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The role of pro-inflammatory peptides in the onset of intestinal inflammation
Immunology, Microbiology, and Inflammatory Disorders A925
April lS9S
CC n = 88 LC = 70 1,2 Age I mean (yrs) 64.1 63 range 23 -87 22-91 NS M/F ratio 2 (%) 27/73 49/52 p ---0.005 Diarrhea 2 acute 9 14 intermittent/chronic 13/66 7/49 NS Delay before diagnosis medianI 7.5 months 5 months p25-75 3-24 months 2-11 months p = 0.065 %Associated "auto-immune dis.''2 38 36 NS Aspirin2Y/N 11/65 15/53 NS NSAID2Y/N 14/62 11/57 NS Abdominal pain2: slight/severe 17/5 16/0 intermitt cramps 17 3 p ---0.011 Weight loss2 35 33 NS $oaoking2: current/former/never 23/6/34 10/16/23 p = 0.017 Followup n = 57 n = 37 median (months) 7 5 p25-75 3-24 3-22 Symptoms2 gone 22 22 improved 26 10 same 17 5 p = 0.040 Wilcoxon I and Chi-square 2 were used for statistical comparison. Conclusions: Lymphocytic and collagenous colitis are similar but not identical clinical syndromes. Unlike CC pts half of the LC pts are males. LC pts present somewhat earlier and are less likely to be active smokers. Their symptoms are milder and are more likely to disappear with or without therapy. Overall after a median follow-up of 6 months only 24% of patients (LC + CC) have persistent important symptoms. G3792 CYTOKINES AND ULCERATIVE COLITIS: MUCOSAL BALANCE BETWEEN PRO AND ANTI-INFLAMMATORY CYTOKINES IN PATIENTS WITH AND WITHOUT POUCHITIS. A Balzan0, N Bevilacqua*, Nappo C*, P Gionchetti*, M Campieri*, G Grande', A Bove, Mancino D*. Dip. di Gastroenterologia - *Lab.Centr.di Analisi Cliniche -ASO A.Cardarelli. Napoli *Cattedra di Immunologia - lst.di Pat.Generale - II Universith degli Studi - Napoli *Dip. di Medicina Intema e Gastroanterologia, Universit~ di Bologna. Pouchitis is the major long-term complication after restorative proctocolectomy with ileal pouch-anal anastomosis for ulcerative colitis (UC). In UC, is well documented a disturbed balance between pro- and antiinflammatory cytokines: In puochitis, the mucosal levels of pro-inflammatory cytokines have been shown to be increased. Aim of this study was to evaluate mucosal balance of pro- and anti-inflammatory cytokines in patients with ileal pouch-anal anastomosis with and without ponchitis. Concentration of interleukin (IL-) 113, IL-1 receptor antagonist (ra), IL-4, IL-8, IL-10, interferon y (INF'/) and tumor necrosis factor ct (TNFct) were determined by ELISA in tissue homogenates of mucosal biopsy specimens. Biopsies were obtained from pelvic ileal pouches in 8 patients with pouchitis and 10 with pouches without pouchitis. Diagnosis and severity of pouchitis were evaluated with the Pouchitis Disease Activity Index (PDAI) which includes clinical sigmoidoscopic and histological criteria. pg/mg tissue protein IL-113 IL-lra IL-4 IL-8 IL-10 INF-y TNF-a
Pouch
Pouchitis
p
8.5 304.7 0.15 23.6 0.28 0.10 5.5
20.0 331.3 0.17 59.5 0.30 0.19 5.5
0.008
0.027
IL-113 and IL-8 mucosal levels were significantly increased in pouchitis compared with normal pouches, and correlated with disease activity (PDAI) [ r=-0.69 and r=0.82 respectively and (p<0.01)]. This study shows that also in pouchitis there is a disturbed balance between pro- and anti-inflammatory cytokines as previously shown in UC.
