Prediction of relapse after treatment with infliximab in patients with Crohn's disease by increased peripheral production of TNF-α and mucosal levels of activated NFκB

Abstracts

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65 DISTINCT CYTOKINE PATTERNS DIFFERENTIATE EARLY VS. LATE COLITIS IN IL-IO-DEFICIENT MICE Spencer, D.M., Levine. A.D. Case Western Reserve University School of Medicine, Cleveland, OH, USA Background: We reported previously that local synthesis of IL-12 and IFN-y by lamina propria mononuclear cells (LPMC) is elevated during the early phase (1 l-22 weeks) of the spontaneous colitis that develops in mice genetically deficient in IL-lo. Neutralizing IL-12 in viva is effective in preventing and reversing early disease. Yet, production of both cytokines precipitously decreases at 24 weeks of age and remains low during the late phase of this disease (24-40 weeks). Aims: The absence of mucosal IL-12 and IFN-y synthesis during the late phase of colitis raises three specific questions about the immunological mediators that initiate and maintain chronic intestinal inflammation in this model: (1) What stimulates the synthesis of IL-12 in early disease? (2) Is antiIL-12 therapy effective during the chronic phase of inflammation? (3) What cytokines are expressed preferentially during the late phase of colitis? Methods and Results: Activating LPMC from the mucosa of mice with early and late disease through the CD40 receptor stimulated a 3-fold increase in IL-12 synthesis over unstimulated levels. Yet, activation through CD40 induced an SO-fold increase in IFN-y production by LPMC from inflamed tissue of mice only with early disease, far greater than the levels predicted by the threefold increase in IL-12. Treatment of 35-40 week old IL-lo”’ mice with either anti-IL-12 mAb or rat IgG had no effect on weight, fecal blood, stool consistency, and inflammatory infiltration of colonic mucosa and submucosa. This lack of response correlated with decreased spontaneous IL-12 and IFN-y production by LPMC observed in mice with late disease. Colonic expression of mRNA for IL-la, IL-lp, and IL-18 increased 2-3-fold at 24-29 weeks, during late disease. IL-Ra mRNA levels increased starting at 32-35 weeks. Conclusion: These results suggest that local syntbesls of IL-12 is induced by CD40 engagement in early colitis and enhanced by the marked elevation in IFN-y production. The dramatic stimulation in IFN-y synthesis by anti-CD40 in inflamed tissue indicates that its regulation may be dependent on expression or signaling through the IL12 receptor. The lack of efficacy of anti-IL-12 antibody treatment during late disease suggests that distinct therapeutic approaches may need to be developed for early vs. late intestinal inflammation, possibly focused on IL- 1 or IL- 18 during chronic IBD.

