A 7 7 6 A G A A B S T R A C T S G A S T R O E N T E R O L O G Y , Vol . 1 0 8 , No. 4

REDIRECTED T CYTOTOXICITY OF INTESTINAL LAMINA PROPRIA LYMPHOCYTES A.Bachetoni, P.Mariani, MD.D'Atessandro, P.Lionetti*, D.Lomanto, V.Speranza. Cliniea Chirurgica II and *Clinics Pediatrics I, University of Rome "La Sapienza".

The cytotsxic function of immunocompetent cells in the gut mucosa might be important in the normal immune response and could be relevant to the mucosal damage seen in inflammatory conditions. The humsn intestinal mucosa is constantly exposed to a variety of antigenic stimuli and a relevant presence of primed T lymphocytes would be expected. It has been observed that cells with NK phenotype are present in the intestinal lamina propria in a small number and exhed a low cytolitic activity against natural killer (NK)-sensitive target cells. We evaluated both NK and CTL function of LPL and PBL in Crohn's disease (CD) pts and in controls. Surgical ileal specimens from pts undergoing bowel resection for CD (n=lO) or for intestinal carcinoma (n=10) were used as source of LPL. LPL were isolated using DTT-EDTA-CoJlagenase digestion fonswed by discontinuous Percoll density gradients while PBL were isolated by FJcolI-Hypaque gradient. The cytotoxic activity of freshly isolated LPL and PBL were assessed against the NK-sensifive K562 cell line (NK aCtivity), and the NK-resistant P815 cell line in the presence of anti-CD3 (5 mcg/ml) and PHA (1 mcg/ml) at effeetor/target ratio of 50:1 and incubated for 6 hrs at 37°C. Data are shown in Fig. 1.

PBL ~ LPL

50 50

30 30 20 20

- - 0 1<562 P815 PSlS+arlti PSI5+pHA K562 P8[5 PSl5+an=i PSI5~-PHA

CD3 CD3

Freshly isolated LPL, in contrast to PBL, exhibited no significant NK function against K562 target cel~s, It is of interest the finding that LPL displaied a significant T cell cytotoxicity against P815 in the presence of either anti-CD3 or PHA in short term assays; in contrast PBL exhibited an elevated CTL activity against P815 targets only when sensitized by PHA, but not by anti-CD3. In all experiments the cytotoxic activity against P815 alone was not present.Our data confirm that LPL of both groups have a low number and a low activity of NK cells. Our data clearly indicate that CTL in LPL are in an activated state and can kill B7- (P815) target cells, previously sensitized with anti-CD3, in an indipendent fashion upon aostimulation. Indeed resting T cells, but not activated T cells, require costimulafion by CD28-CTLA-4/B7.1-B7.2 intersctionl besides the engagement of the CD3/TCR complex, for triggering cell-mediated cytotoxicity. These in vivo activated CTL may play a pivotal role in tissue injury, that occurs in certain intestinal inflammatory disorders.

~WHY ARE RECURRENT CROHN'S LESIONS AFTER ILEAL RESECTIONS ALWAYS AT THE ILEAL SIDE OF THE ANASTOMOSIS? F. Baert, K. Geboes e, P. Rutgeerts. Departments of Gastroenterology and Pathology ~ at the University HOspital Gasthuisberg Leuven, Belgium.

It is well known that postoperative recurrence of Crohn's disease (CD) is located at the ileal side of the anastomosis. In 29 patients (14M, 15F) undergoing a curative ileocolonic resection for CD, we examined the status of ileum, anastomosis and colon with endoscopy and biopsies 3 months after surgery. 13 patients had preoperative colonic involvement; 9 endoscopic, 4 only at histology of the resection specimen. In an additional 2 patients there was fibrous obliteration of the appendix. Results: At three months 25/29 patients had histological ileal recurrence; 29 endoscopic and 2 patients symptomatic recurrence. Macroscopic and microscopic lesions were seen at the anastomotic site in 7 patients while none of the 29 patients had endoscopic colonic recurrence. In on~y5 (2 with preoperative colitis), an increased infiltrate was seen on histology. The microscopic lesions in colonic biopsies were milder than in ileal biopsies. The distribution of the infiltrate was segmentary, the composition was mixed with numerous eosinophils (3/5). Immunohistochemical staining for macrophages (KPI) and MHC class II antigens (HLA-DR) revealed major differences between the colon and the small intestine. In the small intestine KPI macrophages were focally distributed and clustering. In the colon macrophages formed a subepithelial layer (as in the normal colon) except for those biopsies showing an increased infiltrate. MHC class II antigens were not expressed by colonic epithelia] cells and poorly by ileal surface epithelia] cells. It is well known that MHC class II positive epithelial cells and macrophages are involved in intestinal antigen handling. The differences between ileum and colon, observed in this study, may be responsible for the difference in recurrence. The lesions at the anastomotie site may be secondary to surgery.