• G3793 THE ROLE OF PRO-INFLAMMATORY PEPTIDES IN THE ONSET OF INTESTINAL INFLAMMATION. G. Barbara, R. DeGiorgio, P. Blennerhassett, S. Sturiale, E. Grady, N. Bunnett and SM. Collins. McMaster University Hamilton, Canada, University of Bologna Italy, and UCSF, San Francisco. Substance P and other pro-inflammatory peptides have been implicated in the genesis of neurogenic inflammation but its role in intestinal inflammatory processes has yet to he precisely identified. In a model of intestinal inflammation following nematode infection, we have shown that immunoreactive substance P is increased in the inflamed intestine, the dorsal root ganglion and in the dorsal horn of the spinal cord. Furthermore, we have shown that there is a profound decrease in neutral endopeptidase (NEP EC 3.424.11) in this model, implying an increased bioavailability of SP. To elucidate the role of pro-inflammatory peptides in the inflammatory process, we have examined the effect of Trichinella spiralis infection in mice in which the NEP gene had been deleted by homologous recombination (NEP-/-), in gene expressing mice (NEP+/+) and in the background strain C57/B16 (B16). We measured the activity of myeloperoxidase (MPO) as an index of acute inflammation, and stained for the adhesion molecule ICAM-1 which is required for neutrophil recruitment, as well as for SP. Mice were infected with 350-400 T.spiralis larvae by gavage and studies conducted at 6, 12, 24, 48, and 72 hours post-infection (PI). In non-infected mice, MPO activity was higher in NEP"/', than in NEP+/+ or B16 mice (0.7 +0.1 v 0.4 ±0.1 v 0.3 ±0.1 Units/rag). However, following infection there was a significant increase in MPO activity in the NEP / mice compared to the two control strains. In the NEP -/- mice, the increase in MPO was evident as early as 12h PI, when values in NEP expressing controls were still within normal limits, and peaked at 24h PI (2.6-+ 0.4 v 0.9-+ 0.4 and 0.8 ± 0.3 Units/rag P<0.05). This was accompanied by increased staining for both ICAM-1 and SP in the NEP"/- mice compared to controls. Infusion of hrNEP via a minipump, compared to the infusion of a non-relevant peptide, significantly reduced MPO activity in NEP-/- mice infected 24h previously by T.spiralis (1.9 ± 0.2 v 1.1 ± 0.3 Units/mg; P<0.05). These findings indicate that NEP degraded peptides are involved in the initiation of inflammation following nematode infection and that by increasing the bioavailability of these neuropeptides, the onset of inflammation is accelerated. These data also indicate that NEP is a potential target for modulating intestinal inflammation. Supported by NIH and MRC Canada. G3794 DOSE RAISING OF AZATHIOPRINE BEYOND 2.5 MG/KG/DAY IN CROHN'S DISEASE PATIENTS WHO FAIL TO IMPROVE WITH A STANDARD DOSE. L. Barbe, P. Marteau, M. L6mann, Y. Bouhnik, M. Allez, B. Coffin, J. Bonnet, B Messing, JC Rambaud and R Modigliani. St-Louis and St-Lazare Hospitals, Paris, France. About one third of patients treated with azathioprine (AZA) or 6-mercaptopurine (6-MP) does not respond to this treatment. In a recent study, Colonna et al (Am J Gastroenterol 1994; 89: 362-6) found a correlation between leukopenia and the achievement and maintenance of remission. This result suggests that the dose of AZA/6-MP may be inappropriate in non responders. Methods. Eighteen patients (7M; 21-50 yr) with CD were treated with AZA because of steroid dependence (n=8), early recurrence after surgery (n=5) or refractory disease (n=4). The initial median dose of AZA was 2.0 rag/k/day (range: 1.7-2.5). In all these patients, AZA was considered as unsuccessful because of failure to achieve a complete remission or early relapse during tapering steroids, after a median delay of 11 mo (range : 5-35). The daily dose of AZA was then raised beyond 2.5 mg/kg (median daily dose, 2.7 mg/kg/day; median dose raising, 0.8 mg/kg). All patients had active disease when the dose of AZA was raised, and 14 of the 18 were still on steroids (median dose : 20 rag/day) Results. The median duration of follow-up was 9 mo (range : 5-51). A clinical remission with elimination of steroids was achieved in 14 patients (78%). Among the 10 patients maintained on high dose of AZA, 4 had a relapse. In 4 patients, the dose of AZA was subsequently tapered to 2 mg/kg/day; one of the 4 patients had a relapse and was successfully maintained on remission when the dose of AZA was raised again. No patient had significant bone marrow depression. Two patients experienced a cytomegaiovirus infection; one patient had a mild, transient elevation of aminotransferases. Conclusion. Dose raising of AZA beyond 2.5 mg/kg/day may be successful in CD patients who do not respond to standard dose, and should be considered before concluding drug failure.
