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Bile tolerance and bile salt hydrolase activity of lactobacilli and bifidobacteria isolated from the human intestine
A928
AGA ABSTRACTS
• INDUCTION OF ORAL TOLERANCE (OT) IN T CELL RECEPTOR TRANSGENIC MICE. W.G. Thaggarci, M. Tomasi, P.A. Dean, C.T. Weaver, C.O. EIson. Departments of Medicine, Surgery and Pathology, University of Alabama at Birmingham, AL. B a c k u r o u n d / P u r D o s e The mechanisms underlying oral tolerance are poorly understood. The purpose of this study was to determine whether oral tolerance could be induced in an *x13T cell receptor transgenic mouse ( T g ) i n which most T cells recognize ovalbumin (OVA) in the context of H2 d. Methode: Tg and non Tg BALB/c mice were fed OVA 20 mg in soluble form or 2 mg incorporated in the fluid phase of a multiple emulsion delivery system (MES) on days -14 and -7. Mice were then parenterally immunized with OVA in MES I.P. on day 0, 14, and 28. Samples of serum were taken at day 35 and assayed for IgG anti-OVA by ELISA. At day 56 skin test responses to OVA were tested in the ear. Results: Tg and non-Tg mice which were not fed OVA developed strong and comparable levels of serum IgG anti-OVA, although Tg mice had more rapid responses. Both Tg and non-Tg mice fed soluble OVA or OVA in MES had rriarkedly reduced serum IgG anti-OVA and reduced skin test responses consistent with oral tolerance. Tg mice given OVA plus cholera toxin in MES remained tolerant. Conclusion: OT occurs in TCR Tg mouse for both humoral and cellular immunity, and was similar to that found in non-Tg mice. Interestingly, CT did not abrogate OT in the Tg mice as it has done in non-Tg mice. This Tg model is a valuable tool to dissect mechanisms operative in OT.
• MEASLES VACCINATION: A RISK FACTOR FOR INFLAMMATORY BOWEL DISEASE? NP Thompson, SM Montgomery, N Begg. RE Pounder. AJ Wakefield Inflmnmatory Bmvel Disertse Study Grrmp, Royal Free Hospital School of Medicine: Social Statistics Research Unit. City University; Communicable Diseases Surveillance Centre, London, UK. Aim: To test the hypothesis that measles vaccination is a risk factor for tile development of inflammatory bowel disease (IBD). Introduction: Persistent measles vires infection of intestinal tissue has been demonstrated, particularly in Crohn's disease. Early exposure to measles may be a risk factor for the development of Crohn's. Crohn's disease is increasing in incidence in the UK. especially in children. Measles vaccination was introduced in in the UK in 1968 and vaccine uptake is currently 92% Methods: Children, who received live measles vaccine as part of the Medical Research Council's measles vaccine trial in 1964. have been followed-up annually (n=3,545) The prevalence of Crohn's disease. ulcerative colitis and coeliac disease was deternained via a mailed questionnaire. A longitudinal birth cohort, the National Child Development Survey (NCDS) provided a comparison, unvaccinated cohort (n=l 1,407). Case validation, usmg standard criteria, was determined by contacting tile vaccinee's or NCDS member's hospital doctor - after permission for this had been obtained Statistical analysis was by Yates corrected Chi-squared test. Results: The response rate to questiom3aires in both cohorts was greater than 85% 14 cases of Crohn's disease. I 1 of ulcerative colitis and 2 of coeliac disease occurred in vaccinees, aged 31 years 15 cases of Crohn's disease. 14 of ulcerative colitis and 4 of coeliac disease were reported in NCDS cohort members, aged 33 years. Confirmatiou of the diagnosis was obtained in 72% of cases. Compared to NCDS members, the relative risk of developing Crohn's disease in vaccmees was 3.01 (p=0.004) and of developing ulcerative colitis was 2.53 (p=0.03). There was no significan! increased risk of reporting coeliac disease. Conclusions: These data provide further evidence that measles virus may play a role in the development of Crohn's disease, and also ulcerative colitis. Further studies of other vaccine groups are required urgently.
