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Orally immunized μMT antibody-deficient mice are protected against H. felis infection
April 1998 Results
medicine n i iNOsactivityi cNOsactivityi damagescorei ulcerarea i MPOaetivity control ~5~i~-~.5.~-.a-.~.~8-~i~.89~.~.~42-~i~-°9~-.~.~:5-~L.4~87~33~i-.8.24.3 ± 125.4 AG 5 ~0.16±0.04"~ 0.61±0.40 ! 4.8±0.9** !1,52±0.38"*~347.1 ±59.5"* EIT 5 i0.12±0.04" i 0.44±0.25 i 6.2±0.6** i2.07±0.48"*i 477.0±93.8" L-NAME 5 ! 0.26±0.08 i 0"17±0'06 i 7.8±1.0 i3.43±0.60"i 652.7±71.5 (mean + S.E.M) (compared with control*:p<0.05,**:p<0.01) AG and EIT decreased the damage of TNB colitis compared with control. Moreover, AG decreased the damage compared with L-NAME. Conclusions: The selective inhibition of iNns was useful to prevent the damage of colonic mucosa in TNB colitis. We considered that iNns inhibitor may be one of new agents to ameliorate the damage of colonic mncosa in IBD. • G4291 EPITHELIAL CYTOPROTECTION IS PART OF THE THERAPEUTIC EFFECT OF INTERLEUKIN-10 IN MUCOSAL INFLAMMATION. K. Nally, F. Newton, J. O'Connell, J. Morgan, G.C. O'Sullivan, J.K Collins and F. Shanahan. Depts. Medicine and Microbiology, National University of Ireland, Cork, Ireland.
Background & Aims Deficiency of interleukin- 10 (IL- 10) in murine knockout models is associated with enterocolitis similar to Crohn's disease and IL-10 has been used to treat human inflammatory bowel disease. The mechanism of action of IL-10 is generally considered in the context of downregulatory effects on the immunoinflammatory response. The purpose of the present study was to determine if IL-10 can directly protect colonic epithelia from apoptosis induced by proinflammatory cytokines - tumor necrosis alpha (TNF-a) and interferon-T (IFN-'¢). Methods & Results Expression of receptor mRNA for IL-10 (IL-10-R) and TNF (TNF-R, p75 and p55) was first determined by rt-PCR using the colonic epithelial cell lines, SW620 and HT29. The presence of cellsurface receptor protein in each case was confirmed immunochemically with appropriate peptide-ligand competitive controls for staining specificity, and epithelial expression of IL-10R was also demonstrated in vivo by immunochemical staining of colonic biopsies. A direct functional protective effect of IL-10 on epithelia was then shown by determining whether IL-10 can antagonize the synergistic induction of epithelial apoptosis by TNF-ct and IFN-T. Epithelial cell death in response to cytokine combinations was quantitated using a radiolabelled DNA fragmentation assay (JAM test). Pretreatment of both epithelial cell lines with IFN-3' (1-25ng/ml) resulted in a dose-dependent cell death reaching a plateau of 35-40%, and subsequent exposure of the pretreated cells to TNF-et (50ng/ml) consistently enhanced the level of apoptosis (~ 60% plateau). In contrast, pretreatment with IL-10 (1-10ng/ml) inhibited apoptosis induced by IFN-T and TNF-ct alone or in combination (<10% for both cell lines). Summary & conclusion (i) human colonic epithelial cells express functional receptors for IL-10 and (ii) IL-10 exerts a cytoprotective effect on colonic epithelia, inhibiting apoptosis induction by pro-inflammatory cytokines. This implies that the therapeutic mechanism of IL-10 may not be limited to inhibition of mucosal immunoinflammatory events; endorgan epithelial protection suggests that local or topical therapeutic administration of IL-10 may have particular advantage over the parenteral route. Supported by a grant from the Health Research Board of Ireland • G4292 APPENDECTOMY AND THE RISK OF DEVELOPING ULCERATIVE COLITIS: RESULTS AFTER CONTROL OF SMOKING FACTOR. S.W. Nam, S.-K. Yang, H.-Y. Jung, S.H. Yang, S.Y. Kim, H.J. Kim, W.