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66 CHARACTERIZATIONOF SINGLE NUCLEOTIDE POLYMORPHISM (St@) HAPLOTYPESOF IL4 RECEPTOR c( GENE IN INFLAMMATORYBOWEL DISEASE.Zhana WJ, TilbergAF, Fan R and Koltun WA. Milton Hershey Medical Center,PennsylvaniaStateUniversityCollegeof Medicine,Hershey,PA, USA. Background: Present paradigmson the pathcgenesisof IBD suggest it to be an immunemediateddiseaseoccurringin a geneticallysusceptibleindividual.lnterleukin 4 (114)plays a cwcial regulatoryrole in Th2 and perhapsalso Thl cell responsesano has been shown to down-regulateRe inflammatoryresponsein IBD. IL4 achievesits multifunctionaleffects by binding to and signaling through a receptor complex consistingof the lL4Ru chainand the commony chain.The IL4R.Agene is locatedon chromosome16, within the IBD susceptibilitylocus 1 @Of) and the gene is highly polymorphicwith 12 codingSNPs identified.Aims: This study aimedto characterize2 IURA SNPs @dons 400 and 576) and 2.locusSNP haplotypasin order to establish possible associationsbetween lL4RA SNP haplotypesand IBD. Methods: Using sequencespecificprimersand PCR (SSP-PCR)technique,we initiallygenotyped64 individuals from 13 Caucasoid IBD families of European descent, including 41 unaffectedfamily membersand 23 IBD patients (13 Crohn’s disease [CD] and 10 ulcerativecolitis[UC]).Of 6 SNPscausingaminoacid substitutions,2 SNPs were first investigatedat IL4RAcodons 400 and 576. Two-locusSNP haplotypes(lL4RA:400576) were obtainedby analyzingfamily pedigrees.Linkageand associationanalyses were carded out using genetic analysistest TDT (transmissiondisequilibriumtest) Results: Four distinct 2.locus lL4RA SNP haplotypeswere revealedfrom a total of 126 haplotypesidentifiedfrom 64 individuals.Since our sample size was small, CD and UC were combinedin the analyses.TDT analysessuggestedthat lL4RA:Ala400. Arg576 haplotype was more preferentiallytransmitted to unaffected offsprings (P=O.O25).lL4RA:Glu4CQ-Gln576 haplotype, although statistically not significant, suggesteda bansmissiontrend to affectedIBD offsprings(6 trans vs. 4 non-trans,x* q 1.3) and a simulationby increasingthe sample size by 3 folds would reach a significantassociation(simulationx2 q 3.9, PsO.05).Conclusions: Our preliminary haplotypingdata for lL4R.ASNPs showed a skewed transmissionof IL4RA:Ala400Arg576haplotypeto unaffectedoffspringssuggestingpossibleprotectiverole against IBD. The susceptibilitymle of lL4RA:Glu4CO-Gln576 haplotype to IBD could be establishedwhen a 3-fold or larger sample size were studied.As both IURA SNP haplotypesare common in the population,we favor the hypothesisthat common disease-commonvariant(CD-CV)may be a mechanismin diseaseexpressionof IBD, a situation similar to that of common APOM allele associated with Alzheimeis disease.Furthercharacterizationof such 2-locushaplotypesin a larger sample size of IBD familiesis currentlyunderinvestigation.

PREDICTION OF RELAPSE AFTER TREATMENT WITH INFLIXIMAB IN PATIENTS WITH CROHN’S DISEASE BY INCREASED PERIPHERAL PRODUCTION OF TNF-a AND MUCOSAL LEVELS OF ACTIVATED NFKB S. Nikolaus*, T. Kiihbacher, U.R. F6lsch*, A. Raedler” and S. Schreiber*. I” Med. Dept. Christian Albrechts University Hospital, Kiel’, and Tabea IBD Center, Hamburg’, Germany. Backeround: TNFa is increased in active CD and muwsal NFKB, a major player in pro-intlammatcxy signal tmnsduction, is activated. Iofliximab (Remicade*‘M)bids to TNFc( and results in remission rates of up to 50% in chronic active CD (4 wks after single infusion). bltervals for (re) infusion of infliximab which are necessary to avoid relapses are unknown. Aims: To find predictors of relapse in intlii treated CD patients. Metkcds: 24 pts with active CD (CDAI>200, 18-50 yrs, oxtosigmoidal lesions) were recruited from 35 consecutive patients treated by a single infusion with infliximab (5mgikg). Patients had received steroids/iunosupressives in the past without success. Rectal biopsies were taken before, 1,2,4,8 and 12 wks a&x infusion. Nuclear extracts were prepared for detection of activated NFti-p65 (westem blot). Blood was drawn for whole blood cytokine assay (LPS stimulated). After 24hrs ‘INF-a was assessed by ELISA. Results: 21 of 24 patients were in remission (CDAI 50 points to >I50 in comparison with wkl) were seen in 6/21 pts (29%) at wk 4,13nl pts(62%)atwk8,37~1pts(8l%)atwk12and19~1pts(900/0)atwk16.Patients who relapsed, showed an signiticant increase of stimulated TNFa whole blood cytokine levels and increased levels of activated mucosal NFti-p65 at least 24 wks prior to clinical and endoscopic relapse. Conelusions: Treatment with ifXximab reverts the pro-inflammatory immune regulatory imbalance. Short term remission (one week) is seen in 88% oftmated patients although duration of response is highly variable. Immune activation in the mucosa (i.e. activation of NFKB-p65) as well as in the periphery (production of TNF-cz) can predict clinical relapse.