April lS9S
CC n = 88 LC = 70 1,2 Age I mean (yrs) 64.1 63 range 23 -87 22-91 NS M/F ratio 2 (%) 27/73 49/52 p ---0.005 Diarrhea 2 acute 9 14 intermittent/chronic 13/66 7/49 NS Delay before diagnosis medianI 7.5 months 5 months p25-75 3-24 months 2-11 months p = 0.065 %Associated "auto-immune dis.''2 38 36 NS Aspirin2Y/N 11/65 15/53 NS NSAID2Y/N 14/62 11/57 NS Abdominal pain2: slight/severe 17/5 16/0 intermitt cramps 17 3 p ---0.011 Weight loss2 35 33 NS $oaoking2: current/former/never 23/6/34 10/16/23 p = 0.017 Followup n = 57 n = 37 median (months) 7 5 p25-75 3-24 3-22 Symptoms2 gone 22 22 improved 26 10 same 17 5 p = 0.040 Wilcoxon I and Chi-square 2 were used for statistical comparison. Conclusions: Lymphocytic and collagenous colitis are similar but not identical clinical syndromes. Unlike CC pts half of the LC pts are males. LC pts present somewhat earlier and are less likely to be active smokers. Their symptoms are milder and are more likely to disappear with or without therapy. Overall after a median follow-up of 6 months only 24% of patients (LC + CC) have persistent important symptoms. G3792 CYTOKINES AND ULCERATIVE COLITIS: MUCOSAL BALANCE BETWEEN PRO AND ANTI-INFLAMMATORY CYTOKINES IN PATIENTS WITH AND WITHOUT POUCHITIS. A Balzan0, N Bevilacqua*, Nappo C*, P Gionchetti*, M Campieri*, G Grande', A Bove, Mancino D*. Dip. di Gastroenterologia - *Lab.Centr.di Analisi Cliniche -ASO A.Cardarelli. Napoli *Cattedra di Immunologia - lst.di Pat.Generale - II Universith degli Studi - Napoli *Dip. di Medicina Intema e Gastroanterologia, Universit~ di Bologna. Pouchitis is the major long-term complication after restorative proctocolectomy with ileal pouch-anal anastomosis for ulcerative colitis (UC). In UC, is well documented a disturbed balance between pro- and antiinflammatory cytokines: In puochitis, the mucosal levels of pro-inflammatory cytokines have been shown to be increased. Aim of this study was to evaluate mucosal balance of pro- and anti-inflammatory cytokines in patients with ileal pouch-anal anastomosis with and without ponchitis. Concentration of interleukin (IL-) 113, IL-1 receptor antagonist (ra), IL-4, IL-8, IL-10, interferon y (INF'/) and tumor necrosis factor ct (TNFct) were determined by ELISA in tissue homogenates of mucosal biopsy specimens. Biopsies were obtained from pelvic ileal pouches in 8 patients with pouchitis and 10 with pouches without pouchitis. Diagnosis and severity of pouchitis were evaluated with the Pouchitis Disease Activity Index (PDAI) which includes clinical sigmoidoscopic and histological criteria. pg/mg tissue protein IL-113 IL-lra IL-4 IL-8 IL-10 INF-y TNF-a
Pouch
Pouchitis
p
8.5 304.7 0.15 23.6 0.28 0.10 5.5
20.0 331.3 0.17 59.5 0.30 0.19 5.5
0.008
0.027
IL-113 and IL-8 mucosal levels were significantly increased in pouchitis compared with normal pouches, and correlated with disease activity (PDAI) [ r=-0.69 and r=0.82 respectively and (p<0.01)]. This study shows that also in pouchitis there is a disturbed balance between pro- and anti-inflammatory cytokines as previously shown in UC.
