.05). Owd, no si&icant difference was found m avopbyll.M., however, ammg patients younger tlnm 50, gastric avophy significantly associated with Cag+ve status @=0.02). Atrophy and I.M. were lixnd in 55% of CegA+ve and 16% of CagA-ve patients (KR. 6.5 95% C.L.l.240.2). CagA+ve patients had 8OHdG levels significantly higher than all the dher subgroups @=O01) and in particular rban C&Aye patmds @=0.05), also in parients younger than 50 @=O05). No QfFerencein c&dative DNA damagewar deterred betweenH.pylonErad. and H.pylori-vs paients. Higherr levels of 8OHdG were detected in pauenrs who were CagA+ve and hadab-cphy/l.M. Cooelurions: CagA+ve patients were characterizedby I, tugher diseaseacnwty at du corpus, 2. higher RR for atrqhy when younger than 50, 3. l@ber DNA oxnfatm damage,overall andis particular at younger age. Highest DNA oridative damage levels were found in H.pylori+ve CagA+ve patients wnh &c&c metaplsria. This sating may representP cancer-pronebiological ccmtext
NONSTEROIDAL ANTI-INFLAMMATORY DRUGS COUNTERACT H. pylon’ VncA TOXIN-INDUCED VACUOLATION OF GASTRIC EPITHELIAL CELLS IN VITRO. V. Ricci,’ B.A. Manzo,*t P. Sommi,** C. Tuccillo,t M. SsritarigeIo,~ R. Zarrilli,t. M. Romanot ‘Istituh di Fisiologia Umsna e “Patologia Generale, Universit& DI Pavia; tGsr.troenterologia, Seconda Univenitii. di Napoli; **Diwrtimento di Bioloda e Patolonia Cellulare e Molecolare, Univ&sitB Fed&co II, Nipoli BACKGROUND: VacA toxin from H. p$ori induces cytoplasmic vacuolation in gastric epithelial cells in v&o and seems to play a major role in gastric damage. The relationship between nansteroidal antiinflammatory drugs (NSAIDs) and H. pylori-induced gastric mucosal cell injury is controversial. AIM: I) To evaluate whether NSAIDs interfere with VacA-induced vacuole formation in gastric epitbelial cells in culture and 2) to assess the mechanism of any such effect. METHODS: Cell Q&&x8: MKh’ 28 cells &urn a human well differentiated gastric adenocarcinoma; Cell Vacuolation: by neutral red dye uptake; QII :by Pmllferatlon: by [‘HI-tbymidine uptake; VacA confocal microscopy. EXPERIMENTAL DESIGN: I) MKN 28 cells were incubated for 16-24 h with VacA+ broth culture filtrate (BCF) without (contra!) or with aspirin (AP 4) (n C-10 mM). indorncrhncin (IND) (0.1-2.5 mM), NS-398 (I-100 pM), and with the DNA synthes.s inhibitor I-b-D-arabinofUranosylcytosine (ABA-C) (0.05-l mg/ml); 2) MKN 28 cells were incubated with VacA+ BCF for I6 h and then IND at different concentrations was added for 8 more h; 3) MKN 28 cells were incubated with 16,16 dimethyl (dm) PGE2 10”-2.5~10.‘M and ASA 1 mM for 30 min, VacA+ BCF was then added and cells incubated for 24 h. RESULTS: I) NSAIDs, but not ABA-C, prevented dose-dependently and significantly (pcO.05) VacA-induced vacuole formation; 2) NSAIDs and ARA-C dose-dependently and significantly (p
2 INTERLEUKIN (IL)-12 RECEPTOR f32 SUBUNIT EXPRESSION IS UP-REGULATED IN HELICOBACTER PYLORI INFECTED GASTRIC MUCOSA T. Parr&o, F. Luua, L. Sebkova, P. Pingitore, G. Mont&one, M. Imeneo, F. Pallone University of Catanzaro;SLBartholomsw’s and Royal London School of Medicine and Dentistry, London, UK, University of Rome Tor Vergata, Italy Bsckgroud. lnterleukin (IL)-12 is up-regulated in H.py/on (HP) infection and contributes to the Thl-type polarisation in this condition. Biological functions of IL-12 are mediated through interaction with a specific receptor foned by two subunlts: 31 and p2. IL-12RS2 seems crucial in maintaining IL-12 responsiveness and controlling Thl lineage commitment. Aim. To investigate whether IL-12R92 expression is up-regulated in Hp gastritis. Methods. Fourteen patients (8 M. aae ranoe 18-69 vrs. median 