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Holicobacter pylori (H.p) infection: comparison of two serological tests
A894
AGA ABSTRACTS
HeHcobacter pylori (H.p.) infection: comparison of two serological tests. A.S.Pinto.T.Lopes, A.S.C-nerreiro, M.G.Quina. Cllnica Universit~ia de Medi¢ina Intema • de GasUenterologia do Hospital de Pulide Valeme- Lisboa- Portugal Aim: To compare Elisa and Inmnmoblottlng (IB) methods in H.p. detection. Material sad methods: It was performed the seric estimation of the H.p. antibodies (IsG) in 24 samples by Elisa method (Cobas-Core-Roche) and IB (Helicoblot KitDiagnostic Biotechnology P.T.E.). The samples were divided in 2 groups based in the optical densities (OD) by Elisa method: .GroupI- Elisa(-)-<90; n=ll(SF;6M)ages(>3, <30)(average: 10.6y) .Group II- Eiisa (+)- >120; n= 13 (7F; 6M) ages (>25, <83) (average: 53.8y). Stati~'cai analysis: Chi-square test, positive predictive value (PPV) and negative predictive value (NPV). Resalts: The average number of bsads which were detected by IB was 0.9 (0-2) in the group I and 5.5 (0-9) in the Stoup II (p< 0.0001). All the samples with three or more bands were H.p.(+) by Elisa method (PPV-I00%; NPV-92%). The bands corresponding to the molecular weight proteins <40KDa and >66KDa(l) were the most associated with positivity to H.p. by Etisa method (PPV-92%; NPV-83%). In the 8xoup 1I, 69% of the samples had the II6KDa protein. Its presence was associated with high values in optical density (OD>1000) by Elisa method (PPV-100%; NPV-50%).
Conclusions: 1.There is a strong correlation between the two serological tests. 2~The number of bands (E3) by I]3 is strongly correlated with H.p. positivity by Elisa method. &Protein bands with molecular weight <40KDa and >66KDa were the most specific for H.p: positivityby Elisa method. 4.116 KDa protein band correlates with the highest OD values by Elisa method, seSgest~ ~s hopor~ant tmnumo~=ty. (t)
l~iEeu M et tl
Cli~Dia~Lab.l~mmol.May1994:310-7
ULCERATIVE COLITIS AND PORCINE PROLIFERATIVE ENTEROPATHY: A COMMON BACTERIAL ETIOLOGY?. MC.L. Pitcher*, M. Goddardt, S. McOrist:~, J.H. Cummings*. MRC Dunn Clinical Nutrition Centre, Cambridge*; Addenbrooke's Hospital, Cambddget; Royal (Dick) School of Veterinary Studies, University of Edinburgh~, U.K. 95% of patients with active ulcerative colitis (UC) carry sulfatereduc ng bacter a (SRB) in their feces compared to only 55% of those in a state of disease remission and 46% of patients with ileo-anal pouches without evidence of pouchitis. Over 90% of UC-associated SRB belong to the genus Desulfovibrio and are more readily adaptable to the environmental conditions that prevail inside the colitic colon where they produce h gh concentratons of hydrogen sulfide. Reducing sulfur compounds are h gh y tox c causing inhibition of n-butyrate oxidation within colonocytes predominantly of distal gut origin. Proliferative enteropathy (PE) is an nf ammatory intestinal disorder of pigs, ferrets and hamsters associated with the presence of an ntracellular organism, IS intracellulads, in affected mucosa. These are morphologically similar to SRB and 16s rDNA sequence analysis has shown a 91% homology with the genome to Desulfovibrio desulfuricans. Since intramural bacteria have been demonstrated in both inflamed UC tissue and in ileo-anal pouches a novel histochemical study was conducted to determine whether PE, UC and pouchitis share a common bacterial etiology. 12 rectal biopsies from UC patients (5 active, 7 remission) and 8 biopsies from ileo-anal pouches (5 pouchitis, 3 mild acute inflammatory changes only) were dewaxed trypsin digested at pH 7.8 and treated with mouse monoclonal antibody IG4 targeted to IS intracellularis derived from mucosa of a pig with confirmed hemorrhagic PE. Histological sections were stained with rabbit anti-mouse FITC and observed for fluorescing curved bacilli within the mucosa. Sections of intestinal tissue from a pig with PE and a healthy pig were used as positive and negative controls respectively. Fluoresc ng bacilli were observed within the apical cytoplasm of enterocytes in the positive control specimen but in none of the co on c or ileal pouch biopsy specimens. t s conc uded that although Desulfovibrio spp. are present within the feces of UC and ileo-anal pouch patients, monoc ona antibodies directed against porcine PE could not demonstrate evidence of mucosal invasion in this study. Antigenic variation between human and porcine bacterial strains limiting antibody specificity may be an explanation for these findings. The development of specific monoclonal antibodies may prove usefu n future studies particularly since similar bacteria are now being clearly identified in similar lesions in animals. It is postulated that SRB exert toxic effects on the intestinal muc0sa by luminal production of hydrogen sulfide in individuals with genetically-determined risk factors. (Acknowledgements to NACC, Peel Medical Research Trust, and Addenbrooke's NHS Trust Endowment Fund for financial support).
