April 1995 Immunology, Microbiology, and Inflammatory Disorders A783

USEFULNESS OF SCINTIGRAPEY USING 99mTo-~LMPAO LABELLED GRANULOCYTES (PMN) AB EARLY INDICATOR OF POSTOPERATIVE ASYMPTOMATIC RECURRENCE IN CROHNeSiDISEASE (CD).

Bianeons, L. Di Naoio, A. Marohsggiano, M.C. Di Paolo, P. Paolusi, F. S=opinaro & F. Pallone. Clinioa Msdisa2 & Radiologia, Univ. La Sapiensa, Rome; Dipt. End 8perimemtaln, univ. R. Calabriar catamsaro, Italy

Background: Postoperative asymptomatic recurrence occurs after bowel resection in most patients with CD. Since drug regimens effective in preventing clinical relapse in asymptomatic patients are available and recurrence itself may be delayed (Gastroenterology 1992,103,363; Alim Pharm Ther 1994,8,35), non invasive methods for detecting asymptomatic gross lesions may well be of use. Scintigraphy using 99mTc-HMPAO iabelled PMN proved to reliably assess CD site, extent and activity. Aim: To prospectitively evaluate the usefulness of scin- tigraphy using 99mTc-HMPAO labelled PMN in detecting postoperative asymptomatic recurrence in CD. Methods: ii patients with ileal CD undergoing elective ileo-cecal re- Section were enrolled. 7 patients had never been resected before while 4 had previous resections (i resection 3 pts; 2 resections 1 pt). Patients were prospectively followed up and 99mTc scan performed at 6 (5 pts) and 12 mths (all pts) after surgery. ColonosCopy with biopsies was blindly performed whithin 2 wks. Changes detected by endoscopy (Gastroenterology 1990, 99,956) and histology were graded. PMN were isolated by dextrane sedimentation and Ficoll gradient, labelled with 99mTc-HMPAO (10uCi) and injected i.v. in autologous plasma (4cc). Planar and toeogranhic r-camera images were aquired at 1 and 3 hrs and 99mTc uptake graded (score 0-4). Results: All patients were asymptomatic during the study (CDAI<150). Anastomotic recurrence was detected by colonoscopy in 10/11 patients at 12 mths. 99mTc scintigraphy detected a pMN infiltration at the perianastomotic area in 11/11 patients at 12 mths. The onlypatient free from endoscopic recurrence was the one with 2 previous resections. At 6 mths 99mTc scinti- graphy was positive in all 5 patients while recurrence was detected by endoscopy in 3/5 CD (no recurrence in the 2 pts with >i resection). Both at6 and 12 mths scintigraph- ic score was poorly related to both histologic (r = 0.72; p= 0.04) and colonscopic score (~=-0.21; pc0.6). conclusions: Scintigraphy using 99mTc-HMPAO labelled PMN is a sensitive technique for detecting postoperative asymptomatic recurrence in CD, and may well be considered as an alternative to colon0scopy in the regular supervision of resected patients,

• 123I-.T-tiTERZ~ITJ~.~-2 (123Z-ZL-2) 8 C Z ~ Z ~ FOR ~ V1"70~ DETECTION OF GUT LY1~OCY' I~ IlrJ'ZLTRR, TION ZN CROHN'E DIEEE, SE (CD). L, Bia~cone~ A. sicJ:lcre, E. Ferret~i t G. Ronga, P. Pozsilli & F. Pallone. Clinica Medicn 2, Univ. La Sapisnza, Roma; Dipt Med. Bperim., Univ. R. Calabria, Catanzaro, Italy.

Background: The involved gut in CD is heavily infiltra- ted by activated lymphocytes expressing IL-2 receptors and capable of binding IL-2. 123I-IL-2 is a new radio- pharmaceutical that specifically binds to activated !ymphocytes allowing their localization by r-camera imaging (NUcl Med Commun 1992,13,713). Kimsz To examine whether 1231-IL-2 binds to the gut inflamed tissue thus allowing the in vivo detection of intestinal lymphocy- tic infiltration in CD. We also aimed at exploring whe- ther 123I-IL-2 scintigraphy may he useful in the clinical management of CD. Methods: Nine patients with ileal CD were studied, 6 active (CDAI >200) and 3 inac- tive (CDAI <150). Four active patients were longitudi- nally studied before and after 12 wks prednisone (PDN) therapy (I mg/Kg). Controls were i0 healthy volunteers. 123I-IL-2 (2 mCi) were injected i.v. and SPET r-camera images aquired at 1 hr. To quantitate bowel radioactiv- ity a transaxial section below kidney poles was consid- ered and the bowel/hone radioactivity ratio calculated. Data were expressed as maximal bowel/bone ratio. Results: Intestinal 1231-IL-2 uptake was higher in CD than in controls (2.1Z0.33 vs 1.62±0.24; pc0.01). 123I-IL-2 imaging appeared to closely reflect site and extent of bowel involvement, as assessed by X-ray. In the 3 longitudinall~ followed up patients achieving remission with PDN, 231-!L-2 uptake was significantly lower after treatment (CDAI <150) than before (CDAI >200)(1.46±0.3 vs 1.9120.3; pc0.04). ~23I-IL-2 uptake was significantly higher in the 3 CD patients enrolled while in clinical remission (>3 mths with no therapy) than in the 3 CD with PDN-induced remission (2.45±0.37 VS 1.46±0.3; p<0.OS). Summary E conclusion: Intestinal 123I-IL-2 uptake was increased in CD paralleling disea- se site and extent. Steroid-induced remission was associated with significantly lower 123I-iL-2 uptake,

12~ suggesting that I-IL-2 scintigraphy may sensitively reflect the lesional activity of CD. 123I-IL-2 may proveto be a useful too ! in both investigational an clinical settings for CD assessment and monitoring.