39) who underwent aastrosooov for byspepiic complaints &re studied. Six patients were ;e-endoszped after eradication therapy. During endoscopy. four antral biopsies were obtained from each patient. Two samples were used for gastric histology (Sydney system score) and to assess Hp status (urease quick test and Giemsa staining). Hp eradication was further assessed by urea Cl3 breath test. Autologous peripheral blood mononuclear cells (PBMC) were, also, obtained from each patient IL-f2RSl and 82, IL12@40. IFN-y, IL4, and IL-5 mRNA were tested by semiquantitetive RT-PCR. Levels of RNA transcripts were compared with S-actin by laser densitomehy and expressed as arbitrary units. Results. IL-1ZRSl and IL-12R92 transcripts were found in all samples tested. However, a more pronounced expression of IL-12RS2, but not IL-12RSl. was detected in gastric mucosa of Hp positive (n=9) than negative (n=5) patients (0.58 r 0.11 vs 0.33 r 0.08, p-zO.05). In Hpinfected gastric mucosa. up-regulation IL-12RS2 was associated with an increased expression of IL-12/p40 and IFN-y. IL-4 was expressed at low level in all samples tested, whereas IL-5 transcripts were not detectable. According to the improvement of gastritis score, levels of IL-12Rp2, IL-12/p40 and IFN-y expression significantly (~~0.05) decreased after HD eradication while levels of IL-4 did not change. Both IL-12RSl anoIL-12RE2 were barely detectable in PBMC. ” Conclusion. IL-12RS2 is up-regulated in Hoinfected gastric mucosa. further supporting the concept that IL-12 is involved in modulating local immune response during gastric Hp colonisation.
ANTI-VACA AM) CAGA
ANTIBODIBS
CBOSS-REACT
WITH
VASCULAR
ANTIGENS. F Fmncescbi,
AR Sepulveda, A Gasbarrioi, EC Nista, G Gasbanini, DY Gmham,RMGcDta. Baylor College of Medicine, VA Medical Center, Houston, TX and Catholic University
of Rome, Italy.
Objective sad aims: H. pykwi-CagA+ strains have been proposed to affect some organic and fimctional cardiovascular diseases. However, the pathogeoesis is still m&ennined. Aim was to dctcrmine whether speciiic anti-CagA and anti-VacA antibodies cross-react with antigens of normal and atherosclerotic wteriea. Mat&ah and Methods: Eight umbilical cord sections, 14 atbemsclemtic attety sections and 10 gastrointestinal tract sections were studied by immunohistcchemishy. Polyclooal anti-C&A and monoclonsl anti-VacA antibodies were used as primary antibodies. Intensity of immunoreactivity was scored on a 4-point scale: 0 (absence), 1 (weak), 2 (moderate) and 3 (intense). Remits: Anti-VacA antibodies strongly reacted with cytoplasmic antigens of smooth muscle cells in all sections of umbilical cord (mean score 2.5M. 19) ami athemsclemtic vessels (mean score 2.6M.2). Anti-G@ reacted with cytoplasm and nuclei of smooth muscle cells in umbilical
cord (mean score
2.37M. 18) sod atbe.rosclerotic
vessel
sections
(mean score 2.4?0.19), cytoplasm of fibroblasts-like cells in intimal atherosclerotic plaques (mean score 2.5io.2), and cell membrane of cndothelial cells (mean score 2.6kO. 18). Interestingly, smooth muscle cells from the gastrointestinal tract did not react with both anti-VacA and CagA antibodies. Conclusions: Anti-CagA and -VacA antibodies cross-react with antigens form both oormal and athemsclemtic blood vessels. Smooth muscle cells, fibmblasts-like cells and endothelial cells represent the target of immttrtorcactivity. tlsnmely, neither antibody reactcd with smooth muscle cells of the gastmhteshal tract. Binding of anti-VacA and-C&A antibodies to those vasdar antigens, if it occurs in viva, could affect vascular physiology and infIue.nce the progression of atherosclerosis in H. pyloriinfected patients.
A65