GASTROENTEROLOGY, Vol. 108, No. 4
• GENETICS VS. ENVIRONMENT IN INFLAMMATORY BOWEL DISEASE: CONCOR, DANCE RATES IN 130 TWIN PAIRS. NP Thompson, R Driscoll, RE Pounder, AJ Wakefield. lnflamnmtory Bowel Disease Study (;roup, Royal Free Hospital School of Medicine; National Assoc. for Colitis and Crohn's Disease, London, UK Aim: T o determine: the level of concordance for Crohn's disease and ulcerative colitis in identical and non-identical twin pairs. Introduction: There has been only one previous inflammatory bowel disease (IBD) study using twin s. In Crohn's disease coficordance was greater in monozygotic than dizygotic twins; in ulcerative colitis concordance was low in both groups. Linkage analysis studies have not shown an HLA association with either Crohn's or ulcerative-cofitis? There is a 10-20% incidence oflBD in family members of those with these d senses. Methods: The National Association for Colitis and Crohn's Disease (NACC) is a U K patient support organisation, with about 16,000 members of whom about half have Crohn!s disease. All were sent an invitation to be involved in a study of twins. Those who replied were sent a follow-up questionnaire, and if necessary a reminder. This de[ermined the nature of the disease and zygosity, using a validated questionnaire. Concordance rates were compared using MaenteI-Haenszel Chi-squared tests. Results: 216 N ACC mouthers replied to the initial invitation, of whom 184 (85%) replied to the questionnaire 130 twin pairs were identified in whom at least one had IBD and twin concordance was known (68 with Crohn's disease and 62 with ulcerative colitis). The mean age at diagnosis was 30 years in those with Crohn's disease and 35 years in those with ulcerative colitis. The mean duration of IBD was 10 years, in Crohn's 5/23 identical and 2143 non-identical: twins were concordant for IBD and in ulcerative colitis 5131 identical and 1/28 non-identical twins were concordant; in 5 twin pairs zygosity was uncertain. Concordance was significantly ~ e a t e r in identical than non-identical twins, p=0.02. There was no difference in concordance rates between Crohn's disease and ulcerative colitis, p=0.8. Conclusions: This twin study, the largest to date, s u ~ e s t s that there is a small but significant genetic factor in the aetiology of IBD. Environmental factors are likely to be of prime importance in the aetiology of these diseases.
Bile tolerance and bile salt hydrolase activity of lactobacilli and bifidobacteria isolated from the human intestine. G. Thornton 1, M. O'Sullivan 1, G. O'Sullivan 1, A. Weerkamp2, F. Shanahan 1 and J.K. Collins1,1 University College Cork, Ireland and 2NIZO, The Netherlands. The healthy human gastrointestinal tract provides a complex and hostile environment for most microorganisms entering via the oral cavity. These include fluid flow, pH, enzymes, mucous, bile, the mucosal immune system and normal colonising microflora. Little is known about this flora in terms of their metabolic contribution, their role in colonisation resistance against pathogens, overgrowth, inflammation and the production of cytotoxic products or carcinogens. We have isolated crypt adhering lactobacilli and bifidobacteria from freshly resected human small bowel. We have shown that these have the following properties: (a) adherence to epithelial surfaces; (b) acid tolerant; (c) produce inhibitory substances against enteric pathogens; (d) bile tolerant. We report that these gram +ve flora are unusually resistant to human, porcine and bovine bile. Furthermore, we have found that de,conjugation (expression of bile salt hydrolase activity) is not essential for resistance. In addition, we present the first report of a novel inducible bile tolerance mechanism in human GI lactobacilli. Using reverse phase HPLC coupled with pulsed amperometric detection at a gold electrode we have developed a rapid system for the analysis of bile acid metabolic products. We have found that 25% of bile tolerant lactobacilli isolated were capable of deconjugation using a bacterial bile salt hydrolyase. This is significant since all small bowel deconjugation appears to be microbially derived. All bifidobacteria deconjugated bile efficiently. T h e r e was a higher substrate preference for glycine conjugates rather than taurine conjugates. Further metabolised bile acid products have also been identified. In conclusion, we have shown that some human small bowel colonising lactobacilli and bifrdobacteria are extremely bile tolerant. Possession of bile salt hydrolase activity is not an essential component for tolerance. Based on these and previously reported findings on bacterial pathogen inhibitor activities appropriate manipulation of the GI flora should have an important role in immune, inflammatory and other metabolic activities in the human GI tract.