• C U L T U R E O F H U M A N I N T E S T I N A L M U C O S A L M 1 C R O V A S C U L A R E N D O T H E L I A L CELLS: I S O L A T I O N A N D P R E L I M I N A R Y C H A R A C T E R I Z A T I O N D.G. Binion, G.A. West, K. Ina, K. Fukushima, G.M. Dobrea, E. Poptic, S.N. Emancipator, and C. Fiocchi. Case Western Reserve University and Cleveland Clinic Foundation, Cleveland, OH.

Mucosal microvascular endothelial cells are involved in tissue-specific lymphocyte homing, local immune ~ homeostasis , and intestinal inflammation. Investigation into the biology of gut mucosal endothelium and its role in the above events has been prevented by lack of a practical in vitro system for cellular and molecular experimentation. We developed a reproducible method to successfully grow primary cultures of human microvascular endothelial cells from small and large intestinal mucosa.

Mucosal strips were obtained from surgically resected specimens of normal and inflamed bowel. After removal of mucus and epithelial cells by DTF and EDTA, strips were submitted to short-term collagenase digestion. Microclusters of endothelial cells were obtained by squeezing followed by sequential filtration with nitex membranes. Cell patches grew from individual clusters adherent to fibronectin-coated plates, using heparin-, endothelial growth factor- and trace element-enriched medium (MCDBI31). Ring cloning of patches was then employed to obtain totally pure cultures. These were readily expanded to large volume confluent monolayers for up to three months.

Immunofluorescence revealed strong cytoplasmic staining for factor VIII while the epithelial cell marker cytokeratin was negative. Electron microscopy demonstrated Weibel-Palade bodies and tight junctions characteristic of endothelial cells. PDGF production was assayed as a typical endothelial cell product. When supernatants from autologous mucosal endothelial, smooth muscl6 and fibroblast cell cultures were tested, endothelial cells produced an average of 550 pg/ml, whereas muscle and fibroblast levels were undetectable. Expression of adhesion molecules was investigated after recombinant IL-113 (100 U/ml) induction. Northern blot analysis of total RNA revealed marked expression of ELAM-1 transcripts exclusively in stimulated endothelial but not in muscle and fibroblast cells. ICAM-1 and VCAM-1 mRNA were constitutively expressed in the three cell types and were all up-regulated by IL- 113.

This novel and practical in vitro culture system generates large quantities of pure human muc0sal micro,)ascular endothelial cells that express specific markers while retaining broad functional activity. The availability of such cells will allow investigation of their role in regulation of mucosal immunity under normal and pathological conditions.

• FURTHER EVIDENCE FOR A GENETIC ASSOCIATION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST WITH ULCERATIVE COLITIS IN A NORTHERN AND A MEDITERRANEAN POPULATION. G. Bioeue, G. Monteleon¢**, J.B.A. Crusius, A. Dominianni**, N. Perrotti**, P.J. Kostense*, S.G.M. Meuwissen, F. Pallone**, and A.S. Pefia. Departments of Gastroenterology, Epidemiology and Biostatisties*, Free University Hospital, Amsterdam, The Netherlands and Department of Clinical and Experimental Medicine**, Catanzaro, Italy

The importance of the interleukin-I receptor antagonist (IL-lra) in inflammatory bowel diseases has been pointed out by recent studies. It has been suggested that a defecXive production of IL-lra may exist in patients with inflammatory conditions. The characterization of a 86 base pair of identical tandem repeats within the intron 2 of the IL-Ira gene resulted in the first reported association outside the HLA region between ulcerative colitis and the IL-lra allele 2 in a Northern European Caucasian population. Our aim was to study this polymorphism in a Dutch and an Italian population of patients with ulcerative colitis (UC) as well as healthy controls. Methods: Genotype and allelic frequencies of the polymorphic region in the IL- Ira gene were determined in patients with ulcerative colitis and healthy controls. DNA fragments reflecting the 3 alleles were generated by PCR amplification and analyzed by agarose gel electrophoresis (Tarlow et al. Hum Gen 1993;91:403). Results: IL-lra genotype in healthy controls (HC) and UC patients groups from The Netherlands (NL) and Italy (IT)

Genotype 1,1 1,2 2,2 1,3 2,3

HC (NL) n=65 40 20 5 0 0 UC (NL) n=88 40 35 8 3 2 }-IC (IT) n=27 22 4 1 0 0 UC (IT) n=20 II 5 4 0 0 Carriage of at least one copy of allele 2 in the Dutch population gave an odds ratio (OR) of 1.67 (95% CI: 0.83 -3.39) for UC. In the Italian population an OR of 3.60 (0.82-16.8) was found, thus confirming the recent findings in the United Kingdom OR 2 (1.3-3.2) (Mansfield et al. Gastro 1994;106:637). In conclusion: our data indicate that the polymorphism in intron 2 of the IL-lra gene is a genetic marker for UC.