Searching for measles virus in Crohn's disease

A936 AGA ABSTRACTS • G3834

INTESTINAL EPITHELIAL CELL DIFFERENTIATION IMPAIRS CHEMOKINE EXPRESSION INDUCED BY THE INTERLEUKIN-1 SIGNALLING PATHWAY. U. Bticker, C. Jobin, L. Holt, L. Licato, D. Brenner, R.B. Sartor. Center for GI Biology and Disease, Univ. of North Carolina, Chapel Hill. Background: Intestinal epithelial cells (IEC) differentiate morphologically

and functionally as they migrate from the crypt to the villus. Recent studies indicate that cellular differentiation alters the immunoregulatory potential of IEC. This study analysed the mechanisms of a differential proinflammatory cytokine response in undifferentiated vs differentiated HT-29 cells. Methods: Parental (HT-29/p) and Methotrexate-differentiated HT-29/ MTX10-3 cells (provided by Dr. T. Lesuffleur, INSERM u17g, France) were stimulated with IL-113, TNF-~ or PMA. Dose-dependent secretion of IL-g was assessed by ELISA, degradation of IKB~ by Westem blot analysis, activation of transcription factors by Jun kinase assay and EMSA, and IL-8 gene expression by Northern blot analysis. Transient cotransfection with an expression vector for IL-1RI and a reporter gene for the IL-8 promoter (gift from Immunex, Seattle and Dr. G. Wu, Philadelphia) was achieved by lipotransfection, and promoter activity was assessed by luciferase assaY. IL-1 receptor density was determined by radiobinding of t25I-IL-l[3. Results: Differentiated HT-29/MTX10-3 cells stimulated with IL-I[~ secreted 80% less IL-8 than HT-29/p cells, while no significant differences were found with induction by TNF-ct or PMA. IKB~ was not degraded in either cell line following IL-113 stimulation, but NF-KB binding activity was only increased 1.5fold in IL-113 stimulated HT-29/MTX10-3 compared to a 9fold increase in HT-29/p cells. NF-~:B activation was comparable when TNF-~t was used as a stimulus. Furthermore JNK activation and IL-g gene expression were greatly reduced in IL-113 stimulated HT-29/MTX10-3. Although HT-29/MTX10-3 cells contained increased intracellular IL-1 receptor antagonist (IL-1Ra) type I, receptor blockade by secreted extracellular IL-1Ra was not responsible for differences since pretreatment with anti-IL-1Ra did not increase IL-8 secretion. Transfection with an IL-1R expression vector restored the IL-113 stimulated IL-8 promoter activity in HT-29/MTX10-3 cells to the level found in HT-29/p, suggesting differences of IL-1R density and/or function between the two cell lines. However, binding of l~I-IL-1 [3was comparable in both cell lines. Conclusion: Permanent cellular differentiation of HT-29 cells selectively impairs the IL-1 signaling pathway. This alteration may explain 1. the perpetuation of intestinal inflammation in conditions where high epithelial cell turnover leads to an increased number of undifferentiated cells and 2. indicate a potential mode of pharmacological action of Methotrexate in IBD. • G3835 INCREASE IN TGF-131 LEVELS IN RESPONSE TO ANTIINFLAMMATORY THERAPY IN ULCERATIVE COLITIS. Boerr L. Sambuelli A, Sugay E, Felstiner D, Negreira S, Gil A, Camartino G, Torres A, Huemos S, Kogan Z, Cavanne A, Graziano A, Bosisio O, Lumi M, Masciangelli J, Gualdrini H, Gutierrez A, Valero J, Cavanne A, Sautd, Gonzalez O, Doldfin I, Diez R. Hospital B. Udaondo. Buenos Aires - Argentina. In recent years, Interferon "/ (IFN-~/) and Transforming Growth Factor 131 (TGF-131) have arisen as the major mediators of TH1 and TH2 patterns of immune response in the gastrointestinal mucosa. The aim of this study was to determine the involvement of TGF-131 and IFN in untreated active ulcerative colitis (UC) and to investigate their changes during treatment. M A T E R I A L A N D M E T H O D S : 56 determinations of serum TG131 were performed in 20 patients with active UC (10 left-sided and 10 extensive colitis) mean age 38 yrs (range 35-66) and 19 healthy controls in a prospective longitudinal six-week study. Disease activity was scored by a clinical and endoscopic 12-point index (DAI, Sandbom et al, Gastroenterology 1994; 106:1429-1435). Severe activity was defined as DAI scores > 9 (at entry, 9 patients) and mild to moderate disease as scores 3-8 (at entry, 11 patients). Serum TG[31 concentrations were measured by ELISA (GENZYME) at baseline, on the 7m and 42 aa days of standard treatment (severe: 7 days intravenous steroids; mild to moderate: sulfasalazine; oral prednisone was added when required after the first week; 3 severe patients needed surgery soon after 7th day. Improvement (responder patients) on the 7th day was defined by a decrease in the DAI of at least 3 points. R E S U L T S : (ng/ml, X ± SEM).: Basal levels of TGI31 were significantly increased in pretreatment UC (n=20, DAI: 8.2 ± 0.4) compared with controls (58.8 -+ 3.3 ng/ml vs 48.6± 2.5 ng/ml, p<0,02, Mann Withney). After 1 week two patterns of variation were found: responder patients (decrease in DAI _> 3, n=8) increased TGF-131 levels (64.6 ± 5.0 ng/ml) significantly compared to their basal value (52.0 ± 4.3, p<0.02:, Wilcoxon signed test). On the contrary, unresponsive patients (n=12, Basal TGF-[31 values of 58.7 ± 4.5 ng/ml did not show any clear pattern of variation (TGF-131 at 7~ 56.0 ± 3.9 ng/ml=NS). Variation in TGF-131 levels and DAI were significantly correlated (Spearman -0.6835, p 0.001). On 6th week of treatment, responder patients (n=3, DAI < 2 vs. DAI>3) had normal values of TGF-131 (48.8 ± 5.03 ng/ml). On the contrary, non-responders (n=16, DAI>3 had deceased values of TGF-131 (43.3 ± 3.9 ng/ml, p=0.06 vs. responders) IFN-y values were difficult to understand: it was undetectable in normal controls but very heterogeneous in patients before treatment (7.5 ± 2.5 ng/ml) and after treatment (18.6+6.1 INTRODUCTION:

GASTROENTEROLOGYVol. 114, No. 4 ng/ml on the 7th day). No difference between corticoids and sulfasalazine was observed. C O N C L U S I O N : These results are consistent with a pathogenic role of TGF-131 and IFN-T in ulcerative colitis, at least in some degree. Further studies are required to determine whether changes in these cytokines reflect the attempt of the organism to cope with the disease activity or are responsible for production of the lesions. G3836

SERUM TGF-131 AND INTERFERON-y LEVELS IN ILEAL POUCHANAL ANASTOMOSIS IN ULCERATIVE COLITIS. Boerr L, Sambuelli A, Sugay E, Felstiner D, Negreira S, Gil A, Camartino G, Torres A, Huemos S, Kogan Z, Cavanne A, Graziano A, Bosisio O, Lumi M, Gualdrini H, Masciangelli J, Gutierrez A, Valero J, Gonzalez O, Dold~in I, Diez R. Hospital B. Udaondo. Buenos Aires - Argentina. Mechanisms of the physiological and pathological changes of pouch ileal mucosal of ileal pouch-anal anastomosis (IPAA) in ulcerative colitis (UC), remains to be elucidated. A subset of these operated patients are affected by acute inflammatory changes in the pouch ("pouchitis"). As it has been proposed in UC, TH1/TH2 cells dichotomy producing Interferon "t (IFN-T) and Transforming Growth Factor 131 (TGF-131) could be important in the pouchitis pathogenesis. Conversely a chemotactic activity for neutrophils described for TGF-131 could be relevant. On the other hand, an heterogeneous pattern of adaptative histological response is observed in the IPAA and a role of TGF-131 in the chronic adaptative changes has been recently suggested. A I M S : 1) to investigate serum concentrations of TGF-I31 and IFN-y in UC patients who underwent IPAA, compared with healthy controls and with untreated active UC, 2) to compare levels of these cytokines in IPAA with and without acute pouchitis and to correlate these levels with a pouchitis activity index, 3) to investigate whether levels of these cytokines are in relation with the adaptative response of the IPAA. M A T E R I A L A N D M E T H O D S : 29 IPAA (mean evolution time from ileostomy closure 4 ys), 22 UC and 19 healthy controls were included. Serum TGF-131 and IFN-y were measured by ELISA (GEMZYME). Pouchitis was defined by a clinical, endoscopic and histologic index "Pouchitis Disease Activity Index" (PDAI) (Sandbom, Gastroenterology 1994;107:1856-60). Chronic adaptative response assessed according to validated scores (Moskowitz RL Int J Color Dis 1986;1:167-74) consisted of chronic infiltration and villous atrophy. R E S U L T S : ( X ± SEM) INTRODUCTION:

IPAA UC Controls TGF-131 ng/ml 68.6 ± 6.1 (N=29)* 59.3 ± 3.0 (N=22)** 48.6 ± 2.5 (N=19) IFN-)' p g / m l 31.5± 10.1 (N=22)** 7.9:1:2.5 (N=14)*** Non detectable *vs. controls = p < 0.01, vs UC = NS **vs. controls = p < 0.03, vs CU = N S *** vs. controls = p < 0.03

(Mann-Whitney)

As a trend, pouchits subgroup (N=19) have mean TGF-131 levels (73.4 ± 8.9) higher than the non-pouchitis (N=10, 59.2±3.8, p=0.06). A positive correlation was observed between PDAI and TGF-131 levels (r=-0.54,p<0.03) for pouchitis patients. As regards chronic adaptative changes a positive correlation was observed between TGF-131 levels and the degree of villous atrophy (r=-0.52, p<0.02, Spearman). IF/q-'/levels were significantly higher in pouchitis (46.6 ± 15.3) than in controls (p<0.02), but again, the difference between patients with or without acute changes did not reach statistical significance (46.5 ± 15.3 pg/ml vs 13.4 ± 10.6 p=0.19, NS). C O N C L U S I O N : our results suggest that TGF-131 and IFN-y are involved in the pathogenesis of both inflammatory acute injury (pouchitis) and adaptative changes in the IPAA, but the exact role of these cytokines is still unclear probably due to their wide variety of functional effects which may contribute both to inflammation and to tissular repair. G3837 S E A R C H I N G FOR MEASLES VIRUS IN CROHN'S DISEASE. LAR Boerr, A Sambuelli, E. Baumeister, S Negreira, A Gil, G Camartino, J Bai, A Torres, S Gon~alves, I Dold~tn, J Valero, MA Bartelini, G Srur, D Felstiner, A. Pontoriero, Kogan Z., Chertcoff A., V. Savy. Bonorino Udaondo Hospital and INEI-ANLIS "Carlos Malbr~in". Buenos Aires. Argentina.

Persistent measles virus (MV) intestinal infection has been proposed as etiological in Crohn's disease (CD). Results of serum MV-specific IgM antibody studies, MV antigen immunodetection and MV genomic sequence detection in intestinal tissues are controversial. A I M S : 1) to investigate the presence of MV genomic sequence by RT/Nested-PCR in intestinal biopsies of CD patients, 2) to investigate the presence of MV antigens in biopsies, 3) to study the serological MV-IgM antibodies prevalence in patients with CD 4) to correlate the findings with localization and activity of the disease. M A T E R I A L A N D M E T H O D S : Sera of 34 patients with CD (colonic=18, ileocolonic=12 and ileal=4) and 27 healthy controls were tested for MV-IgM by an immunofluorescence assay (IFA), removing the total IgG antibodies with goat anti IgG human serum (Bio Wittaker~), commercial slides (Bion TM) and fluorescein anti-human IgM (mu chain specific) (DAKOTM). A commercial enzyme-linked immunosorbent assay (ClarkTM) was used to detect specific MV-IgM. An RT/Nested-PCR was carded out in biopsy samples obtained from 31 CD and the viral antigens

INTRODUCTION:

Immunology, Microbiology, and Inflammatory Disorders A937

April 1998

were detected by IFA (Ag-IFA) using a monoclonal anti-MV nucleoprotein antibody in formalin fixed paraffin wax embedded tissue sections of 33 CD. Six healthy controls have been analyzed for RT/Nested PCR and 16 for antigen IFA R E S U L T S : CD Controls * p < 0.02

