April 1995 Immunology, Microbiology, and Inflammatory Disorders A821

• INCRIMINATION OF ANAEROBIC BACTERIA IN THE PATHOGENESIS OF EXPERIMENTAL COLITIS. A. Garcia-Lafuente, M. Antolfn, E. Crespo, A. Salas, M. Laguarda, F. Guarner, P, Forcada, J. Gavald~, J-R. Malagelada. Digestive System Research Unit & Microbiology, Hospital General Vail d'Hebron, Barcelona; Pathology, Hospital Mutua, Terrasa, Spain.

We investigated the role of enteric bacteria in the pathogenesis of chronic transmural inflammation induced by trinitrobenzenesulfonic acid (TNB). First, 6 bacteria species were identified in cultures of colonic tissue from 4 rats 24 h after TNB administration and stored at -80°C for subsequent use, In 6 groups of rats (n=4-6), a colonic segment (ascending to mid-descending colon) was surgically excluded and brought out through 2 colostomies, the cecum' removed, and continuity of the tract achieved by ileocolic anastomosis. Colostomies were closed by ligature to preclude entry of air into the colonic lumen. Facultative aerobes (Streptococcus vir., Klebsiella pneum, and Enterobacter aero., 2x101°) were instilled into the segment in 2 groups of rats (ae and tnb- Be); same aerobes plus anaerobes (Clostridium ram., Bacteroides frag. and Bacteroides uni,) were instilled in another 2 groups (aria and tnb- anal and broad spectrum antibiotics (imipenem+vancomycin) were instilled in nil and tnb-nil groups. Three days later, segment washings were cultured (all species but Streptococcus were recovered; no growth in nil groups), and tnb groups instilled with TNB (45 rag, 10% EtOH). On the following day, all groups were perfused with warmed saline at 18 mL/h, effluents assayed for eicosanoid (PGE2, TXB2 and LTB 4) release (pg/min), and segments removed for tissue myeloperoxidase activity determination (U/mg) and histological assessment of damage. Results:

PGE 2 LTB4 TXB= MPO nil 71 +12 106±8 23±5 7±2 ae 61±7 116±7 21±5 5±1 ana 80±14 115±7 27±6 7±2 tnb-nil 97±13 110±6 55±9§ 14±2§ tnb-ae 517+87# 138±14 154±23# 20±2 tnb-ana 1100±237" 206±48 371 ±77* 47±8* §p<O.05 vs.nU #p<0.05 vs.nil&tnb-nil *p<0.05 vs.nil, tnb-nil&tnb-ae No mucosal lesions were observed in nil, ae and aria; tnb-nil rats showed edema or mild mucosal necrosis, tnb-ae rats showed mucosal necrosis and all mb-ana showed deep mucosal and submucosal necrosis.

In conclusion, transmural TNB colitis only develops in the presence of luminal anaerobic bacteria.

ADEQUATE IL-10 PRODUCTION OF PERIPHERAL BLOOD - AND LAMINA PROPRIA MONONUCLEAR CELLS IN INFLAMMATORY BOWEL DISEASE. C. Gasch6 W. TilUnger, C. Dejaco, S. Bakos, W. Reinisch, R. P6tzi, T. WaldhOr, H. Lochs, A. Gangl. Dept. of Gastroenterology and Hepetology; University of Vienna; Austda.

Back,qround: IL-10 has important regulatory effects on inflammatory responses deactivating monocytes and macrophages. IL-10 deficiency in gene knockout mice leads to chronic enterocolitls. It was hypothesized that a deficient IL-10 production might cause an imbalance between pro- and antiinflammatory mechanisms and induce chronic inflammation in IBD. Patients and Methods: Serum samples and peripheral blood mononuclear cells (PBMC) of untreated patients with active CD (CDAI >200, n=22), with inactive CD (CDAI <150, n=12), with ulcerative colitis (UC; n=19) and of matched normal controls (NC, n=19)) were studied. In addition, lamina propda mononuclear cells (LPMC) were isolated from colonoscopic biopsies (CD: n=5, UC: n=4, NC: n=4). Cells were cultured with or without activation using PMA (lng/ml) and anti-CD3 monoclonal antibody (moAb: 20ng/ml) for PBMC and PHA (!% v/v) for LPMC. IL-10 concentrations in serum samples and supematants were determined by a specific ELISA using the anti-lL-10 moAb JES3-9D7 as a coating Ab and the anti-lL-10 rabbit polyclonal Ab (rabbit 170) as a tracer Ab. There is no cross-reactivity to BCFR-1 or other known cytokines. Results: IL-10 concentrations in all serum samples and in all PBMC and LPMC supematants without activation were below the detection limit (<10pg/ml). Maximum concentrations of IL-10 were detected in supematantS 24 hours after cell activation. IL-10 production of activated PBMC revealed no difference between active CD, inactive CD, UC or NC (table). IL-10 production of activated LPMC was insignificantly lower and did not differ between the stud~/~lroups as well (table).

active CD inactive CD UC NC p PBMC 115+39 92+_.28 75_+30 73+_26 n.s. LPMC 44_+34 n.d. 24_+7 27_+12 n.s. (Mean_+SEM, n.d.: not done, n,s.: not significant) Conclusion: Our data show that PBMC and LPMC from both active and inactive CD or UC produce normal amounts of IL-10 after activation. Therefore, the imbalance between pro- and antiinfiammatory mechanisms in IBD is not caused by deficient IL-10 production.