-S. Hong, and Y.I. Min. Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea. Background: It is well-known that current smokers have the decreased risk for the development of ulcerative colitis (UC) than non-smokers, while former smokers have the increased risk. Recently, some authors have suggested that the development of UC has a strong negative association with appendectomy, while others have found a weaker or no association. This difference can be explained by many factors including the influence of confounding variables. We postulated that this discrepancy might be partly explained by the smoking status, a well-known confounding variable, because previous studies did not match the smoking status. Aim: This study was conducted to evaluate the relationship between appendectomy and the risk for the development of UC after control of smoking status. Methods: A total of 159 patients with UC (67 males and 92 females, mean age 41.7 years) and 159 controls recruited from the orthopedic-traumatology clinic were included in this study. For the selection of controls, we matched smoking status as well as age ( -+ 1 year) and sex. Smoking status of each case and control was categorized as 'never' (n=110), 'ex' (n=25), or 'current' (n=24) at the onset of UC symptoms. For all patients and controls, data collection about previous appendectomy was performed after inclusion in the study by history taking, examination of the
Immunology, Microbiology, and Inflantmatory Disorders A1049
appendectomy scar and, if available, review of the medical records. Results: Appendectomy had been performed in one (0.6%) out of 159 patients with UC and 19 (11.9%) of 159 controls prior to the development of UC. The risk for the development of UC was significantly lower after appendectomy (odds ratio, 0.06; 95% CI, 0.01-0.39) Conclusion: Even after control of the smoking factor, appendectomy is negatively associated with ulcerative colitis. G4293
ORALLY IMMUNIZED ~VIT ANTIBODY-DEFICIENT MICE ARE PROTECTED AGAINST H. FELIS INFECTION. J. G. Nedrud. T. G. Blanchard, R. Redline, N. Sigmund, and S. J. Czinn. Case Western Reserve University, Cleveland, Ohio, USA. Immunization of mice with Helicobacter antigens plus cholera toxin can protect them from either H. pylori of H. felis infection. Based on other models of mucosal immunity it has been assumed that mucosal IgA responses are important in this protection. However, we recently showed that IgA deficient mice can be protected from Helicobacter infection (Nedrud et.al, Gut 39 suppl 2, A45, 1996) and other investigators have suggested a role for IgG antibodies in protection (Ferrero et. al. Gastroenterology 113: 185-194, 1997). In addition, we have shown that adoptive transfer of spleen ceils from immunized mice can reduce the bacterial load in H. felis challenged mice (Mohammadi et.al. Gastroenterology 113: 1848-57, 1997). These results have suggested that antibody independent, cell-mediated immune responses could play an important role in protection from Helicobacter infection. To directly test this hypothesis we orally immunized and challenged antibody deficient, ~tMT/mice with H. felis. In 2 separate experiments, antibody deficient mice were protected from infection as assessed by rapid urease or examination of silver stained sections (Exp#1:6/8 laMT and 8/8 wild type mice protected, Exp#2:5/5 ~MT and 7/7 wild type mice protected.) We conclude that antibody independent, cell-mediated immune mechanisms can contribute to protection from Helicobacter infection. G4294