GASTROENTEROLOGY, Vol. 108, No. 4
SULFATE-REDUCING BACTERIA: PREVALENCE IN ACTIVE AND INACTIVE ULCERATIVE COLITIS. M.C.L. Pitcher. E.R. Beatty, G.R. Gibson, J.H. Cummings. MRC Dunn Clinical Nutrition Centre, Hills Road, Cambddge, U.K. Reducing sulfur compounds within the colonic lumen are highly toxic causing inhibition of n-butyrate oxidation within colonocytes, predom nant y n the distal gut. A major source of hydrogen sulfide !s from the reduction of sulfate of dietary ano mucinous origin 10y me actmn or sulfate-reducing bacteria (SRB) and it has been postulated that they may be important in the pathogenesis of ulcerative colitis (UC) and inflammatory bowel diseases in animals. Anaerobic fecal slurres were prepared from 29 UC patients (20 active, 9 remission), 13 patients with ileo-anal pouches and 10 healthy controls. SRB were enumerated by the agar shake dilution method, sulfatereduct on rates measured by the Jorgensen radiotracer method, sulfide by the methylene blue method modified by Strocch and sulfate by anion exchange chromatography with conductivity detection. No patient had received ant m crobials for at least 1 month previously. Disease activity was graded clinically and histologically from rectal biopsies. Results were statistically assessed by analysis of variance. The incidence of SRB carriage in the active and remission UC groups was 95% and 55% respectively with mean Iog~pcounts/g (SE).of 8.15 (0 7) and 5.02 (1.6) (p=0.041). All controls naroourea ~HU w=m counts of 7 9 (0 7) (p=0.61). 46% of the pouch group .carried SRB but in lower counts of 3.64 (1.26) (p=0.011). Mean sulfate-reauction rates nmol/g/h (SE) were not significantly different from controls (0.13 (2.5)). Median fecal sufldes i~nol/g (SE) in the UC group did not differ significantly from controls (0,24 (0.08)) but were lower in pouches (0.09 (0.01)), (p=0.034). Mean free fecal sulfate itmol/g (SE) in the UC group (1.38 (0.25)) was higher than in controls (0.58 (0.16)) (p=0.055). Mean bound fecal sulfate was undetectable in the pouch group (0 (0.19)) compared to controls (0.81 (0.09)) (p=O.011). The ratio of free/total mean fecal suffate was higher in the UC group (0.75 (0.09)) (p=0.009) and pouch group (1 05 (0.15)) (p=0.001) than controls (0.36 (0.09)). It is concluded that SRB are more prevalent in active UC although their activities do not appear to differ from controls. SRB growth in the ileoana pouch is reported for the first time. The higher ratios of free/total fecal sulfate in UC patients may be due to increased enzymatic desulfatation of mucin by feca bacteria and in pouch patients to the lesser degree of su fate ncorporation of mucin with a higher proportion appearing as free sulfate. Sulfate availability is of crucial importance to ox dative, as opposed to fermentative, growth of SRB and the resultant luminal production of sulfide could trigger colonic inflammation in individuals with genetically determined risk factors. (Acknowledgements to NACC, Peel Medical Research Trust & Addenbrooke's NHS Trust Endowment Fund).