• DOUBLE-BLIND, PLACEBO CONTROLLED TRIAL OF ERYTHROPOIETIN AND IRON SACCHARATE FOR ANEMIA IN CROHN'S DISEASE. C. Gasche C. Dejaco, T. Waldh~r, W. Reinisch, W. Tillinger, H. Vogelseng, A. Gangl, H. Lochs. Dept. of Gastroenterology and Hepatology; University of Vienna, Austria.

Backqround: Both iron deficiency and inadequate endogenous erythropoietin (EPO) production are related to the development of anemia in Crohn's disease (CD). Patients and Methods: We treated 31 patients with CD and anemia (hemoglobin (Hb) _<10.5 g/dl) with recombinant human EPO (r-HuEPO; 150 IE/kg, 3 x /week s.c.) or placebo (PL) in a randomized, double-blind, two-phase trial. All patients received iron saccharate (Fe-Sac) as an infusion (200 mg in 250 ml NaCI) twice a week for 14 days and subsequently once a week until the end of the trial. A response was defined as an increase in Hb >2 g/dl after 8 weeks. In addition, the impact of treatment on quality of life (QOL; range: 9 - 45, 45 indicating lowest QOL), CDAI, C-reactive protein (CRP) and iron deficiency parameters (serum iron, transferrin, transferdn saturation, ferritin) was studied. At the end of the blinded study phase the code was broken and an open label treatment phase was initiated. PL nonresponders received r-HuEPO, 150 IE/kg, and r- HuEPO nonresponders, 300 IE/kg (3 x /week, s.c.), respectively, for another8 weeks. Results: Both groups were matched in sex, Hb, CDAI, CRP and ferritin. 15116 patients of the r-HuEPO group and 10115 of the PL group responded within the first study phase (p<O.05). The increase in Hb in the r-HuEPO group was higher (8.8-+0.4 to 13,5i-0.4 g/all) than in the PL group (8.3_+0.4 to11.7_+0.5 g/dl; p<O.05). Both groups had a significant improvement in QOL (r-HuEPO: 22_+1 to 17+1, p<O.O01; PL: 21_+1 to 16_+1, p<O.05), and in the CDAI (r-HuEPO: 225_+19 to 112_+12, p<O.O01; PL: 228_+15 to 121_+19, p<O.O01). Ferritin and transferrin saturation increased while CRP showed no change. In the second phase of the study, all previous nonresponders (n=6; 5 previous PL and 1 previous f-HuEPO) showed a response (9.5_-+0.6 to 13.1_+0.7 g/dl, p<O.O01). Both drugs were well tolerated. Side effects were related to FeSac with local burning at the site of venipuncuture (n=3), bitter taste (n=l), fever <38°C (n=2) and temporary hypotension (n=2). During the study no patient received blood transfusions. Conclusion: Treatment of anemia in CD with r-HuEPO and Fe-Sac is highly effective and leads to an improvement in QOL

• TROPOMYOSIN ISOFORMS IN HUMAN COLONIC MUCOSA AND THE AUTOANTIBODIES AGAINST SPECIFIC ISOFORMS IN ULCERATIVE COLITIS (UC) SERA. X.Genq*, J. Lin+, A. Dasgupta*, P.M. Gregory*, K.M. Das*, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ* and University of Iowa, Iowa City, IA+.

We earlier reported a putative autoantigen, Mr 40K protein (P4O), associated with UC. Recently, we purified P40 and sequenced two tryptic peptides and demonstrated that P40 belongs to tropomyosin (TMs) family (J Immunol 150:2487,1993). TMs are cytoskeletal protein present in all eukaryotic cells with organ specific isoforms attributed to alternative RNA splicing and polyadenylation. At least 8 TM isoforms have been identified in human fibroblast. However, colonic TMs are unknown, and which tropomyosin isoform is related to the putative autoantigen remains to be determined. Colonic mucosa (including epithelium and stromal tissue) was collected by stripping the mucosa at submucosal plane from operative specimens of normal colon. Mucosal TMs were extracted by homogenization, boiling, ultracentrifugation, and differential ammonium sulfate precipitation. TM isoforms were analyzed by ELISAs using the anti-TM isoform specific monoclonal antibodies and by 2D gel analysis. Human colonic mucosal extract contained all of the 5 TM isoforms, hTMI-hTM5. We further investigated the immunoreactivity of sera from inflammatory bowel disease (58 UC, 25 Crohn's disease, CD) and 25 healthy subjects against 9 recombinant TM isoforms and chimeras by ELISAs. These recombinant TM isoforms were purified from several bacterial clones harboring respective full-length cDNAs encoding human fibroblast TM isoforms, such as hTMI, hTM2, hTM3, hTM5, and some chimeras, hTM3/5, hTM5-gly/3, hTM5-gly, hTM5/2, hTM5/3. The immunoreactivity (OD values) of UC sera against hTMI, 3&5 isoforms and chimeras derived therefrom was significantly (p<.001) higher than CD sera and normal subjects. There was no difference in the immunoreactivity with hTM2, mutated hTM5 (hTMS-gly) and hTM5/2 among UC, CD and normal sera. CD and normal sera had similar OD values against all 9 recombinant proteins. These results demonstrate the presence of several TM isoforms in colonic mucosa. UC sera, but not CD sera, preferentially reacted with certain specific TM isoform(s). Further analysis using tryptic fragments of hTMs and additional chimeras may help to identify the immunoreactive peptide(s) associated with UC.