THE ROLE OF MYCOPHENOLATE MOFELTIL IN THE MANAGEMENT OF REFRACTORY INFLAMMATORY BOWEL DISEASE (IBD). O.S. Nehme, C.A. Overley, J.I. O'brien. Division Of Gastroenterology, Indiana University Medical School, Indianapolis, Indiana. Immunomodulatory Agents (IA) have been an alternative option in some IBD patients who have failed to respond to conventional medical therapy or who have been resistant to tapering their steroid doses. However some patients are either intolerant or non-responsive to the currently used IA, i.e. azathioprine (AZA), 6-mercaptopurine (6-MP), cyclosporine A (CyA), methotrexate (MTX). Mycophenolate Motetil (MMF) is an immunosuppressive agent that has been used for organ transplant recipients. Reports have also suggested that MMF is beneficial for autoimmune disorders such as rheumatoid arthritis. AIM: The purpose of this study was to assess the efficacy and compliance of using MMF in those patients who are either intolerant or non-responsive to the use of other IA. METHOD: In this ongoing prospective study (3 month duration), 4 patients have been enrolled with refractory IBD [Ulcerative Colitis (UC) - 1; Crohn's (CD) - 3]. They received MMF after demonstrating an inability to either tolerate other IA or a lack of efficacy with other agents. Two pts had refractory perineal CD with extensive fistulization; another pt had refractory Crohn's Disease; the final pt had refractory UC. These patients received MMF 1 g daily (divided in two doses). All patients were continued on (5-aminosalicylic acid) agents. All patients were also on steroids and were able to taper their steroid dose over the course of MMF therapy. For baseline and subsequent serial assessment of IBD severity, an inflammatory activity index was calculated. UC patients were followed with the Truelove-Witt criteria and the CD pts were followed with a modified Harvey Bradshaw Index. In addition physical exam and laboratory tests (ESR, C-RP, etc.) were obtained. Most patients also had endoscopies and/or CT scans at baseline and for follow-up. All patients had been on at least two or more of the other IA (AZA, 6-MP, CyA, MTX) prior to starting MMF. The pts had discontinued the other IA secondary to lack of efficiency, intolerance, or a quick relapse after lowering the dose. To date all 4 patients have demonstrated clinical improvement. All 4 patients have also been able to lower their daily dose of steroids they had started with. No significant upper or lower GI side effects have been detected. All patients have complied with MMF administration and required follow-up. CONCLUSION: MMF appears to be effective in refractory IBD pts who are either intolerant or non-responsive to other IA. GI side effects were unremarkable and compliance was good. We plan to continue to study the efficacy of MMF in additional IBD pts.
medicine n i iNOsactivityi cNOsactivityi damagescorei ulcerarea i MPOaetivity control ~5~i~-~.5.~-.a-.~.~8-~i~.89~.~.~42-~i~-°9~-.~.~:5-~L.4~87~33~i-.8.24.3 ± 125.4 AG 5 ~0.16±0.04"~ 0.61±0.40 ! 4.8±0.9** !1,52±0.38"*~347.1 ±59.5"* EIT 5 i0.12±0.04" i 0.44±0.25 i 6.2±0.6** i2.07±0.48"*i 477.0±93.8" L-NAME 5 ! 0.26±0.08 i 0"17±0'06 i 7.8±1.0 i3.43±0.60"i 652.7±71.5 (mean + S.E.M) (compared with control*:p<0.05,**:p<0.01) AG and EIT decreased the damage of TNB colitis compared with control. Moreover, AG decreased the damage compared with L-NAME. Conclusions: The selective inhibition of iNns was useful to prevent the damage of colonic mucosa in TNB colitis. We considered that iNns inhibitor may be one of new agents to ameliorate the damage of colonic mncosa in IBD. • G4291 EPITHELIAL CYTOPROTECTION IS PART OF THE THERAPEUTIC EFFECT OF INTERLEUKIN-10 IN MUCOSAL INFLAMMATION. K. Nally, F. Newton, J. O'Connell, J. Morgan, G.C. O'Sullivan, J.K Collins and F. Shanahan. Depts. Medicine and Microbiology, National University of Ireland, Cork, Ireland.