TRANSDUCTION OF THE COLONIC MUCOSA BY RETENTION E N E M A DELIVERY OF A RETROVIRAL REPORTER GENE FOLLOWING EXPERIMENTALLY-INDUCED RABBIT COLITIS. T.T. Pizarro a, V. Casini-Raggia, M. Gordon b, and F. Cominellia. Division of Gastrointestinal and Liver Diseases a and The Vector Production Unitb, USC, School of Medicine, Los Angeles, CA. The aim of the present study was to develop a gene therapy model that can successfully and specifically transduce colonic cells with the ability to locally produce and deliver recombinant anti-inflammatory cytokines into the colonic microenvironment following experimentallyinduced colitis. To determine if successful transduetion of replicating c e l l s w i t h i n the i n f l a m e d colonic mucosa could be achieved, inflammation was induced in the distal colon using the model of rabbit immune-complex colitis. A 4.0 In] retention enema solution (10 p g / m l protamine sulfate) with a titer of 6 X 105 c f u / m l of a retroviral vector containing a neomycin resistant (Neo R) reporter gene was then administered 6 hr post-colitis and twice daffy thereafter for two days. Control animals treated with saline retention enemas underwent the same experimental protocol. Healthy rabbits given either retroviral or saline enemas (same protocol as above), wherein colitis was not induced, were used for safety precautions; these animals were sacrificed and analyzed using the same criteria as colitis animals. 48 hrs after the induction of colitis, rabbits were sacrificed and the distal colon was removed. Ten cross-sectional specimens from the distal 10 cm of colon were obtained and processed for the presence of Neo R using RT-PCR methodologies. We report that only colon specimens obtained from colitis rabbits receiving retroviral vector retention enemas containing the Neo R reporter gene showed positive bands for the Neo R gene (424 b.p.). We therefore have developed a model for the successful transduction of the colonic mucosa following experimentally-induced rabbit colitis. This model can be used to locally deliver anti-infiammatory factors to the colonic microenvironment, and may serve as a novel therapy for the treatment of chronic intestinal inflammation, such as IBD.
AGA ABSTRACTS
HeHcobacter pylori (H.p.) infection: comparison of two serological tests. A.S.Pinto.T.Lopes, A.S.C-nerreiro, M.G.Quina. Cllnica Universit~ia de Medi¢ina Intema • de GasUenterologia do Hospital de Pulide Valeme- Lisboa- Portugal Aim: To compare Elisa and Inmnmoblottlng (IB) methods in H.p. detection. Material sad methods: It was performed the seric estimation of the H.p. antibodies (IsG) in 24 samples by Elisa method (Cobas-Core-Roche) and IB (Helicoblot KitDiagnostic Biotechnology P.T.E.). The samples were divided in 2 groups based in the optical densities (OD) by Elisa method: .GroupI- Elisa(-)-<90; n=ll(SF;6M)ages(>3, <30)(average: 10.6y) .Group II- Eiisa (+)- >120; n= 13 (7F; 6M) ages (>25, <83) (average: 53.8y). Stati~'cai analysis: Chi-square test, positive predictive value (PPV) and negative predictive value (NPV). Resalts: The average number of bsads which were detected by IB was 0.9 (0-2) in the group I and 5.5 (0-9) in the Stoup II (p< 0.0001). All the samples with three or more bands were H.p.(+) by Elisa method (PPV-I00%; NPV-92%). The bands corresponding to the molecular weight proteins <40KDa and >66KDa(l) were the most associated with positivity to H.p. by Etisa method (PPV-92%; NPV-83%). In the 8xoup 1I, 69% of the samples had the II6KDa protein. Its presence was associated with high values in optical density (OD>1000) by Elisa method (PPV-100%; NPV-50%).