IgM-IFA IgM-ELISA 10/34 (29%)* 6/34 (18%)@ 1/27 (3.7%) 1/27 (3.7) @NS (Fisher Exact test)

RT/PCR Ag-IFA 3/31 (10%)@ 16/33(48%)** 0/6 (0%) 0/16 (0%) ** p < 0.003 (Chi-Square)

No relation with the activity or localization of the disease was found. CONCLUSION: We detected positive serum and tissular findings for the measles virus by serum antibodies, genomic sequence and viral antigen tissular detection with variable prevalence according to the method used. MV-IgM IFA and MV antigen tissular immunodetection findings have been previously reported, but these subjects are still controversial. To our knowledge no positive MV RT/PCR results have been reported but positive nucleid acid hybridization findings have. Further studies with a larger control and CD population will be necessary to relate a persistent measles infection with the etiology of Crohn's disease. G3838

IMMUNE RESPONSE TO FOOD IN CROHN'S DISEASE: I N VIVO A N D I N VITRO ASSESSMENT. B0gaerde JB 1, Kamm MA, Cahill J, Emmanuel A, Vaizey C, Knight S 2. St Mark's Hospital and 2Antigen Presenting Research Group, NPIMR London. 1Dept of Physiology, University of Pretoria, Pretoria. Background. Disease improvement in response to purified liquid diets and food exclusion in Crohn's disease suggests that food antigens perpetuate inflammation. We aimed to (i) determine if patients are sensitised to specific food antigens (ii) to determine if this is restricted to the gut and (iii) develop an in vivo test for sensitisation using laser doppler rectal blood flow as a quantitative measure. Methods. Peripheral blood lymphocyte proliferative responses were measured in response to 6 purified food antigens (cereal, cabbage, citrus, milk, yeast and peanut groups) and saline control in 10 Crohn's patients with predominantly terminal ileal disease and 10 healthy controls. Skin scratch testing and rectal exposure (flexible sigmoidoscopy) to the same antigens were then assessed in a double blind study, by immediate and 3.5 hour laser doppler blood flow, and rectal biopsies taken from each antigen site. Results. Peripheral blood lymphocyte proliferation was observed in 32/60 allergen/lymphocyte combinations in patients and 8/60 controls (p<0.0001). The Crohn's patient group demonstrated abnormal rectal blood flow response to yeast (p=0.036) and citms fruits (p=0.038). Individual Crohn's patients showed an abnormal response to all antigens: rectal blood flow increased in 24/60 patient and 6/60 control antigen sites (p<0.0001) after 3.5 hours. Skin blood flow changes in response to food allergens were not observed in patients or controls. Conclusion. Crohn's patients demonstrate in vitro and in vivo sensitisation to food antigens, which may play a part in perpetuating the inflammatory process. The lack of skin response demonstrates that this immune activation is gut specific. Laser doppler rectal fiowmetry may allow the rapid identification of specific antigens. Yeast and citrus fruits may play a particularly important role, although patients responded abnormally to all food antigen groups. This is the first in vivo technique described for assessing the immune response to food in patients with Crohn's disease. G3839

IMMUNE SENSITIZATION TO FOOD AND BACTERIA IN CROHN'S DISEASE. Bo~aerde JB.* Kamm MA, Knight S**. St Mark's Hospital and **Antigen Presentation Research Group, NPIMR London. *Dept of Physiology, University of Pretoria, Pretoria. Background. Diets whose sole protein source consists of amino acids or simple proteins are therapeutically beneficial in Crohn's disease, suggesting that complex food proteins may contribute to inflammation. Enteric bacteria are an alternative antigenic source. The presence of oligoclonal T cell populations in Crohn's disease further supports the hypothesis that antigens drive the inflammatory process. We have assessed the prevalence and magnitude of lymphocyte priming to food and bacterial antigens in patients with Crohn's disease and healthy controls. Methods. Thirty-one Crohn's disease patients (mean age 46, range 23 to 73), and 22 healthy controls (mean age 33, range 25 to 45) were studied. Peripheral blood lymphocytes were collected, and incubated with antigens in hanging drop culture for 4 days. Antigens tested were cows milk, cereals, cabbage group, citrus group, peanut group, Saccharomyces (yeast), Bacteroides, E. coli, and Klebsiella. On the fourth day 3H-thymidine incorporation was measured after a 4 hour pulse. Responses to allergens were considered positive if mean proliferative values were above the 99% confidence interval for background proliferation.