Background & Aims Deficiency of interleukin- 10 (IL- 10) in murine knockout models is associated with enterocolitis similar to Crohn's disease and IL-10 has been used to treat human inflammatory bowel disease. The mechanism of action of IL-10 is generally considered in the context of downregulatory effects on the immunoinflammatory response. The purpose of the present study was to determine if IL-10 can directly protect colonic epithelia from apoptosis induced by proinflammatory cytokines - tumor necrosis alpha (TNF-a) and interferon-T (IFN-'¢). Methods & Results Expression of receptor mRNA for IL-10 (IL-10-R) and TNF (TNF-R, p75 and p55) was first determined by rt-PCR using the colonic epithelial cell lines, SW620 and HT29. The presence of cellsurface receptor protein in each case was confirmed immunochemically with appropriate peptide-ligand competitive controls for staining specificity, and epithelial expression of IL-10R was also demonstrated in vivo by immunochemical staining of colonic biopsies. A direct functional protective effect of IL-10 on epithelia was then shown by determining whether IL-10 can antagonize the synergistic induction of epithelial apoptosis by TNF-ct and IFN-T. Epithelial cell death in response to cytokine combinations was quantitated using a radiolabelled DNA fragmentation assay (JAM test). Pretreatment of both epithelial cell lines with IFN-3' (1-25ng/ml) resulted in a dose-dependent cell death reaching a plateau of 35-40%, and subsequent exposure of the pretreated cells to TNF-et (50ng/ml) consistently enhanced the level of apoptosis (~ 60% plateau). In contrast, pretreatment with IL-10 (1-10ng/ml) inhibited apoptosis induced by IFN-T and TNF-ct alone or in combination (<10% for both cell lines). Summary & conclusion (i) human colonic epithelial cells express functional receptors for IL-10 and (ii) IL-10 exerts a cytoprotective effect on colonic epithelia, inhibiting apoptosis induction by pro-inflammatory cytokines. This implies that the therapeutic mechanism of IL-10 may not be limited to inhibition of mucosal immunoinflammatory events; endorgan epithelial protection suggests that local or topical therapeutic administration of IL-10 may have particular advantage over the parenteral route. Supported by a grant from the Health Research Board of Ireland • G4292 APPENDECTOMY AND THE RISK OF DEVELOPING ULCERATIVE COLITIS: RESULTS AFTER CONTROL OF SMOKING FACTOR. S.W. Nam, S.-K. Yang, H.-Y. Jung, S.H. Yang, S.Y. Kim, H.J. Kim, W.-S. Hong, and Y.I. Min. Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea. Background: It is well-known that current smokers have the decreased risk for the development of ulcerative colitis (UC) than non-smokers, while former smokers have the increased risk. Recently, some authors have suggested that the development of UC has a strong negative association with appendectomy, while others have found a weaker or no association. This difference can be explained by many factors including the influence of confounding variables. We postulated that this discrepancy might be partly explained by the smoking status, a well-known confounding variable, because previous studies did not match the smoking status. Aim: This study was conducted to evaluate the relationship between appendectomy and the risk for the development of UC after control of smoking status. Methods: A total of 159 patients with UC (67 males and 92 females, mean age 41.7 years) and 159 controls recruited from the orthopedic-traumatology clinic were included in this study. For the selection of controls, we matched smoking status as well as age ( -+ 1 year) and sex. Smoking status of each case and control was categorized as 'never' (n=110), 'ex' (n=25), or 'current' (n=24) at the onset of UC symptoms. For all patients and controls, data collection about previous appendectomy was performed after inclusion in the study by history taking, examination of the
Immunology, Microbiology, and Inflantmatory Disorders A1049
appendectomy scar and, if available, review of the medical records. Results: Appendectomy had been performed in one (0.6%) out of 159 patients with UC and 19 (11.9%) of 159 controls prior to the development of UC. The risk for the development of UC was significantly lower after appendectomy (odds ratio, 0.06; 95% CI, 0.01-0.39) Conclusion: Even after control of the smoking factor, appendectomy is negatively associated with ulcerative colitis. G4293