AGA ABSTRACTS
• INDUCTION OF ORAL TOLERANCE (OT) IN T CELL RECEPTOR TRANSGENIC MICE. W.G. Thaggarci, M. Tomasi, P.A. Dean, C.T. Weaver, C.O. EIson. Departments of Medicine, Surgery and Pathology, University of Alabama at Birmingham, AL. B a c k u r o u n d / P u r D o s e The mechanisms underlying oral tolerance are poorly understood. The purpose of this study was to determine whether oral tolerance could be induced in an *x13T cell receptor transgenic mouse ( T g ) i n which most T cells recognize ovalbumin (OVA) in the context of H2 d. Methode: Tg and non Tg BALB/c mice were fed OVA 20 mg in soluble form or 2 mg incorporated in the fluid phase of a multiple emulsion delivery system (MES) on days -14 and -7. Mice were then parenterally immunized with OVA in MES I.P. on day 0, 14, and 28. Samples of serum were taken at day 35 and assayed for IgG anti-OVA by ELISA. At day 56 skin test responses to OVA were tested in the ear. Results: Tg and non-Tg mice which were not fed OVA developed strong and comparable levels of serum IgG anti-OVA, although Tg mice had more rapid responses. Both Tg and non-Tg mice fed soluble OVA or OVA in MES had rriarkedly reduced serum IgG anti-OVA and reduced skin test responses consistent with oral tolerance. Tg mice given OVA plus cholera toxin in MES remained tolerant. Conclusion: OT occurs in TCR Tg mouse for both humoral and cellular immunity, and was similar to that found in non-Tg mice. Interestingly, CT did not abrogate OT in the Tg mice as it has done in non-Tg mice. This Tg model is a valuable tool to dissect mechanisms operative in OT.
• MEASLES VACCINATION: A RISK FACTOR FOR INFLAMMATORY BOWEL DISEASE? NP Thompson, SM Montgomery, N Begg. RE Pounder. AJ Wakefield Inflmnmatory Bmvel Disertse Study Grrmp, Royal Free Hospital School of Medicine: Social Statistics Research Unit. City University; Communicable Diseases Surveillance Centre, London, UK. Aim: To test the hypothesis that measles vaccination is a risk factor for tile development of inflammatory bowel disease (IBD). Introduction: Persistent measles vires infection of intestinal tissue has been demonstrated, particularly in Crohn's disease. Early exposure to measles may be a risk factor for the development of Crohn's. Crohn's disease is increasing in incidence in the UK. especially in children. Measles vaccination was introduced in in the UK in 1968 and vaccine uptake is currently 92% Methods: Children, who received live measles vaccine as part of the Medical Research Council's measles vaccine trial in 1964. have been followed-up annually (n=3,545) The prevalence of Crohn's disease. ulcerative colitis and coeliac disease was deternained via a mailed questionnaire. A longitudinal birth cohort, the National Child Development Survey (NCDS) provided a comparison, unvaccinated cohort (n=l 1,407). Case validation, usmg standard criteria, was determined by contacting tile vaccinee's or NCDS member's hospital doctor - after permission for this had been obtained Statistical analysis was by Yates corrected Chi-squared test. Results: The response rate to questiom3aires in both cohorts was greater than 85% 14 cases of Crohn's disease. I 1 of ulcerative colitis and 2 of coeliac disease occurred in vaccinees, aged 31 years 15 cases of Crohn's disease. 14 of ulcerative colitis and 4 of coeliac disease were reported in NCDS cohort members, aged 33 years. Confirmatiou of the diagnosis was obtained in 72% of cases. Compared to NCDS members, the relative risk of developing Crohn's disease in vaccmees was 3.01 (p=0.004) and of developing ulcerative colitis was 2.53 (p=0.03). There was no significan! increased risk of reporting coeliac disease. Conclusions: These data provide further evidence that measles virus may play a role in the development of Crohn's disease, and also ulcerative colitis. Further studies of other vaccine groups are required urgently.
