Problems which address health related quality of life (HRQOL) in inflammatory bowel disease (IBD)

GASTROENTEROLOGY Vol. 114, No. 4

A1002 AGA ABSTRACTS

G4102 MUCOSAL G R O W T H RELATED ONCOGENE ALPHA IN INFLAMMATORY BOWEL DISEASE. A. Imada, K. Ina, T. Hosokawa, M. Ohsuga, M. Shirnada, T. Ando, and K. Kusugami. First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan. Introduction: Interleukin-8 (IL-8) and growth related oncogene alpha (GRO-a) are included in the ~t-chemokine superfamily and have chemotactic and activatory properties on inflammatory cells. In this study, we examined mucosal IL-8 and GRO-~ secretion, and expression of IL-8 and GRO-ot protein and their specific mRNA using mucosal specimens of inflammatory bowel diseases (IBD). Methods: Colonic mucosal biopsies or surgical specimens were obtained from 17 patients with Crohn's disease (CD), 24 with ulcerative colitis (UC), and 18 with other inflammatory conditions (ischemic colitis, drug-induced colitis, etc.). In 18 patients, normal mucosa was obtained as control specimens from patients with colonic polyps or carcinoma. Colonoscopic biopsies were cultured on a culture insert for 24 hours and the activity of IL-8 and GRO-~t in the culture supernatant was measured by ELISA methods. IL-8 and GRO-ct gene expression was analyzed by in situ hybridization (ISH). Positive cell types for IL-8 and GRO-~x were determined by immunohistochemistry and multi-immunofluorescence (using a kryptonargon ion laser microscopy) with anti-IL-8, anti-GRO-c~, CD68, and MPO antibodies. Results: In the organ cultures, significantly higher levels of chemokines, IL-8 and GRO-ct were secreted in UC patients (p<0.05 versus control). The content of IL-8 in CD was also significantly higher than controls (13<0.05). IBD specimens contained significantly higher number of cells expressing IL-8 and GRO-tx mRNA than controls. IL-8 mRNA was localized in epitheriai cells, CD68 + macrophages and neutrophils, while GRO-a mRNA was found mainly in epitherial ceils and neutrophils.

IL-8 (ng/mg protein) GRO-ct (pg/mg protein)

Control UC CD 51.9a 192.4" 121.8" (35-108)b (57-520) (52-352) 502.1 1044.3" 586.1 (282-685) (237-2512) (239-934) amedian, brange, * p < 0.05 versus control

inflammatory control 65.4 (7-273) 531.5 (174-2512)

Conclusion: Incresed IL-8 and GRO-~ activity may be involved in IBD mucosai damage through their chemotactic and stimulatory activities on inflammatory ceils.

• G4103 CYCLOSPORIN A HAS SUPPRESSIVE EFFECTS ON NEUTROPHILS AND MUCOSAL T-CELLS. K. Ina, K. Kusugami, T. Ando, T. Hosokawa, A. Imada, M. Ohsuga, M. Shimada. First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan. Introduetion: Cyclosporin A (CsA), an immunosuppressive agent that is capable of modulating T-cell function, is known to be beneficial to treatment for patients with steroid-resistant ulcerative colitis (UC). Since polymorphonuclear neutrophils (PMN) infiltration in the intestinal mucosa is the hallmark of active UC, we examined the effects of CsA on the action of T-cells and PMN. Methods: Mucosal T-cell lines were established from lamina propria mononuclear ceils isolated from endoscopic specimens. Peripheral blood mononuclear cells and PMN were isolated by dextran sedimentation and centrifugation on a Ficoll-Hypaque cushion. Ceils were pretreated with physiologically relevant concentration of CsA, 5-aminosalicylic acid (5-ASA), and prednisolone (PSL). In some experiments, AA-861, inhibitory agent of 5-1ipoxygenase, was used to examine the effect of leukotrienes. Apoptosis was assessed by acridine orange/ethidium bromide staining. Cytotoxic activity against HT-29 target ceils exhibited by mucosal T-cell line was determined by -~lCr-release assay. Gene expression for cytokines (IFN-'/ and TNF-ct) was analyzed with RT-PCR. Chemotactic activity of T-cells and PMN was measured by modified Boyden chamber using interleukin (IL)-15, IL-8 and organ culture supernatants. Superoxide anion (02) production of PMN stimulated with fMLP and platelet-activating factor (PAF) was examined with luminol-dependent chemiluminescence. Results: CsA suppressed both killing activity against HT-29 cells and gene expression of IFN-T and TNF-o~ by mucosal T-cells in a dose-dependent manner, while neither 5-ASA nor PSL did. PMN migration induced by IL-8 and UC supernatants was substantially inhibited by CsA and PSL, but less by 5-ASA. These treatment did not alter the viability and apoptosis of PMN. Peripheral blood T-cell migration was also remarkably reduced after CsA treatment. CsA and PSL decreased O 2 production by PMN stimulated with fMLP and PAF, but 5-ASA had no inhibitory effects. (-) migrated PMN (/ram2)