Conclusions: 1.There is a strong correlation between the two serological tests. 2~The number of bands (E3) by I]3 is strongly correlated with H.p. positivity by Elisa method. &Protein bands with molecular weight <40KDa and >66KDa were the most specific for H.p: positivityby Elisa method. 4.116 KDa protein band correlates with the highest OD values by Elisa method, seSgest~ ~s hopor~ant tmnumo~=ty. (t)
l~iEeu M et tl
Cli~Dia~Lab.l~mmol.May1994:310-7
ULCERATIVE COLITIS AND PORCINE PROLIFERATIVE ENTEROPATHY: A COMMON BACTERIAL ETIOLOGY?. MC.L. Pitcher*, M. Goddardt, S. McOrist:~, J.H. Cummings*. MRC Dunn Clinical Nutrition Centre, Cambridge*; Addenbrooke's Hospital, Cambddget; Royal (Dick) School of Veterinary Studies, University of Edinburgh~, U.K. 95% of patients with active ulcerative colitis (UC) carry sulfatereduc ng bacter a (SRB) in their feces compared to only 55% of those in a state of disease remission and 46% of patients with ileo-anal pouches without evidence of pouchitis. Over 90% of UC-associated SRB belong to the genus Desulfovibrio and are more readily adaptable to the environmental conditions that prevail inside the colitic colon where they produce h gh concentratons of hydrogen sulfide. Reducing sulfur compounds are h gh y tox c causing inhibition of n-butyrate oxidation within colonocytes predominantly of distal gut origin. Proliferative enteropathy (PE) is an nf ammatory intestinal disorder of pigs, ferrets and hamsters associated with the presence of an ntracellular organism, IS intracellulads, in affected mucosa. These are morphologically similar to SRB and 16s rDNA sequence analysis has shown a 91% homology with the genome to Desulfovibrio desulfuricans. Since intramural bacteria have been demonstrated in both inflamed UC tissue and in ileo-anal pouches a novel histochemical study was conducted to determine whether PE, UC and pouchitis share a common bacterial etiology. 12 rectal biopsies from UC patients (5 active, 7 remission) and 8 biopsies from ileo-anal pouches (5 pouchitis, 3 mild acute inflammatory changes only) were dewaxed trypsin digested at pH 7.8 and treated with mouse monoclonal antibody IG4 targeted to IS intracellularis derived from mucosa of a pig with confirmed hemorrhagic PE. Histological sections were stained with rabbit anti-mouse FITC and observed for fluorescing curved bacilli within the mucosa. Sections of intestinal tissue from a pig with PE and a healthy pig were used as positive and negative controls respectively. Fluoresc ng bacilli were observed within the apical cytoplasm of enterocytes in the positive control specimen but in none of the co on c or ileal pouch biopsy specimens. t s conc uded that although Desulfovibrio spp. are present within the feces of UC and ileo-anal pouch patients, monoc ona antibodies directed against porcine PE could not demonstrate evidence of mucosal invasion in this study. Antigenic variation between human and porcine bacterial strains limiting antibody specificity may be an explanation for these findings. The development of specific monoclonal antibodies may prove usefu n future studies particularly since similar bacteria are now being clearly identified in similar lesions in animals. It is postulated that SRB exert toxic effects on the intestinal muc0sa by luminal production of hydrogen sulfide in individuals with genetically-determined risk factors. (Acknowledgements to NACC, Peel Medical Research Trust, and Addenbrooke's NHS Trust Endowment Fund for financial support).