• G3793 THE ROLE OF PRO-INFLAMMATORY PEPTIDES IN THE ONSET OF INTESTINAL INFLAMMATION. G. Barbara, R. DeGiorgio, P. Blennerhassett, S. Sturiale, E. Grady, N. Bunnett and SM. Collins. McMaster University Hamilton, Canada, University of Bologna Italy, and UCSF, San Francisco. Substance P and other pro-inflammatory peptides have been implicated in the genesis of neurogenic inflammation but its role in intestinal inflammatory processes has yet to he precisely identified. In a model of intestinal inflammation following nematode infection, we have shown that immunoreactive substance P is increased in the inflamed intestine, the dorsal root ganglion and in the dorsal horn of the spinal cord. Furthermore, we have shown that there is a profound decrease in neutral endopeptidase (NEP EC 3.424.11) in this model, implying an increased bioavailability of SP. To elucidate the role of pro-inflammatory peptides in the inflammatory process, we have examined the effect of Trichinella spiralis infection in mice in which the NEP gene had been deleted by homologous recombination (NEP-/-), in gene expressing mice (NEP+/+) and in the background strain C57/B16 (B16). We measured the activity of myeloperoxidase (MPO) as an index of acute inflammation, and stained for the adhesion molecule ICAM-1 which is required for neutrophil recruitment, as well as for SP. Mice were infected with 350-400 T.spiralis larvae by gavage and studies conducted at 6, 12, 24, 48, and 72 hours post-infection (PI). In non-infected mice, MPO activity was higher in NEP"/', than in NEP+/+ or B16 mice (0.7 +0.1 v 0.4 ±0.1 v 0.3 ±0.1 Units/rag). However, following infection there was a significant increase in MPO activity in the NEP / mice compared to the two control strains. In the NEP -/- mice, the increase in MPO was evident as early as 12h PI, when values in NEP expressing controls were still within normal limits, and peaked at 24h PI (2.6-+ 0.4 v 0.9-+ 0.4 and 0.8 ± 0.3 Units/rag P<0.05). This was accompanied by increased staining for both ICAM-1 and SP in the NEP"/- mice compared to controls. Infusion of hrNEP via a minipump, compared to the infusion of a non-relevant peptide, significantly reduced MPO activity in NEP-/- mice infected 24h previously by T.spiralis (1.9 ± 0.2 v 1.1 ± 0.3 Units/mg; P<0.05). These findings indicate that NEP degraded peptides are involved in the initiation of inflammation following nematode infection and that by increasing the bioavailability of these neuropeptides, the onset of inflammation is accelerated. These data also indicate that NEP is a potential target for modulating intestinal inflammation. Supported by NIH and MRC Canada. G3794 DOSE RAISING OF AZATHIOPRINE BEYOND 2.5 MG/KG/DAY IN CROHN'S DISEASE PATIENTS WHO FAIL TO IMPROVE WITH A STANDARD DOSE. L. Barbe, P. Marteau, M. L6mann, Y. Bouhnik, M. Allez, B. Coffin, J. Bonnet, B Messing, JC Rambaud and R Modigliani. St-Louis and St-Lazare Hospitals, Paris, France. About one third of patients treated with azathioprine (AZA) or 6-mercaptopurine (6-MP) does not respond to this treatment. In a recent study, Colonna et al (Am J Gastroenterol 1994; 89: 362-6) found a correlation between leukopenia and the achievement and maintenance of remission. This result suggests that the dose of AZA/6-MP may be inappropriate in non responders. Methods. Eighteen patients (7M; 21-50 yr) with CD were treated with AZA because of steroid dependence (n=8), early recurrence after surgery (n=5) or refractory disease (n=4). The initial median dose of AZA was 2.0 rag/k/day (range: 1.7-2.5). In all these patients, AZA was considered as unsuccessful because of failure to achieve a complete remission or early relapse during tapering steroids, after a median delay of 11 mo (range : 5-35). The daily dose of AZA was then raised beyond 2.5 mg/kg (median daily dose, 2.7 mg/kg/day; median dose raising, 0.8 mg/kg). All patients had active disease when the dose of AZA was raised, and 14 of the 18 were still on steroids (median dose : 20 rag/day) Results. The median duration of follow-up was 9 mo (range : 5-51). A clinical remission with elimination of steroids was achieved in 14 patients (78%). Among the 10 patients maintained on high dose of AZA, 4 had a relapse. In 4 patients, the dose of AZA was subsequently tapered to 2 mg/kg/day; one of the 4 patients had a relapse and was successfully maintained on remission when the dose of AZA was raised again. No patient had significant bone marrow depression. Two patients experienced a cytomegaiovirus infection; one patient had a mild, transient elevation of aminotransferases. Conclusion. Dose raising of AZA beyond 2.5 mg/kg/day may be successful in CD patients who do not respond to standard dose, and should be considered before concluding drug failure.