15.0±3.8

PMN0252.4±9.8 production (kcpm)

CsA (100 ng/ml) 5-ASA (10.4 M) PSL (10 ~tg/ml) 8~2 ± 3.6"

10.0±3.5

6.1±3.1'

22.0±5.6**

61.2± 15.8

31.0±7.2"

* p < 0.05, ** p < 0.01

Conclusions: CsA has more suppressive effects on the biological functions of PMN as well as T-cells than PSL and 5-ASA. The present study suggested that CsA could be used for the treatment of severe active UC, because of its potent dual effects on PMN and T-cells. G4104 NON-DISEASE RELATED FACTORS AFFECTING HEALTH RELATED QUALITY OF LIFE (HRQOL) IN INFLAMMATORY BOWEL DISEASE (1BD), E. Jan Irvine, E. Grace, GD Kerr, E. Lyrenas, T. Bolin, C. O'Moraln, M. Korman, Q. Zhou and the International Quality of Life Study Group. Coordinating Center McMaster University, Hamilton, Ont. Background: Using a disease specific instrument, the Inflammatory Bowel Disease Questionnaire (IBDQ), we have previously observed that IBD patients have impaired HRQOL and that disease extent in ulcerative colitis (UC), severity (in both UC and Crohn's disease (CD)) and treatment efficacy determine degree of HRQOL impairment. Aim: To explore non-disease related determinants of HRQOL in 13 countries, we evaluated 1904 IBD patients (851 with indeterminate colitis (IC) or UC; 1053 with Crohn's disease) at a single visit. Methods: Patients completed demographic data, survey disease activity index (DAI; range 15 to 65) and quality of life questionnaires (the IBDQ; score 32 to 224; higher score=better HRQOL). Univariate analysis was used to explore potential non-disease related determinants of HRQOL. Factors significant at the p<.05 level were then assessed using multivariate modeling. Results: Female gender, (in both CD and UC), smoking status (CD), age (<60yr,CD and UC), lower level of education (CD), unemployment (CD and UC), major life event occurrence in prior 12 months (CD and UC), prior use of immunosuppressives (UC and CD), or corticosteroids (UC and CD) or 5-ASA (CD), prior surgery (CD) all predicted poorer HRQOL (p<.02). Clinical pattern in CD (inflammatory, stricturing, fistulizing) did not predict IBDQ score. After adjusting for disease activity index (44% of IBDQ variance explained), forward stepwise regression was performed. Features which remained significant at the p<.05 level, in descending order were: employment status (1% variance explained), education level (0.5%), life event in the prior 12 months (0.4%), steroids previously used (0.4%), female gender (0.3%) and prior use of immunosuppressives (0.1% of variance explained). Conclusion: Disease activity was the single variable which accounted for the greatest proportion of the variance of the IBDQ score. However, other demographic variables also predicted poor HRQOL. These features should be considered for adjustment when assessing or analyzing data in clinical trials. They may also be used to identify subgroups of patients who might benefit from intensive psychosocial supportive interventions. Funded by grants from Astra International (12 sites); U K site funded by Schering Plough USA.