GASTROENTEROLOGY, Vol. 108, No. 4
SULFATE-REDUCING BACTERIA: PREVALENCE IN ACTIVE AND INACTIVE ULCERATIVE COLITIS. M.C.L. Pitcher. E.R. Beatty, G.R. Gibson, J.H. Cummings. MRC Dunn Clinical Nutrition Centre, Hills Road, Cambddge, U.K. Reducing sulfur compounds within the colonic lumen are highly toxic causing inhibition of n-butyrate oxidation within colonocytes, predom nant y n the distal gut. A major source of hydrogen sulfide !s from the reduction of sulfate of dietary ano mucinous origin 10y me actmn or sulfate-reducing bacteria (SRB) and it has been postulated that they may be important in the pathogenesis of ulcerative colitis (UC) and inflammatory bowel diseases in animals. Anaerobic fecal slurres were prepared from 29 UC patients (20 active, 9 remission), 13 patients with ileo-anal pouches and 10 healthy controls. SRB were enumerated by the agar shake dilution method, sulfatereduct on rates measured by the Jorgensen radiotracer method, sulfide by the methylene blue method modified by Strocch and sulfate by anion exchange chromatography with conductivity detection. No patient had received ant m crobials for at least 1 month previously. Disease activity was graded clinically and histologically from rectal biopsies. Results were statistically assessed by analysis of variance. The incidence of SRB carriage in the active and remission UC groups was 95% and 55% respectively with mean Iog~pcounts/g (SE).of 8.15 (0 7) and 5.02 (1.6) (p=0.041). All controls naroourea ~HU w=m counts of 7 9 (0 7) (p=0.61). 46% of the pouch group .carried SRB but in lower counts of 3.64 (1.26) (p=0.011). Mean sulfate-reauction rates nmol/g/h (SE) were not significantly different from controls (0.13 (2.5)). Median fecal sufldes i~nol/g (SE) in the UC group did not differ significantly from controls (0,24 (0.08)) but were lower in pouches (0.09 (0.01)), (p=0.034). Mean free fecal sulfate itmol/g (SE) in the UC group (1.38 (0.25)) was higher than in controls (0.58 (0.16)) (p=0.055). Mean bound fecal sulfate was undetectable in the pouch group (0 (0.19)) compared to controls (0.81 (0.09)) (p=O.011). The ratio of free/total mean fecal suffate was higher in the UC group (0.75 (0.09)) (p=0.009) and pouch group (1 05 (0.15)) (p=0.001) than controls (0.36 (0.09)). It is concluded that SRB are more prevalent in active UC although their activities do not appear to differ from controls. SRB growth in the ileoana pouch is reported for the first time. The higher ratios of free/total fecal sulfate in UC patients may be due to increased enzymatic desulfatation of mucin by feca bacteria and in pouch patients to the lesser degree of su fate ncorporation of mucin with a higher proportion appearing as free sulfate. Sulfate availability is of crucial importance to ox dative, as opposed to fermentative, growth of SRB and the resultant luminal production of sulfide could trigger colonic inflammation in individuals with genetically determined risk factors. (Acknowledgements to NACC, Peel Medical Research Trust & Addenbrooke's NHS Trust Endowment Fund).
TRANSDUCTION OF THE COLONIC MUCOSA BY RETENTION E N E M A DELIVERY OF A RETROVIRAL REPORTER GENE FOLLOWING EXPERIMENTALLY-INDUCED RABBIT COLITIS. T.T. Pizarro a, V. Casini-Raggia, M. Gordon b, and F. Cominellia. Division of Gastrointestinal and Liver Diseases a and The Vector Production Unitb, USC, School of Medicine, Los Angeles, CA. The aim of the present study was to develop a gene therapy model that can successfully and specifically transduce colonic cells with the ability to locally produce and deliver recombinant anti-inflammatory cytokines into the colonic microenvironment following experimentallyinduced colitis. To determine if successful transduetion of replicating c e l l s w i t h i n the i n f l a m e d colonic mucosa could be achieved, inflammation was induced in the distal colon using the model of rabbit immune-complex colitis. A 4.0 In] retention enema solution (10 p g / m l protamine sulfate) with a titer of 6 X 105 c f u / m l of a retroviral vector containing a neomycin resistant (Neo R) reporter gene was then administered 6 hr post-colitis and twice daffy thereafter for two days. Control animals treated with saline retention enemas underwent the same experimental protocol. Healthy rabbits given either retroviral or saline enemas (same protocol as above), wherein colitis was not induced, were used for safety precautions; these animals were sacrificed and analyzed using the same criteria as colitis animals. 48 hrs after the induction of colitis, rabbits were sacrificed and the distal colon was removed. Ten cross-sectional specimens from the distal 10 cm of colon were obtained and processed for the presence of Neo R using RT-PCR methodologies. We report that only colon specimens obtained from colitis rabbits receiving retroviral vector retention enemas containing the Neo R reporter gene showed positive bands for the Neo R gene (424 b.p.). We therefore have developed a model for the successful transduction of the colonic mucosa following experimentally-induced rabbit colitis. This model can be used to locally deliver anti-infiammatory factors to the colonic microenvironment, and may serve as a novel therapy for the treatment of chronic intestinal inflammation, such as IBD.