• THE EFFECT OF ULCERATIVE COLITIS ASSOCIATED ANTI- NEUTROPHIL CYTOPLASMIC ANTIBODIES ON RESPIRATORY BURST ACTIVITY OF NORMAL HUMAN NEUTROPHILS H.I. Balkovitz*, A.B. Borlet, * * ' " R.H: Duerr . Departments of Medicine and tPhysiology, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Background/Aims: Anti-neutrophil cytoplasmic antibodies (ANCA) that exhibit a perinuclear pattern of immunofluorescence (p-ANCA) are found in the majority of sera from patients with ulcerative colitis (UC). ANCA in sera from patients with necrotizing vasculitides have been reported to stimulate neutrophil respiratory burst activity in vitro. The purpose of this study was to determine whether p-ANCA positive UC sera stimulate neutrophil respiratory burst activity in vitro. Methods: Sera from 10 healthy controls and 10 p-ANCA positive UC patients were diluted h l 0 in plastic scintillation vials containing RPMI 1640, 25 mM HEPES, and 1% FBS; 1 x 106 neutrophils isolated from a healthy individual; and luminol 2 laM for a total volume of 1 mL. A positive control vial contained PMA 1 ~tg per mL instead of serum. The healthy control and p-ANCA positive UC samples, as well as the PMA positive control, were placed in a scintillation counter set to count out of coincidence. Counts per minute (cpm) were continuously recorded for 12 second periods. The results at 30 and 60 minutes were extrapolated from a graph containing all Clam points. The results for the healthy control and UC groups at both 30 and 60 minutes were compared using the Mann-Whitney U test, Results:

]cpm (mean+/-SD) x 10-3[cpm (mean+/-SD) x 10 -3 study group [at 30 minutes [a t 60 minutes healthy control (n=10) | 516 +L 199 [ 682 +/- 308 p-~ANCA+ UC (fi= 10) [ 512+/-150 | 671+/-141 PMA positi.ve control [ .. 3667 | 2778

There were no significant differences between the cpm for the healthy control and p-ANCA positive UC sera at both 30 and 60 minutes. Potential for stimulation of neutrophil respiratory burst activity was demonstrated by the dramatic response to PMA. Conclusion: p-ANCA in UC has no significant effect on respiratory burst activity of normal human neutrnphils in vitro.

IgM-SPECIFIC ANTIBODY AGAINST MEASLES VIRUS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE FA Balzola. F Castellino, P Colombatto~ P Manzini~ MR Brunetto, M Astegim~o, G Verme, A Wakefield*, A Pera**, F Bonino. Dip Sper di Gastroenterologia, Osp Molinette; **Div di Gastroenterologia, Osp Mauriziano, Torino, Italy; *Inflammaro~, Bowel Disease Study Group, Royal Free Hospital School of Medicine, London, UK.

Measles vires (MV) has been implicated in file aetiology of Crohn's Disease (CD). Aim: To evaluate the presence of MV-specific IgM antibodies as a potential

marker of viral related disease in Inflammato~' Bowel Disease (IBD). Methods: Sera from patients with CD (n=36:23 male: median age 40 yrs, range

20-66); ulcerative colitis (UC) (n=22:14 male: median age 42 yrs, range 19-65): indeterminate colitis (IC) (n=3; 2 male: median age 45 yrs, range 23-77); chronic hepatitis (CH) (n=19; 10 male; median age 51 yrs, range 38-68), 12 with HCV and 7 with HBV infection, and normal blood donors (n=7; male 4: median age 43 yrs, range 30-59) were studied. Sub-acute Sclerosing Paneneephalitis (SSPE) (n=4 sera) were used as positive controls. Disease activity in CD and UC was determined using Simple Index and CA1 score. No patients had Rheumatoid Factor or extra-intestinal pathology. All had normal levels of total IgM. Sera at high dilution (1:100) were examined by the Indirect Fluorescent Antibody Measles Test System (lgM IFA, Selavo srl).

Results:12 out 13 (92%) with mild activity of CD. 14 out 19 (74%) with moderate activity and 2 out 4 (50%) with severe diseasc respectively, ~ere positive. Overall 28 out 36 with CD (78%: p<0.00h Students T test) showed positive results. All 4 patients (100%) with UC in mild or severe disease were positive. 9 out 18 (50°/.) with moderate disease were also positive. Overall 13 of 22 with UC (59%; p<0,001) were positive. All those with IC, 18 out 19 (95%) with CH and all the blood donors were negative. The only one positive sen~m (5%) was of a type I1 mixed eryoglobulinaemia in an HCV patient.

Conclusion: Raised levels of MV-specific !gM in the majority' of those with CD and about half with UC, compared wifla a very low prevalcece in patients with other chronic inflammatory diseases, support an aefiologieal role for MV m IBD.