G4105 PROBLEMS WHICH ADDRESS HEALTH RELATED QUALITY OF LIFE (HRQOL) IN INFLAMMATORY BOWEL DISEASE (IBD). EJ Irvine, E Grace, GD Kerr, E Lyrenas, T Bolin, C O'Morain, M Korman, Q. Zhou and the International Quality of Life Study Group. Coordinating Center McMaster University, Hamilton, Ont. Background: IBD patients have impaired HRQOL compared to healthy controls when assessed by the Inflammatory Bowel Disease Questionnaire (IBDQ), a validated index to detect changes in health status over time or after treatment. Aim: To determine the frequency of physical, emotional and social problems in IBD patients in other cultural settings and identify whether the IBDQ has missed important HRQOL problems. Methods: 150 problems compiled from HRQOL indices, guided interviews with IBD patients or clinicians were translated and administered to 1904 subjects in 13 countries (Northern and Western Europe, North America, Australia). Problems were reported (Yes/No) as experienced due to IBD and rated on a 5 point Likert scale from 1-"does not bother you much" to 5-"bothers you very much indeed". Problems experienced by>50% of respondents were ranked and compared (chi-square) or by mean impact (range 0 to 5; by ANOVA) between Crohn's disease (CD) and ulcerative (UC) or indeterminate colitis (IC). Results: 851 patients with UC/IC and 1053 with CD participated. Of the 49 most prevalent problems, those identified most frequently were in Bowel (frequent BM (82%) or loose BM (81%)), Systemic (fatigue (83%)) and Emotional (worry about relapse (79%) or anxiety(73%)) dimensions. Social impairment (avoiding events without toilets (58%)) was much less frequent. CD patients identified 42 of 49 problems more frequently (p<.05) than did patients with UC/IC, with a greater mean impact in 44 of the 49 (p<.05). Patients with CD more frequently experienced fear that their children would develop CD while UC patients were more fearful of developing cancer. Generally, however, the same HRQOL problems were identified by both CD and UC. Only 3 items not included in the IBDQ occurred in >50% of subjects with mean impact scores >1.5 (pain in back, pain in joints, fear children will have IBD). There were some geographic differences in problem severity, partially explained by disease severity, with Scandinavia, United Kingdom and Ireland having lower mean impact scores than N. America, Australia and Western Europe. Conclusion: Problems with the greatest impact on HRQOL in 13 countries are already included in the IBDQ. Frequency and severity of

April 1998

lramqnology, Microbiology, and Inflamnaatory Disorders A1003

problems are greater in Crohn's disease than in UC/IC. Further exploration of geographic differences is warranted. Funded by grants from ,~stra International (12 countries); UK site by Schering Plough USA. G4106 OVEREXPRESSION OF B7-1 AND B7-2 ON LFA-1-POSlTIVE LYMPHOCYTES IN INFLAMMATORY BOWEL DISEASE. C. Isbertl, C-T Germer l, D. Albrecht 1, J.-P. Ritz l, K. Thomsen-Mund 2, D. Schuppan 2, H.J. Buhr 1 IDept. of Surgery I, 2Dept. of Gastroenterology, University Hospital Benjamin Franklin, Freie Universitiit Berlin. Purpose: The B7/CD28 pathway is essential for initiating antigen-specific T-cell activation. LFA-1 (CDlla/CD18) is required for sufficient migration into inflammatory tissue. The aim of this study was to evaluate the role of B7-1/7-2 and LFA-1 in inflammatory bowel disease (IBD). Methods: Immunohistological single and double staining (PAP/APAAP) with monoclonal antibodies against HLA I/II, CD4, CD8, CD28, B7-1, B7-2, CD1 la, CD18 (LFA-1) and CD68 were performed in tissue samples from patients with crohn's disease [CD] (n=15), ulcerative colitis [UC] (n=14), colorectal carcinoma [CA] (n=5) and FAP (n=3). Unaffected tissue was used for control [CO] (n=5). Results: The similar expression for B7-1 and B7-2 was generally much higher in UC and CD than in CA and FAP. In over 50% of the cases, B7 expression in CU could be transmurally detected in the area of the muscularis propria. In crohn's disease multinucleated gigant ceils in the granulomas express B7-1 and B7-2. Double staining showed a higher B7-11B7-2 coexpression for CD4 positive than for CD8 positive T cells. In follicular tissue, MC and CU showed an overexpression of B7-1/B7-2 in the follicle center and of CD4/CD8 T cells in the periphery. In contrast to CO, FAP and TM, LFA-l-positive leucocytes demonstrated an increased coexpression of B7-1/B7-2 in MC and CU. In CU and CD LFA-1 positive leucocytes showed a high coexpression of B7-1 and B7-2 in contrast to CD68 )ositive macrophages. PAP/APAAP B7-2/CD4 B7-2/CD8 B7-2/LFA- 1 B7-2/CD68

