- Email: [email protected]
Lidocaine enemas for intractable distal ulcerative colitis (IDUC): Efficacy and safety
April 1998 immunohistochemically examined for the expression of enteroglial markers at several different passages. All 5 cell lines expressed robust GFAP, S-100 and vimentin immunoreactivities, but no cell line expressed the mesenchymal markers desmin (smooth muscle cell differentiation marker) or Thy-l.1 (expressed on rodent fibroblasts). To further confirm these findings, dot blot analysis of the cytosolic and membrane protein fraction of each cell line was performed showing expression of GFAP and S-100 in the cytosolic fraction, while no Thy-l.1 expression could be detected. These findings indicate that immortalized enteroglial cells display the same phenotype as cells from primary cultures, suggesting that they are physiologically resembling primary cells. In summary, we are presenting the first immortalized enteroglial cell lines retaining several of the properties that are typical for primary EGC. These cell lines appear to be useful models for detailed investigations of the functional properties of EGC in vitro. Supported by DFG Ru 528/5. • G4393
PROSTAGLANDIN H SYNTHASE-2 INDUCTION BY HYDROXYEICOSA-TETRAENOIC ACIDS (HETES) IN AN INTESTINAL SUBEPITHELIAL MYOFIBROBLAST CELL LINE. J.I. Saada, J.D. Valentich, R. C. Mifflin, and D.W. Powell. Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX. Hydroxyeicosatetraenoic acids (HETES) are arachidonic acid metabolites synthesized by lipoxygenase, cytochrome P450 enzymatic pathways, and prostaglandin H synthase-2 (PGHS-2) when acetylated by aspirin. HETES and their metabolic byproducts play diverse roles in cellular regulation which include chemotaxis, regulation of vascular tone, and modulation of apoptosis. Increased synthesis of 5-, 11-, 12-, and 15-HETES occurs in inflamed colonic mucosa and inhibition of lipoxygenase activity is efficacious in the treatment of inflammatory bowel disease (IBD). The present study was undertaken when it was discovered that HETES are capable of regulating PGHS-2 expression in the intestinal supepithelial myofibroblast cell line, 18Co. Confluent 18Co monolayers were cultured 24 hours in the presence of various HETES or Lipoxins (5(S)-HETE, 12(R)HETE, 15(S)-HETE, 15(R)-HETE, 5(S)-15(S)-DiHETE, Lipoxin A4 and Lipoxin B4) with or without recombinant human IL-lt~ or aspirin, known inducers of PGHS-2 in these cells. PGHS-2 activity was measured by radioimmunoassay of secreted PGE 2 into culture medium. Cells were harvested for either protein or RNA extraction and PGHS-2 protein and RNA levels were analyzed by Western analysis using PGHS-2-specific antibodies, or Northern analysis using cDNA probes specific for human PGHS-2, respectively. PGHS-2 protein levels were quantitated by densitometry of developed blots and RNA levels were quantitated on a Molecular Dynamics phosphorimaging system. Modest inductions of PGHS-2 activity, protein, and mRNA were seen when 18Co cells were incubated in the presence of 2.0 ~aM 5(S)-, 12(R)-, 15(S)-, or 15(R)-HETE. The observed inductions were more marked following coincubation with IL-lct at 500 pg/ml (2-3X). The highest PGHS-2 protein and mRNA levels were seen when aspirin, 5.0 mM, was coincubated: PGHS-2 levels were ten fold higher than those seen following IL-lct treatment alone and roughly twice those seen in cells treated with IL-1 plus aspirin. No increases in PGHS- 2 protein or mRNA were seen following treatment with 5(S)-15(S)-DiHETE, Lipoxin A4, or Lipoxin B4 at concentrations in the micromolar range. Thus 5-, 12-, and 15-HETES at micromolar concentrations enhance PGHS-2 gene expression 2-3X in the intestinal supepithelial myofibroblast cell line 18Co. These results suggest a novel level of cross talk between the lipoxygenase and cyclooxygenase pathways. Certain lipoxygenase products can enhance synthesis of cyclooxygenase products via enhancement of PGHS-2 expression. Thus inflammatory and epithelial ceils