GASTROENTEROLOGY, Vol. 108, No. 4
• GENETICS VS. ENVIRONMENT IN INFLAMMATORY BOWEL DISEASE: CONCOR, DANCE RATES IN 130 TWIN PAIRS. NP Thompson, R Driscoll, RE Pounder, AJ Wakefield. lnflamnmtory Bowel Disease Study (;roup, Royal Free Hospital School of Medicine; National Assoc. for Colitis and Crohn's Disease, London, UK Aim: T o determine: the level of concordance for Crohn's disease and ulcerative colitis in identical and non-identical twin pairs. Introduction: There has been only one previous inflammatory bowel disease (IBD) study using twin s. In Crohn's disease coficordance was greater in monozygotic than dizygotic twins; in ulcerative colitis concordance was low in both groups. Linkage analysis studies have not shown an HLA association with either Crohn's or ulcerative-cofitis? There is a 10-20% incidence oflBD in family members of those with these d senses. Methods: The National Association for Colitis and Crohn's Disease (NACC) is a U K patient support organisation, with about 16,000 members of whom about half have Crohn!s disease. All were sent an invitation to be involved in a study of twins. Those who replied were sent a follow-up questionnaire, and if necessary a reminder. This de[ermined the nature of the disease and zygosity, using a validated questionnaire. Concordance rates were compared using MaenteI-Haenszel Chi-squared tests. Results: 216 N ACC mouthers replied to the initial invitation, of whom 184 (85%) replied to the questionnaire 130 twin pairs were identified in whom at least one had IBD and twin concordance was known (68 with Crohn's disease and 62 with ulcerative colitis). The mean age at diagnosis was 30 years in those with Crohn's disease and 35 years in those with ulcerative colitis. The mean duration of IBD was 10 years, in Crohn's 5/23 identical and 2143 non-identical: twins were concordant for IBD and in ulcerative colitis 5131 identical and 1/28 non-identical twins were concordant; in 5 twin pairs zygosity was uncertain. Concordance was significantly ~ e a t e r in identical than non-identical twins, p=0.02. There was no difference in concordance rates between Crohn's disease and ulcerative colitis, p=0.8. Conclusions: This twin study, the largest to date, s u ~ e s t s that there is a small but significant genetic factor in the aetiology of IBD. Environmental factors are likely to be of prime importance in the aetiology of these diseases.
Bile tolerance and bile salt hydrolase activity of lactobacilli and bifidobacteria isolated from the human intestine. G. Thornton 1, M. O'Sullivan 1, G. O'Sullivan 1, A. Weerkamp2, F. Shanahan 1 and J.K. Collins1,1 University College Cork, Ireland and 2NIZO, The Netherlands. The healthy human gastrointestinal tract provides a complex and hostile environment for most microorganisms entering via the oral cavity. These include fluid flow, pH, enzymes, mucous, bile, the mucosal immune system and normal colonising microflora. Little is known about this flora in terms of their metabolic contribution, their role in colonisation resistance against pathogens, overgrowth, inflammation and the production of cytotoxic products or carcinogens. We have isolated crypt adhering lactobacilli and bifidobacteria from freshly resected human small bowel. We have shown that these have the following properties: (a) adherence to epithelial surfaces; (b) acid tolerant; (c) produce inhibitory substances against enteric pathogens; (d) bile tolerant. We report that these gram +ve flora are unusually resistant to human, porcine and bovine bile. Furthermore, we have found that de,conjugation (expression of bile salt hydrolase activity) is not essential for resistance. In addition, we present the first report of a novel inducible bile tolerance mechanism in human GI lactobacilli. Using reverse phase HPLC coupled with pulsed amperometric detection at a gold electrode we have developed a rapid system for the analysis of bile acid metabolic products. We have found that 25% of bile tolerant lactobacilli isolated were capable of deconjugation using a bacterial bile salt hydrolyase. This is significant since all small bowel deconjugation appears to be microbially derived. All bifidobacteria deconjugated bile efficiently. T h e r e was a higher substrate preference for glycine conjugates rather than taurine conjugates. Further metabolised bile acid products have also been identified. In conclusion, we have shown that some human small bowel colonising lactobacilli and bifrdobacteria are extremely bile tolerant. Possession of bile salt hydrolase activity is not an essential component for tolerance. Based on these and previously reported findings on bacterial pathogen inhibitor activities appropriate manipulation of the GI flora should have an important role in immune, inflammatory and other metabolic activities in the human GI tract.