Transfer of CD4+ CD45RBhigh T-cells into TCB β×δ double deficient mice induces chronic colitis

injected mice compared to mutant mice receivang PBS (8.50-+2.50 vs 0.17• for CD4+CD45RB~O vs. PBS-injected [3 q x 8-~ mice; p < 0 0005). The chronic gut inflammation observed in the T-cell injected ]3 -i. x 8 / mice correlated with large and significant increases in mRNA levels of TNF-a and IFN-~, compared to their PBS-injected counterparts (6 and 16-fold increases, respectively, when normalized for GAPDH mRNA expression). Summary and Conclusions: PBS-injected TCR ~ / x B"~"mice do not develop colitis when maintained under SPF conditions. However, these same IEL-deficient mice develop moderate to severe colitis at 8-10 weeks following injections with naive CD4+CD45RBh~ghT-cells. Our data suggests that IELs may represent an important regulatory cell population that prevents or limits the development of chronic colitis. (Supported by the Broad Medical Research Program).

Ml160

A novel molecular mechanism of action for azathioprine in IBD: 6-Thio-GTP induces apoptosis of lamina propria T cells via blockade of the GTPase Racl Imke Tiede, Daniela Poppe, Susanne Strand, Dennis Strand, Stefan Wirtz, Christoph Becker, Richard Biumberg, Heiko Iven, Reza Mohammad Ahmadian, Peter R. Galle, Markus F. Neurath Azathioprina and its metabolite 6-mercaptopurine (6-MP) are immuuosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohns disease. However, their molecular mechanism of action is un known. We found that azathioprine, 6-MP and 6-thioguanine induced apoptosis of T cells from patients with Crohns disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac 1 activation via b in drag of azathiopnne generated 6-ThioGTP to Racl instead of GTP. Binding of 6-Thio-GTP to Racl was associated with a strongly reduced exchange rate of hydrolyzed Thio-GDP suggesting that long-term treatment with azathioprine leads to accumulation of G DP-I oaded inactive Racl molecules in T cells. Interestingly, the activation of another GTPase, Ras, was not affected by azathioprine suggesting that the specificity of this drug for Rat1 is mediated by interaction of the sulfur group of 6-Thio-GTP with the GT Pase backbone. This prediction was supported by three dimensional modelling and analysis of van der Waal interaction energys between various GTPases and 6-ThioGTP. The activation of Racl target genes such as MEK, NF-kappaB and bd-xL was suppressed by aza thioprine leading to a mitochondnat pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Racl activity. This idea was supported by clinical data showing that azathioprine respons iveness in IBD pat ients is associated with a significant increase in the number of apoptotic T cells in the lamina propria, whereas non-responders showed no significant changes as compared to control patients. These findings explain the immunosuppressive effects o f azathioprine and suggest that 6-Thio-GTP denvates may be useful as potent novel immunosuppressive agents in autoimmune diseases and organ transplantation.

Ml163 Cross-reactivity between Mycobacterium Avium Paratuberculosis and Human Intestinal Antigens Characterizes Crohn's Disease Dimitrios Polymeros, Dimitrios P. Bogdanos, Ajeya Shetty, Roser Vega, Cinzia Papadia, Diego Vergani, Alastair Forbes

Background: Humoral responses to Mycobacterium avium, subspecies paratuberculosis (MAP) antigens have been frequently found in patients with Crohn's disease(CD). Objective: To investigate the role of molecular mimicry and immunological cross-reactivity between MAP antigens and human intestinal antigens. Materials and methods: The "BLAST 2 sequences" computer program was used to compare the amino acid sequences of 89 MAP and 56 human intestinal proteins deposited in the SWISS-PROT protein database. Twenty three pairs of 15-meric biotinylated peptidyl sequences from 3 MAP antigens (including hsp65) showing high degree of similarity to sequences from 21 human intestinal proteins were selected for synthesis (Mimotopes, Clayton, Australia). Anti-MAP/human antibody responses were tested by ELISA in serum samples from 50 patients with CD (mean age: 40.9 yr), 50 patients with ulcerative colitis (UC) (mean age: 49.4 yr) as pathological controls, and 38 healthy controls (mean age: 34.7 yr). Results: Antibody responses were present in 7 out of 23 MAP/human pairs. Double reactivity to at least 1 MAP/human pair was found in 21/50 (42%) patients with CD, but in only 2/50 (4%) patients with UC (p< 0.00001) and in 3/38 (7.9%) normal controls (p< 0.001). Double reactivity to MAP gsd:302~4/ human glutathione peroxidasem.us was found in 15/50 (30%) CD patients, in 2/50 (4%) UC patients (p<0.0001) and 1/38 (2.6%) normal controls (p = 0.025). Double reactivity to MAP alkylohydroperoxidase C20.3+/ human TOG637.~5~protein was found in 10/50 (20%) CD patients, compared to 0/50 (0%) patients with UC (p= 0.008). Simultaneous reactivity to homologous MAP/ human sequences was cross-reactive as confirmed by inhibition studies. Conclusions: This is the first report demonstrating the presence of cross-reactive humoral responses to homologous Mycobacterium avium paratuberculosis and human intestinal antigens in Crohn's disease. The biological significance of these findings needs further investigation.