can influence production of prostaglandins via synthesis of HETES. Supported by NIH Grant DK15350. G4394 PITUITARY-ADRENAL FUNCTION IMPAIRMENT IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE (IBD) TREATED WITH CORTICOSTEROID THERAPY. J.M. Sabatr, J. Desramr, V. Abitb01, M. Gaudric, M.A. Dugue, D. Couturier, S. Chaussade. Service d'HrpatoGastroent&ologie et Laboratoire d'hormonologie, Hopital Cochin, Paris, France. Corticosteroid (CS) therapy remains the mainstay of acute treatment for moderate-severe IBD, with approximately 90% of patients" achieving remission over 7 weeks at 1 mg/kg equivalent prednisone daily dose. Little is known about the impact of such a treatment on hypothalamo-pituitary-adrenal function, and about the modalities of decreasing CS therapy. The aim of this retrospective study was to evaluate the frequence of pituitaryadrenal function impairement by performing ACTH-test (the current test used in France for hypothaiamo-pituitary-adrenai function assessment) in a group of patients with IBD treated by CS therapy, and to identify risk factors of such side-effect. Patients and methods: ACTH-tests have been performed in 42 attacks of IBD treated by CS therapy corresponding to 34 patients: 8 M and 26 F; mean age 35,4 years, range 20-66 years; 34 attacks in patients with Crohns disease (mean CDAI before the beginning of CS therapy: 287 _+15), and 8 in patients
Immunology, Microbiology, and Inflanxmatory Disorders A1073
with moderate-severe ulcerative colitis. ACTH-tests, with measurement of serum basal cortisol level before (N<100 ng/mL) and 60 min after IM injection of I m g of tetracosactide (N<210 ng/mL), were performed prior stopping CS therapy or if clinical signs of adrenal insufficiency were noted. The response to ACTH-test was considered to be abnormal if plasma cortisol level at 60 min was below 210 ng/mL. R_esults: 25/42 of ACTH-tests (22/34 patients) were abnormal with symptoms of adrenal insufficiency (asthenia and abdominal pain) in 5 patients. Neither duration of the disease, previous surgical treatment or immunosuppressive therapy, association with immune modifiers, nor daily dose, cumulative dose or duration of CS therapy, were associated with an increased risk of impaired pituitary-adrenal function. The only risk factor for pituitary-adrenal function impairement was previous history of CS therapy (54.8% vs. 4.8%; P=0.027). Conclusion: In this retrospective study, 60 % of ACTH-tests (corresponding to 64,7% of patients) performed in IBD patients treated by CS were abnormal, with clinical adrenal insufficiency in 5 patients. These results may suggest to access hypothalamo-pituitary-adrenal function prior stopping CS therapy and the need to supplement with hydrocortisone when pituitary-adrenal function is impaired during decrease of corticosteroid doses. G4395 LIDOCAINE ENEMAS FOR INTRACTABLE DISTAL ULCERATIVE COLITIS (IDUC): EFFICACY AND SAFETY. F.G. Saibil. Division of Gastroenterology, Sunnybrook Health Science Centre, University of Toronto, Toronto, ON. Twenty-two patients with IDUC poorly responsive to oral and/or rectal steroids, and oral and/or rectal 5 aminosalicylate (5-ASA) were treated with 2% lidocaine gel enemas. There were 11 males and 11 females, ages 21-65. The extent of endoscopic disease was 25-100 cm, with a mean of 49 cm. The duration of disease prior to lidocaine therapy was 2-23 years. The daily lidocaine dose was 30 mL in all patients but one, who received 60 mL, Concurrent medication doses were held constant for the first 2 weeks of therapy. Because of the known risk of seizures with oral, intra-urethal and intravenous lidocaine, serum levels were measured in the patient taking 60 midday. Sixteen of 22 patients had a very good (4) or excellent (12) response within 2 weeks; 9/12 (excellent) and 2/4 (very good) were able to stop all steroids; 2/4 (very good) were able to reduce maintenance doses of prednisone. Of 6 nonresponders, 2 stopped lidocalne after 1 week, for non-medical reasons. Four were on lidocaine for 2-7 weeks without benefit. No patient in the series had side effects. In the patient on 30 mL bid, a trough lidocaine level was 3 umol/L just prior to the morning dose. Levels were then measured after a 30 mL dose, at 30, 60, 90, 120, 150, and 180 minutes. Values were 5, 7, 8, 6, 8, and 7 umol/L, respectively; all of these are at or below the acceptable therapeutic range for cardiac indications (6-21 umol/L). Conclusions: 1) Lidocaine enemas are frequently effective in patients with IDUC. 2) Systemic absorption from the from the rectum is minimal. 