CU ++ + +++ ++

CD ++ + +++ ++

CA + + + +

FAP (+) (+) (+) (+)

CO (+) (+) (+) (+)

Conclusions: 1) B7-1/B7-2 are overexpressed in MC and CU. 2) The

overexpression of B7-1/7-2 on LFA-l-positive leucocytes seems to play an important role in the migration and activation of cytotoxic T lymphocytes and thus in the pathogenesis of IBD. 3) On the other hand antigenpresentation via CD68 positive macrophages is less important. G4107

NF-~: B EXPRESSION IN H. PYLORI-INFECTED GASTRIC BIOPSY SPECIMENS. H. Isomoto1), M.Miyazaki 1), Y.Mizuta 1), F.Takeshima 1), K.Murasel), K.Yamasaki 2), T.Koji -~), S.Kohno 1). 1) 2nd. Dept. of Internal Med., Nagasaki Univ. Sch. of Med., 2) Internal Med., Nagasaki Municipal Medical Center, 3)3rd. Dept. of Anatomy, Nagasaki Univ. Sch. of Med., Nagasaki, Japan. Background: Transcription factor NF-~ B plays a pivotal role in immune and

inflammatory responses via regulating mRNA expression of cytokines and adhesion molecules. The aim of this study was to determine the activation of NF-~: B (through the dissociation from I ~: B) and its localization in gastic biopsy specimens of patients with H.pylori infection. Methods: 38 patients (normal, n=6; gastric ulcer, 4; duodenal ulcer, 5; chronic gastritis, 23) were studied. At the time of endoscopy, four biopsy specimens were obtained from both the antrum and the corpus; one for the evaluation of NF-K B expression and three for assessment for H.pylori by histological examination, culture and CLO test. NF-K B expression was examined by Southwestern histochemistry (Koji et al; J Histochem Cytochem 42, 1399), where synthetic doublestranded oligo-DNA with consensus sequence for NF-~ B was labeled with digoxigenin and then reacted with frozen sections of gastric biopsy specimens. NF-K B bound to the probe was visualized by immunohistochemistry. Some of the H.pylori-positive patients were treated with eradication therapy. Results: Activated NF-K B mainly localized in nuclei of gastric epithelial cells. Based upon percentages of the ceils positive for NF-t: B, we categolized into four grades -, +, ++ and +++, corresponding to <5%, 5-25%, 25-50% and >50%, respectively. In H.pylori-infected antral biopsy specimens (n=19), 2 specimens were -, 2 were +, 11 were ++ and 4 were +++, while 13 were -, 4 were + and 2 were ++ in H.pylori-negative antral specimens (n=19). Thus, the grades of NF-~: B was significantly increased in H.pylori-infected antral mucosa compared with that in H.pylorinegative(p<0.01). Activation of NF-t: B was also evident in H.pylori-infected corpus. Moreover, in the biopsy specimens of H.pylori-eradicated patients the grades of NF-): B were sabstantially decreased. Condusions: NF-~: B activation was evident in gastric epithelial cells in the biopsy specimens infected with H.pylori, and it is indicated that NF-K B is involved in the pathogenesis of immune and inflammatory responses to H.pyloriin the gastic mucosa.