Results. Mean background and mitogen stimulated proliferation did not differ between patients and controls. Mean proliferation to antigens was not above background in the control group, but in Crohn's patients proliferative responses to all food and bacterial antigens were significantly higher than background values. Twenty-three of 31 Crohn's patients and five of 21 controls responded to one or more food or bacterial allergens (p = 0.003). Two controls, and 16 Crohn's patients responded to 4 or more allergens (p = 0.0025). Conclusion. Peripheral

lymphocytes of Crohn's disease patients are abnormally sensitised to food and bacterial antigens. These sensitised lymphocytes may be involved in perpetuating the inflammatory process. This is the first demonstration of an immune basis to the clinical effects of food in Crohn's disease. G3840

A NEW MURINE H E L I C O B A C T E R SPECIES. U. R. M. Bohrl§, S. Thoma2§, K. Bonhagen2, G. Adler l, B. Glasbrenner l, H. Meyer3, J. Reimann2, I Department of Internal Medicine I, 2 Institute for Medical Microbiology and Immunology, 3 Laboratory Animal Research Unit, University of Ulm, Germany § authors contributed equally. We isolated a new Helicobacter species from the got of immunodeficient mice using a new PCR screening method for Helicobacter species. Culturing of this unknown Helicobacter species was possible under anaerobic conditions at 37°C, 42°C, and 25°C, but not under microaerophilic conditions. The morphology of the isolates showed smooth, slightly curved rods with a single flagellum at each end. Comparison of the 16S-rRNA sequence with related bacteria indicated that this sequence was most closely related to the Helicobacter hepaticus sequence with a similarity of 96.2%. The results of culture growth conditions, morphology, biochemical tests and 16S-rRNA sequencing showed that this isolate represents a novel Helicobacter species. A definite name for this bacterium will be proposed at the meeting. G3841 MUCOSAL IMMUNE DEPENDENCY ON CD28 CO-STIMULATION.

David Boone. Themistocles Dassopoulos, James Lodolce, Sophia Chai, Shanthi Trettin and Averil Ma. Dept of Medicine, Committee on Immunology, Univ. of Chicago, Chicago, IL. BACKGROUND: During the activation of mature T lymphocytes, multiple signals which provide stimulatory or inhibitory growth signals and pro- or anti-apoptotic survival signals regulate the differentiation, proliferation and survival of responding T cells. In the intestinal mucosa, lymphocytes appear to exist predominantly in an activated state, a condition seen only transiently in the periphery. The signals which maintain this state of chronic activation are yet to be determined. To begin to dissect the novel condition of stably activated cells in the intestinal mucosa, we have examined the role of the costimulatory molecule CD28 in maintaining the intestinal lymphoid compartment. METHODS: Thymocytes, peripheral lymph nodes, mesenteric lymph nodes, Peyer's patch lymphocytes (PPLs), lamina propria lymphocytes (LPLs), and intraepithelial lymphocytes (IELs) were isolated from CD28 deficient (CD284-) and normal mice. Single cell suspensions were prepared from all tissues, quantitated, incubated with monoclonal antibodies specific for B and T lymphocyte specific markers and analyzed by flow cytometry. Lamina propria lymphocytes from CD28-/- and normal mice were also cultured with interleukin-2 or interleukin 15. T ceils were quantitated by staining cultures with T cell specific markers and flow cytometry RESULTS: Remarkably, CD284- mice possess negligible Peyer's patch (PP) lymphocytes. Both T and B lymphocyte compartments are reduced over 10 fold when compared to normal B6 littermates. Despite this profound reduction in PP lymphocyte numbers, lamina propria lymphocytes (LPLs) are present in essentially normal numbers--although they display lower levels of activation markers. Finally, CD28-/- LPLs respond normally to both interleukin-2 and interleukin-15. CONCLUSION: CD28 is essential for the maintenance of PP but not LPLs, mesenteric lymph nodes, or other peripheral lymphoid compartments. Furthermore, CD28 is not required for the proliferative and/or survival effects of IL-2 and IL-15 upon LPLs. These findings have significant implications for the normal activation sequence of mucosal lymphocytes.