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Does close rectal dissection for proctectomy in inflammatory bowel disease protect against impotence?
Abstracts
57 ACTIVIN A MODULATES INTESTINAL EPITHELIAL CELL FUNCflON IN YlTRo ,&g,& Sehulte KM, Wie&nwmn B. Dignass AU Dep. of Cmology, Cbari*Vircbow Clinic, Berlin. Germany T3ackground:The TGF@famii member Activin A has been sbom to a&t growth and differendation in a variety of diftaent mrgei c&t. Recently, a strong induction of A&in A axpr&on hru ban demonstn& after skin injury, sugge&g a function of Activin A in wound fopair. Little is known about the fu&kmal activities of Activin A in intestinal epithelial eel1 populati~ and its potential relevance for intestinal wound repair. Aims:Thcaimofthisstudywastoehau&rizethefllnctional e~ofhotivinAoninttstinrlcpitMirrlccUsandtolookfor Aetiviu rweptor expfassion in intestinal apitbalial cells. f&&o& Effects of A&in A on Intestinal epitbalial cell migration were usessed asing on in vim wounding model. Eff&ts on intestinal epithebd all JJroMwiuu were mcasumd by cololimwle Mmasrays In II&MOO, the expression of type II Aotivin receptors was assesod by RT-FCR in IECI cells. The effects of Aclivin A on TGFB mRNA expressionwem datenuiued by semiquantitative RT-PCR. Results: A&via A caused an anbancement of epithalial c&I m&ration and an inhibition of opithelial c8ll proliferation in IBC-6 cells. The effects of Aotivin A on intestinal opitheliaI cells were etdmced by simultanaous addition of TGFB and could not be blo&cd by addition of i mnumonautralizing TGFB antibodies. Semiquantitntive RT-PCR ahowui no differences in TGFP @on foUowiog Activin A treatment In addition, the expmssion of type II Activin nxeptom (ActRII and AotRBE) was confirmed by RT-F’CR. Cinmlusions: Activin A mod&&es epitheIial ceil migration and prolif&ation, two prassses thai are critical for wuund healing, through a TGFB-independentmechanism suggesting that other TGFB family members besi&s TGFpl-3 may play an @or@nt 1010 for intestinal wound hading and may provide new approachesto modulate intastkmlwwldrepair.
RJXSLENCY OF A MODEL HUMAN INTESTINAL EPITHELIUM TO FAS-MEDIATED APOPTOTIC INJURY Authors: Abreu M.T., Palladino A. A., Arnold E.T., Kwon R. S., and. McRoberts J.A. ’ ; Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center and ‘VA Greater Los Angeles Healthcare System Los Angeles, California Background: Intestinal epithelial cell apoptosis occurs continually without apparent permeability defects and is increased in response to intestinal inflammation. The Fas death receptor has been implicated as an effector of crypt epithelial cell apoptosis in IBD. Aims: To test whether immune-mediated apoptosis results in barrier dysfunction of a model epithelium. Methods: T84 cells were cultured as a polarized monolayer and exposed to agonist Ab to Fas. Apoptosis was assessedmorphologically and biochemically. Barrier function was assessedby transepithelial resistance (TER) and permeability measurements. Immunofluorescent staining (IF) was analyzed by confocal microscopy. Results: T84 cells were found to express a high level of Fas which was predominantly basolateral in polarized T84 monolayers. Basolateral cross-linking of Fas resulted in T84 cell apoptosis and a loss of 50% of the cells within 24h. Apoptosis was coincident with a decrease in TER to 50% of baseline values, increased flux of mannitol, but not relatively small macromolecules t3kD. Preservation of barrier function was associated with dramatic rearrangement of tight junctions and desmosomal junctions in apoptotic monolayers. Triple IF-staining of nuclei, tight junctions and E-cadherin in apoptotic monolayers revealed multiple apoptotic and intact nuclei contained within large tight junction outlines but preservation of individual cell-cell contact through E-cadherin interactions. 3-D reconstruction of the monolayers shows flattening of apoptotic monolayers to 40% of control monolayer height. Conclusion: Immune-mediated apoptosis of intestinal epithelial cells may contribute to the permeability defects associated with inflammatory conditions of the bowel but the intestinal epithelium is remarkably resilient at preserving barrier function in the face of apoptosis.