• G4108

IL-10 REGULATES AUTOREACTIVE T CELL ACTIVITY IN COLITISPRONE MICE. Komei It0 and Peter B. Ernst. Department of Pediatrics and Microbiology & Immunology, University of Texas Medical Branch, Galveston, TX 77555-0366. Back~,round: Absence of the IL-10 gene in mice results in chronic colitis. Previous studies have shown that the presence of luminal bacteria triggers colitis since housing mice in germ free conditions prevents the disease. Antimicrobial therapy may attenuate but not abrogate colitis suggesting endogenous host antigens continue to stimulate sensitized T cells. We hypothesized that bacterial antigens could induce autoreactive T ceils that may contribute to inflammation in the colon. Methods.: Splenocytes from normal or IL-10-deficient (KO) mice were given a primary stimulation in vitro with Staphylococcal enterotoxin B (SEB) for 3 days. After resting for 4 days, the responder ceils were exposed to a secondary stimulation with mitomycin-C treated syngeneic feeder cells for 3 days. Proliferative and cytokine responses were measured after both primary and secondary stimulation. Results: Splenic T cells from IL-10 KO mice proliferated in response to syngeneic feeder cells alone. Unlike the T cells from IL-10 KO mice, spleen cells from control C57BL/10 or 129/SvEv mice treated with media alone did not proliferate in response to syngeneic cells. Spleen cells from IL-10 KO mice had greater proliferation and IL-2 responses after priming with SEB. In addition, responder cells from IL-10 KO or control mice proliferated following a secondary exposure to syngeneic feeder ceils. Administration of IL-10 in the primary or the secondary culture inhibited the secondary autoreactive response in both IL-10 KO and control cells. The specificity of IL-10 activity was confirmed by using an anti-IL-10 receptor antibody (1B1.2), which reversed the suppressive effect of IL-10. To determine if this response was truly associated with autoreactive T cells, mice lacking specific T cell receptors recognizing self antigens were stimulated with SEB. For these experiments, CBA/J mice were used since T cells with the Vb6 or Vb8 T cell receptor that recognize the Mls- 1 viral superantigen are almost entirely deleted because Mls-1 is expressed in these mice. SEB-primed T cells from CBA/J mice proliferated upon secondary exposure to syngeneic feeder cells. Analysis of the T cell receptor in the proliferating cells showed an increase in Vb8+CD4÷ T cells as well as Vb6+CD4÷ T cells. Depletion of CD8÷ cells before priming did not affect the secondary response. Furthermore, the secondary response was blocked by antibodies recognizing Class II MHC. Conclusion: T cells from IL-10 deficient mice tend to be autoreactive while IL-10 can inhibit autoreactive T cells that are induced in response to stimulation with microbial antigens. These data suggest that IL-10 plays a protective role in maintaining self tolerance. IL-10 was kindly provided by Schering-Plough Research Institute. G4109 DIVERSITY OF VACA-FEPC INTERGENIC REGION AND rfaJ GENE OF HELICOBACTER PYLORI ISOLATED IN JAPAN. Y. It0, T. Azuma, S. Ito, H.Suto, H. Miyaji, Y. Yamazaki, M. Kuriyama, Y. Kohli, Y. Keida Fukui Medical University, Fukui, Aiseikai Yamashina Hospital, Kyoto, Okinawa Chubu Hospital, Okinawa, JAPAN.

Background and Aim: Our recent study revealed that the rfaJ gene was inserted into thevacA-JepC intergenic region of H. pylori F37 and contained polyC tract (C13) resulting in prematured termination of RfaJ. To investigate the diversitry of the vacA-fepC intergenic region and rfitJ in Japanese clinical isolates, PCR amplification and direct sequencing were carried out.

Materials and Methods: One hundred and forty-two Japanese isolates from two different area (87 in Fukui and 55 in Okinawa) were included in this study. First, vacA-fepC intergenic region was amplified by PCR to investigate the frequency of insertion. Second, the rfaJ specific primer set was designed to amplify this gene which was not inserted in the vacA-fepC intergenic region. Third, PCR direct sequenceing of the rfaJ gene was performed in 8 Japanese strains. Results: Inserted sequences in vacA-fepC intergenic region were identified in 10 (6:is 605 and 4:rfaJ) of 87 Fukui strains and in 11 (1:is605 and 10:ribJ) of 55 Okinawan strains. Possession of inserted sequence in the vacA-fepC intergenic region was not associated with vacuolating cytotoxin activity or clinical outcome of infected patients. DNA sequencing of the rfaJ gene inserted into vacA-fepC intergenic region from 5 strains revealed the length of polyC tract differed among strains (C7-C14) and all RfaJ examined were truncated because of point mutation. The rfaJ gene from 68 (78.2%) of 87 Fukui strains and 27 (73.0%) of 37 Okinawan strains could be amplified using rfaJ specific primers. Sequencing of the rfaJ from three strains which do not have inserted sequence in the vacA-fepC intergenic region revealed that such strains possess rfaJ containing AG repeat within this gene. The length of AG repeat differed among strains as well as that of polyC tract. Conclusions: We identified two kinds of inserted sequences (is605 and rfaJ) in the vacA-fepC intergenic region. More than at least 70% of isolates examined had rfaJ in the chromosome. The rfaJ gene of these strains contained the different length of polyC tract or AG repeat.