3) The therapy appears to be safe and free of side effects. G4396
INTRAGASTRIC PLASMID DNA IMMUNIZATION INDUCES CD4+ SPLENIC T CELLS PRODUCING INTERFERON-,/ AND INTERLEUKIN-10. A. Saito, A. Irisawa, Y. Sato, K. Obara, T. Nishimaki, R. Kasukawa, Department of Internal Medicine, 1I, Fukushima Medical College, Fukushima 960-12, Japan. Direct injection of naked plasmid DNA (pDNA) either intramuscularly or intradermally causes transfection of cells at the injected site through an unknown mechanism, and induces strong, long-lived systemic immune responses to the antigen encoded by the pDNA. The large majority of studies on DNA immunization, however, focus on systemic immunity and only few reports to date have shown that mucosally delivered pDNA generates mucosal as well as systemic immunity against an encoded antigen. We injected 13-galactosidase (13-gal) or pDNA encoding 13-gal, pACB-Z, intragastricaily (i.g.) or intradermally (i.d.) to the Balb/C mice and compared the mucosal humoral immune response (fecal lgA responses) and cytokine profiles of 13-gal-stimulated CD4+ splenic T cells. Elevated titers of 13 -gal-specific IgA Ab were seen in fecal extracts from mice immunized i.g. with either pACB-Z (152-+ 18 U/ml) or 13-gal (64-+ 8 U/ml), whereas intradermal injection of pDNA did not induce fecal IgA response. CD4+ T ceils from mice immunized i.g. with 13-gal produced IL-4(18 + 4pg/ml) and IL-10 (28 -+ 5pg/ml) but not IFN-y, which cytokine profile was characteristic of Th2 cells. In contrast, i.d. Immunization of pACB-Z induced production of IFN-T (2800 _+ 123 pg/ml) but only trace amounts of IL-4 and IL-10 by CD4+ T cells. This cytokine profile was compatible with that of Thl cells. However, CD4+ T cells from mice immunized i.g. with the same pDNA produced both IFN-'/(1206 -+ 134 pg/ml) and IL-10 (42 + 6 pg/ml) but trace amounts of IL-4. These findings suggest that IL-10 produced by CD4+ T cells may play a crucial role in the induction of mucosal immunity by intragastric DNA immunization.
PROSTAGLANDIN H SYNTHASE-2 INDUCTION BY HYDROXYEICOSA-TETRAENOIC ACIDS (HETES) IN AN INTESTINAL SUBEPITHELIAL MYOFIBROBLAST CELL LINE. J.I. Saada, J.D. Valentich, R. C. Mifflin, and D.W. Powell. Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX. Hydroxyeicosatetraenoic acids (HETES) are arachidonic acid metabolites synthesized by lipoxygenase, cytochrome P450 enzymatic pathways, and prostaglandin H synthase-2 (PGHS-2) when acetylated by aspirin. HETES and their metabolic byproducts play diverse roles in cellular regulation which include chemotaxis, regulation of vascular tone, and modulation of apoptosis. Increased synthesis of 5-, 11-, 12-, and 15-HETES occurs in inflamed colonic mucosa and inhibition of lipoxygenase activity is efficacious in the treatment of inflammatory bowel disease (IBD). The present study was undertaken when it was discovered that HETES are capable of regulating PGHS-2 expression in the intestinal supepithelial myofibroblast cell line, 18Co. Confluent 18Co monolayers were cultured 24 hours in the presence of various HETES or Lipoxins (5(S)-HETE, 12(R)HETE, 15(S)-HETE, 15(R)-HETE, 5(S)-15(S)-DiHETE, Lipoxin A4 and Lipoxin B4) with or without recombinant human IL-lt~ or aspirin, known inducers of PGHS-2 in these cells. PGHS-2 activity was measured by radioimmunoassay of secreted PGE 2 into culture medium. Cells were harvested for either protein or RNA extraction and