St+
60 DISCORDANT SIBLING PAIR LINKAGE ANALYSIS PROVIDES FVRTHER B"PWRT FOR THE caRoM3.soME 12 INFLAMMATORY BOWEL DISEASE S"SCEPTlBILITY Locus van Heel Wellcome
DA, Marlow A, Satsangi J, Carey Truet Centre for Human Genetics,
Iui, Jewel1 DP Oxford, UK
Background: Evidence for an inflammatory bowel disease (IBD) susceotibilitv locus on chromosome 12 has now been shown using affected sibling/relative pair linkage studies from five independent centres world-wide. Increased allele sharing in siblings could also be due to other factors, including prenatal genetlc selection which may cause over 30% of conceptions to be lost. Aims: To use discordant sibling par llnkage analysis as and a control for our effected sibling pair linkage study, as a new patient group to confirm/refute this region. Methods: Eight microsatellite markers spanning the mly reported regions of linkage on chromosome 377 12913-14 30cM) were genotyped on ABI (approx. Seq"e"CeIS zn 236 affected sibling pair pedigrees. Multipoint non-parametric linkage analysis was performed 163 discordant sibling pairs using ASPEX sib-phase ~2.0. disease only pedigrees, 21 from 28 Crohn'e (dep) ulcerative colitis only pedigreees and 16 mxed pedigrees were analysed. Independent dsp were formed using the 1" affected sibling and all unaffected siblings. ReSUlt5: Genotype success rate was >99%. Reduced mean allele sharino (below 50%) was observed in the reaion around previousl; reported peaks of linkage at d12s83-and d12s85: n of
All dep Independent
163
dsp
80
dsp
Peak reduced sharing D12S90 D12S90
%sharing
mlod
p
45.1%
0.65
41.6%
0.93
0.04 0.02
Conclusion: Discordant sibling pairs show significantly reduced allele sharing around this previously reported locus. Discordant elbling pair analysis provides an important negative control for affected sib-pair studies, and provides new evidence for an IBD susceptibility locus on chromosome 12.
DOES CLOSE RECTAL DISSECTION FOR PROCTECTOMY IN INFLAMMATORY BOWEL DISEASE PROTECT AGAINST IMPOTENCE? J)indsep I, George BD, Kettlewell MG, Mortensen NJMcC. John Radcliffe Hospital, Oxford, UK. Background: Close rectal dissection of the rectum (CRD) is a surgical technique used by some surgeons in inflammatory bowel disease (IBD). It is carried out within the mesorccmm close to the rectal muscle tube to avoid damaging the pelvic sexual nerves. Other surgeons dissect in the more anatomical mesorectal plane using mesorectal dissection (MRD). It is not known whether CRD protects the sexual nerves better than MRD. Aims: We aimed to determine the difference in impotence rates using these two surgical dissection techniques. Method: Patients undergoing IBD surgery were entered on a prospective database. Male patients were mailed a standardised validated urological impotency questionnaire, the International Index of Erectile Function. Results: There was an 81% response rate. 6 of 156 assessable patients were totally impotent (3.8%). They were all in the 50-70 year age group, with no impotence in patients under 50.21 patients complained of minor diminution of erectile function (13.5%), where sexual activity was still possible. There was no statistical difference in the rate of complete (2.2% vs 4.5%, p=O.67) or partial (13.5% vs 13.38, p=O.99) impotence between close rectal and mesorectal dissection (Fisher’s exact test). There were no ejaculatory difficulties. The time elapsed since surgery ranged from 2.7 to 192.7 months with a median of 74.5 months. Conclusions: Rectal excisional surgery for IBD can be conducted with low rates of impotence. Minor difficulties with erections may be more common than currently recognised. We could not show that CRD significantly protects the patient from impotence compared to operating in the anatomical mesorectal plane.