ORALLY IMMUNIZED ~VIT ANTIBODY-DEFICIENT MICE ARE PROTECTED AGAINST H. FELIS INFECTION. J. G. Nedrud. T. G. Blanchard, R. Redline, N. Sigmund, and S. J. Czinn. Case Western Reserve University, Cleveland, Ohio, USA. Immunization of mice with Helicobacter antigens plus cholera toxin can protect them from either H. pylori of H. felis infection. Based on other models of mucosal immunity it has been assumed that mucosal IgA responses are important in this protection. However, we recently showed that IgA deficient mice can be protected from Helicobacter infection (Nedrud et.al, Gut 39 suppl 2, A45, 1996) and other investigators have suggested a role for IgG antibodies in protection (Ferrero et. al. Gastroenterology 113: 185-194, 1997). In addition, we have shown that adoptive transfer of spleen ceils from immunized mice can reduce the bacterial load in H. felis challenged mice (Mohammadi et.al. Gastroenterology 113: 1848-57, 1997). These results have suggested that antibody independent, cell-mediated immune responses could play an important role in protection from Helicobacter infection. To directly test this hypothesis we orally immunized and challenged antibody deficient, ~tMT/mice with H. felis. In 2 separate experiments, antibody deficient mice were protected from infection as assessed by rapid urease or examination of silver stained sections (Exp#1:6/8 laMT and 8/8 wild type mice protected, Exp#2:5/5 ~MT and 7/7 wild type mice protected.) We conclude that antibody independent, cell-mediated immune mechanisms can contribute to protection from Helicobacter infection. G4294
THE ROLE OF MYCOPHENOLATE MOFELTIL IN THE MANAGEMENT OF REFRACTORY INFLAMMATORY BOWEL DISEASE (IBD). O.S. Nehme, C.A. Overley, J.I. O'brien. Division Of Gastroenterology, Indiana University Medical School, Indianapolis, Indiana. Immunomodulatory Agents (IA) have been an alternative option in some IBD patients who have failed to respond to conventional medical therapy or who have been resistant to tapering their steroid doses. However some patients are either intolerant or non-responsive to the currently used IA, i.e. azathioprine (AZA), 6-mercaptopurine (6-MP), cyclosporine A (CyA), methotrexate (MTX). Mycophenolate Motetil (MMF) is an immunosuppressive agent that has been used for organ transplant recipients. Reports have also suggested that MMF is beneficial for autoimmune disorders such as rheumatoid arthritis. AIM: The purpose of this study was to assess the efficacy and compliance of using MMF in those patients who are either intolerant or non-responsive to the use of other IA. METHOD: In this ongoing prospective study (3 month duration), 4 patients have been enrolled with refractory IBD [Ulcerative Colitis (UC) - 1; Crohn's (CD) - 3]. They received MMF after demonstrating an inability to either tolerate other IA or a lack of efficacy with other agents. Two pts had refractory perineal CD with extensive fistulization; another pt had refractory Crohn's Disease; the final pt had refractory UC. These patients received MMF 1 g daily (divided in two doses). All patients were continued on (5-aminosalicylic acid) agents. All patients were also on steroids and were able to taper their steroid dose over the course of MMF therapy. For baseline and subsequent serial assessment of IBD severity, an inflammatory activity index was calculated. UC patients were followed with the Truelove-Witt criteria and the CD pts were followed with a modified Harvey Bradshaw Index. In addition physical exam and laboratory tests (ESR, C-RP, etc.) were obtained. Most patients also had endoscopies and/or CT scans at baseline and for follow-up. All patients had been on at least two or more of the other IA (AZA, 6-MP, CyA, MTX) prior to starting MMF. The pts had discontinued the other IA secondary to lack of efficiency, intolerance, or a quick relapse after lowering the dose. To date all 4 patients have demonstrated clinical improvement. All 4 patients have also been able to lower their daily dose of steroids they had started with. No significant upper or lower GI side effects have been detected. All patients have complied with MMF administration and required follow-up. CONCLUSION: MMF appears to be effective in refractory IBD pts who are either intolerant or non-responsive to other IA. GI side effects were unremarkable and compliance was good. We plan to continue to study the efficacy of MMF in additional IBD pts.