PGHS-2 protein and RNA levels were analyzed by Western analysis using PGHS-2-specific antibodies, or Northern analysis using cDNA probes specific for human PGHS-2, respectively. PGHS-2 protein levels were quantitated by densitometry of developed blots and RNA levels were quantitated on a Molecular Dynamics phosphorimaging system. Modest inductions of PGHS-2 activity, protein, and mRNA were seen when 18Co cells were incubated in the presence of 2.0 ~aM 5(S)-, 12(R)-, 15(S)-, or 15(R)-HETE. The observed inductions were more marked following coincubation with IL-lct at 500 pg/ml (2-3X). The highest PGHS-2 protein and mRNA levels were seen when aspirin, 5.0 mM, was coincubated: PGHS-2 levels were ten fold higher than those seen following IL-lct treatment alone and roughly twice those seen in cells treated with IL-1 plus aspirin. No increases in PGHS- 2 protein or mRNA were seen following treatment with 5(S)-15(S)-DiHETE, Lipoxin A4, or Lipoxin B4 at concentrations in the micromolar range. Thus 5-, 12-, and 15-HETES at micromolar concentrations enhance PGHS-2 gene expression 2-3X in the intestinal supepithelial myofibroblast cell line 18Co. These results suggest a novel level of cross talk between the lipoxygenase and cyclooxygenase pathways. Certain lipoxygenase products can enhance synthesis of cyclooxygenase products via enhancement of PGHS-2 expression. Thus inflammatory and epithelial ceils can influence production of prostaglandins via synthesis of HETES. Supported by NIH Grant DK15350. G4394 PITUITARY-ADRENAL FUNCTION IMPAIRMENT IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE (IBD) TREATED WITH CORTICOSTEROID THERAPY. J.M. Sabatr, J. Desramr, V. Abitb01, M. Gaudric, M.A. Dugue, D. Couturier, S. Chaussade. Service d'HrpatoGastroent&ologie et Laboratoire d'hormonologie, Hopital Cochin, Paris, France. Corticosteroid (CS) therapy remains the mainstay of acute treatment for moderate-severe IBD, with approximately 90% of patients" achieving remission over 7 weeks at 1 mg/kg equivalent prednisone daily dose. Little is known about the impact of such a treatment on hypothalamo-pituitary-adrenal function, and about the modalities of decreasing CS therapy. The aim of this retrospective study was to evaluate the frequence of pituitaryadrenal function impairement by performing ACTH-test (the current test used in France for hypothaiamo-pituitary-adrenai function assessment) in a group of patients with IBD treated by CS therapy, and to identify risk factors of such side-effect. Patients and methods: ACTH-tests have been performed in 42 attacks of IBD treated by CS therapy corresponding to 34 patients: 8 M and 26 F; mean age 35,4 years, range 20-66 years; 34 attacks in patients with Crohns disease (mean CDAI before the beginning of CS therapy: 287 _+15), and 8 in patients
Immunology, Microbiology, and Inflanxmatory Disorders A1073
with moderate-severe ulcerative colitis. ACTH-tests, with measurement of serum basal cortisol level before (N<100 ng/mL) and 60 min after IM injection of I m g of tetracosactide (N<210 ng/mL), were performed prior stopping CS therapy or if clinical signs of adrenal insufficiency were noted. The response to ACTH-test was considered to be abnormal if plasma cortisol level at 60 min was below 210 ng/mL. R_esults: 25/42 of ACTH-tests (22/34 patients) were abnormal with symptoms of adrenal insufficiency (asthenia and abdominal pain) in 5 patients. Neither duration of the disease, previous surgical treatment or immunosuppressive therapy, association with immune modifiers, nor daily dose, cumulative dose or duration of CS therapy, were associated with an increased risk of impaired pituitary-adrenal function. The only risk factor for pituitary-adrenal function impairement was previous history of CS therapy (54.8% vs. 4.8%; P=0.027). Conclusion: In this retrospective study, 60 % of ACTH-tests (corresponding to 64,7% of patients) performed in IBD patients treated by CS were abnormal, with clinical adrenal insufficiency in 5 