A19
57 ACTIVIN A MODULATES INTESTINAL EPITHELIAL CELL FUNCflON IN YlTRo ,&g,& Sehulte KM, Wie&nwmn B. Dignass AU Dep. of Cmology, Cbari*Vircbow Clinic, Berlin. Germany T3ackground:The TGF@famii member Activin A has been sbom to a&t growth and differendation in a variety of diftaent mrgei c&t. Recently, a strong induction of A&in A axpr&on hru ban demonstn& after skin injury, sugge&g a function of Activin A in wound fopair. Little is known about the fu&kmal activities of Activin A in intestinal epithelial eel1 populati~ and its potential relevance for intestinal wound repair. Aims:Thcaimofthisstudywastoehau&rizethefllnctional e~ofhotivinAoninttstinrlcpitMirrlccUsandtolookfor Aetiviu rweptor expfassion in intestinal apitbalial cells. f&&o& Effects of A&in A on Intestinal epitbalial cell migration were usessed asing on in vim wounding model. Eff&ts on intestinal epithebd all JJroMwiuu were mcasumd by cololimwle Mmasrays In II&MOO, the expression of type II Aotivin receptors was assesod by RT-FCR in IECI cells. The effects of Aclivin A on TGFB mRNA expressionwem datenuiued by semiquantitative RT-PCR. Results: A&via A caused an anbancement of epithalial c&I m&ration and an inhibition of opithelial c8ll proliferation in IBC-6 cells. The effects of Aotivin A on intestinal opitheliaI cells were etdmced by simultanaous addition of TGFB and could not be blo&cd by addition of i mnumonautralizing TGFB antibodies. Semiquantitntive RT-PCR ahowui no differences in TGFP @on foUowiog Activin A treatment In addition, the expmssion of type II Activin nxeptom (ActRII and AotRBE) was confirmed by RT-F’CR. Cinmlusions: Activin A mod&&es epitheIial ceil migration and prolif&ation, two prassses thai are critical for wuund healing, through a TGFB-independentmechanism suggesting that other TGFB family members besi&s TGFpl-3 may play an @or@nt 1010 for intestinal wound hading and may provide new approachesto modulate intastkmlwwldrepair.
RJXSLENCY OF A MODEL HUMAN INTESTINAL EPITHELIUM TO FAS-MEDIATED APOPTOTIC INJURY Authors: Abreu M.T., Palladino A. A., Arnold E.T., Kwon R. S., and. McRoberts J.A. ’ ; Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center and ‘VA Greater Los Angeles Healthcare System Los Angeles, California Background: Intestinal epithelial cell apoptosis occurs continually without apparent permeability defects and is increased in response to intestinal inflammation. The Fas death receptor has been implicated as an effector of crypt epithelial cell apoptosis in IBD. Aims: To test whether immune-mediated apoptosis results in barrier dysfunction of a model epithelium. Methods: T84 cells were cultured as a polarized monolayer and exposed to agonist Ab to Fas. Apoptosis was assessedmorphologically and biochemically. Barrier function was assessedby transepithelial resistance (TER) and permeability measurements. Immunofluorescent staining (IF) was analyzed by confocal microscopy. Results: T84 cells were found to express a high level of Fas which was predominantly basolateral in polarized T84 monolayers. Basolateral cross-linking of Fas resulted in T84 cell apoptosis and a loss of 50% of the cells within 24h. Apoptosis was coincident with a decrease in TER to 50% of baseline values, increased flux of mannitol, but not relatively small macromolecules t3kD. Preservation of barrier function was associated with dramatic rearrangement of tight junctions and desmosomal junctions in apoptotic monolayers. Triple IF-staining of nuclei, tight junctions and E-cadherin in apoptotic monolayers revealed multiple apoptotic and intact nuclei contained within large tight junction outlines but preservation of individual cell-cell contact through E-cadherin interactions. 3-D reconstruction of the monolayers shows flattening of apoptotic monolayers to 40% of control monolayer height. Conclusion: Immune-mediated apoptosis of intestinal epithelial cells may contribute to the permeability defects associated with inflammatory conditions of the bowel but the intestinal epithelium is remarkably resilient at preserving barrier function in the face of apoptosis.
St+
60 DISCORDANT SIBLING PAIR LINKAGE ANALYSIS PROVIDES FVRTHER B"PWRT FOR THE caRoM3.soME 12 INFLAMMATORY BOWEL DISEASE S"SCEPTlBILITY Locus van Heel Wellcome
DA, Marlow A, Satsangi J, Carey Truet Centre for Human Genetics,
Iui, Jewel1 DP Oxford, UK
Background: Evidence for an inflammatory bowel disease (IBD) susceotibilitv locus on chromosome 12 has now been shown using affected sibling/relative pair linkage studies from five independent centres world-wide. Increased allele sharing in siblings could also be due to other factors, including prenatal genetlc selection which may cause over 30% of conceptions to be lost. Aims: To use discordant sibling par llnkage analysis as and a control for our effected sibling pair linkage study, as a new patient group to confirm/refute this region. Methods: Eight microsatellite markers spanning the mly reported regions of linkage on chromosome 377 12913-14 30cM) were genotyped on ABI (approx. Seq"e"CeIS zn 236 affected sibling pair pedigrees. Multipoint non-parametric linkage analysis was performed 163 discordant sibling pairs using ASPEX sib-phase ~2.0. disease only pedigrees, 21 from 28 Crohn'e (dep) ulcerative colitis only pedigreees and 16 mxed pedigrees were analysed. Independent dsp were formed using the 1" affected sibling and all unaffected siblings. ReSUlt5: Genotype success rate was >99%. Reduced mean allele sharino (below 50%) was observed in the reaion around previousl; reported peaks of linkage at d12s83-and d12s85: n of
All dep Independent
163
dsp
80
dsp
Peak reduced sharing D12S90 D12S90
%sharing
mlod
p
45.1%
0.65
41.6%
0.93
0.04 0.02
Conclusion: Discordant sibling pairs show significantly reduced allele sharing around this previously reported locus. Discordant elbling pair analysis provides an important negative control for affected sib-pair studies, and provides new evidence for an IBD susceptibility locus on chromosome 12.
DOES CLOSE RECTAL DISSECTION FOR PROCTECTOMY IN INFLAMMATORY BOWEL DISEASE PROTECT AGAINST IMPOTENCE? J)indsep I, George BD, Kettlewell MG, Mortensen NJMcC. John Radcliffe Hospital, Oxford, UK. Background: Close rectal dissection of the rectum (CRD) is a surgical technique used by some surgeons in inflammatory bowel disease (IBD). It is carried out within the mesorccmm close to the rectal muscle tube to avoid damaging the pelvic sexual nerves. Other surgeons dissect in the more anatomical mesorectal plane using mesorectal dissection (MRD). It is not known whether CRD protects the sexual nerves better than MRD. Aims: We aimed to determine the difference in impotence rates using these two surgical dissection techniques. Method: Patients undergoing IBD surgery were entered on a prospective database. Male patients were mailed a standardised validated urological impotency questionnaire, the International Index of Erectile Function. Results: There was an 81% response rate. 6 of 156 assessable patients were totally impotent (3.8%). They were all in the 50-70 year age group, with no impotence in patients under 50.21 patients complained of minor diminution of erectile function (13.5%), where sexual activity was still possible. There was no statistical difference in the rate of complete (2.2% vs 4.5%, p=O.67) or partial (13.5% vs 13.38, p=O.99) impotence between close rectal and mesorectal dissection (Fisher’s exact test). There were no ejaculatory difficulties. The time elapsed since surgery ranged from 2.7 to 192.7 months with a median of 74.5 months. Conclusions: Rectal excisional surgery for IBD can be conducted with low rates of impotence. Minor difficulties with erections may be more common than currently recognised. We could not show that CRD significantly protects the patient from impotence compared to operating in the anatomical mesorectal plane.
A19