patients. These results may suggest to access hypothalamo-pituitary-adrenal function prior stopping CS therapy and the need to supplement with hydrocortisone when pituitary-adrenal function is impaired during decrease of corticosteroid doses. G4395 LIDOCAINE ENEMAS FOR INTRACTABLE DISTAL ULCERATIVE COLITIS (IDUC): EFFICACY AND SAFETY. F.G. Saibil. Division of Gastroenterology, Sunnybrook Health Science Centre, University of Toronto, Toronto, ON. Twenty-two patients with IDUC poorly responsive to oral and/or rectal steroids, and oral and/or rectal 5 aminosalicylate (5-ASA) were treated with 2% lidocaine gel enemas. There were 11 males and 11 females, ages 21-65. The extent of endoscopic disease was 25-100 cm, with a mean of 49 cm. The duration of disease prior to lidocaine therapy was 2-23 years. The daily lidocaine dose was 30 mL in all patients but one, who received 60 mL, Concurrent medication doses were held constant for the first 2 weeks of therapy. Because of the known risk of seizures with oral, intra-urethal and intravenous lidocaine, serum levels were measured in the patient taking 60 midday. Sixteen of 22 patients had a very good (4) or excellent (12) response within 2 weeks; 9/12 (excellent) and 2/4 (very good) were able to stop all steroids; 2/4 (very good) were able to reduce maintenance doses of prednisone. Of 6 nonresponders, 2 stopped lidocalne after 1 week, for non-medical reasons. Four were on lidocaine for 2-7 weeks without benefit. No patient in the series had side effects. In the patient on 30 mL bid, a trough lidocaine level was 3 umol/L just prior to the morning dose. Levels were then measured after a 30 mL dose, at 30, 60, 90, 120, 150, and 180 minutes. Values were 5, 7, 8, 6, 8, and 7 umol/L, respectively; all of these are at or below the acceptable therapeutic range for cardiac indications (6-21 umol/L). Conclusions: 1) Lidocaine enemas are frequently effective in patients with IDUC. 2) Systemic absorption from the from the rectum is minimal. 3) The therapy appears to be safe and free of side effects. G4396
INTRAGASTRIC PLASMID DNA IMMUNIZATION INDUCES CD4+ SPLENIC T CELLS PRODUCING INTERFERON-,/ AND INTERLEUKIN-10. A. Saito, A. Irisawa, Y. Sato, K. Obara, T. Nishimaki, R. Kasukawa, Department of Internal Medicine, 1I, Fukushima Medical College, Fukushima 960-12, Japan. Direct injection of naked plasmid DNA (pDNA) either intramuscularly or intradermally causes transfection of cells at the injected site through an unknown mechanism, and induces strong, long-lived systemic immune responses to the antigen encoded by the pDNA. The large majority of studies on DNA immunization, however, focus on systemic immunity and only few reports to date have shown that mucosally delivered pDNA generates mucosal as well as systemic immunity against an encoded antigen. We injected 13-galactosidase (13-gal) or pDNA encoding 13-gal, pACB-Z, intragastricaily (i.g.) or intradermally (i.d.) to the Balb/C mice and compared the mucosal humoral immune response (fecal lgA responses) and cytokine profiles of 13-gal-stimulated CD4+ splenic T cells. Elevated titers of 13 -gal-specific IgA Ab were seen in fecal extracts from mice immunized i.g. with either pACB-Z (152-+ 18 U/ml) or 13-gal (64-+ 8 U/ml), whereas intradermal injection of pDNA did not induce fecal IgA response. CD4+ T ceils from mice immunized i.g. with 13-gal produced IL-4(18 + 4pg/ml) and IL-10 (28 -+ 5pg/ml) but not IFN-y, which cytokine profile was characteristic of Th2 cells. In contrast, i.d. Immunization of pACB-Z induced production of IFN-T (2800 _+ 123 pg/ml) but only trace amounts of IL-4 and IL-10 by CD4+ T cells. This cytokine profile was compatible with that of Thl cells. However, CD4+ T cells from mice immunized i.g. with the same pDNA produced both IFN-'/(1206 -+ 134 pg/ml) and IL-10 (42 + 6 pg/ml) but trace amounts of IL-4. These findings suggest that IL-10 produced by CD4+ T cells may play a crucial role in the induction of mucosal immunity by intragastric DNA immunization.