Ml161 Lactobacillus Casei Can Overcome Resistance to Apoptosis in Lymphocytes from Patients with Crohn's Disease Monica Carol, Natalia Borruel, Maria Antolin, Francesc Casellas, Francisco Guamer, Juan R. Malagelada

Background: Crohn's disease (CD) is characterized by mucosal infiltration by activated T lymphocytes resistant to apoptosis. The resistance appears to be associated with an imbalance in the expression of Bcl-2/Bax (anti- and pro-apoptosis proteins, respectively). Increased production of IL-6 actively contributes to lymphocyte resistance to apoptosis and, in fact, blocking the synthesis of IL-6 has been suggested as a potential treatment for CD. Aim: Our aim was to study the effect of L. case/ on the resistance of CD intestinal lymphocytes to undergo apoptosis. Methods: Explants of ileal mucosa (20-30rag) surgically obtained from 12 patients with active CD were incubated for 24 h in the presence of either L. case/DN114 001 (Lc), a non pathogenic E. coli ATCC 35345 (Ec), or no bacteria (Blank). The levels of IL-6 were determined in the culture supernatants. After the incubation, intra-epithehal (IEL) and lamina propria mononuclear cells (LPMC) were isolated, and populations analyzed to characterize lymphocyte phenotype (CD3, CD4, CD8). The percentage of LPMC positive for Bcl-2 and Bax and mean fluorescence intensity was determined by flow cytometry. Apoptosis was measured by the TUNEL technique and analysed by a laser scan cytometer in LPL. Data are median (range) or mean+/-sem Results: In Crohn's disease, incubation with L casei significantly decreased the IEL population (CD3 +) (Blank: 5.4(1.6-129), Ec: 58(1.1-141), Lc: 39(09-7), Blank vs. Lc p(0.05) and in LPMC (Blank: 4.6(1.%14), Ec: 5.7(1.1-11 8), Lc: 4.3(09-8.9), Ec vs. Lc p<0.05). The Lc induced decrease was associated with a significant increase in the percentage of apoptotic LPL (Blank: 4.6 +/- 1.5, Ec: 5.2 +/1.7, Lc: 85+/-2.7, blank vs. Lc p(0.05). Although there was no changes in the ratio Bcl2/Bax, Bcl-2 mean fluorescence intensity significantly decreased after incubation of tissues with Lc (Blank: 152 +/-1.9, Ec:12.1 +/-2.8, Lc: 10.1 +/-2.6, Blank vs. Lc p(0.05). Furthermore, after incubation of diseased tissues with Lc, there was a significant decrease in the levels of IL-6 (Blank: 24.2+/-4.8 ng/mL, Ec: 34.8+/-56 ng/mL, Lc: 15.8+/-5.4 ng/mL, Blank vs. Lc p(0.05). Conclusion: We report a novel action of a specific Lactobacillus strain, that is capable of decreasing the resistance of Crohn's disease lymphocytes to undergo apoptosis. Supported in part by a grant from Danone Vitapole (Palaiseau, France).

Ml164

Local Expansion of Plasma-Lineage Cells Without Formation of Germinal Centers Characterizes Ulcerative Colitis Yoshio Jinno, Hamo Otain, Shiro Nakamura, Motoji Oki, Hiroshi Nagura, Nobuhide Oshitani, Takaynkimatsumoto Matsumoto, Tetsno Arakawa Abnormalities in humoral immunity are implicated in the pathogenesis of ulcerative colitis (UC). However, detailed mechanism of B cell activation in the locale remained unraveled. We attempted to clarify the immune abnormality of UC using Ki-67, one of the representative proliferation-associated antigens to reveal that it was plasma-lineage cells that proliferate actively in UC Intestinal specimens obtained at surgery from 30 patients with UC and 15 Crohn's disease (CrD) were analyzed by immunohistochemistry and immunoelectron microscopy. UC, particularly in ulcer base, was characterized by an abundance of K1-67 + small round ceils. These proliferating cells were identified mostly as CD19 +, CD20-, and CD138 + immature plasma-lineage ceils, lmmunoelectron microscopy for CD19 confirmed this. This change was specific for UC, since CrD or ishemic colitis did not show such changes even in ulcer base. Furthermore, the labehng index of Ki-67 among CD19+ ceils was positively correlated with the clinical activity of UC (the correlation coefficient =0.74, p<0.005). The present data indicated that proliferation/expansion of CD19 +, CD20-, and CD138 + immature plasma-lineage ceils take place in ulcer base of UC as one of important processes associated with the severity of UC.

Ml162

The labeling index of Ki-67 a m o n g C D19 + cells

Transfer of CD4+CD45RB b~ShT-Cells into TCR ~ x fi Double Deficient Mice Induces Chronic Colitis Dmitry V Ostanin, Stephen Laroux, Sulaiman Bharwain, Matthew Grisham

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Introduction: Recent data suggests that intraepithelial lymphocytes (IELs) may play an important role in preventing or suppressing the development of chronic gut inflammation in mice. The objective of this study was to assess the role that IELs may play in regulating the onset and/or severity of chronic colitis using TCR ]3 x 8 double-deficient (13 J x ~") mice These mice lack CD4" and CD8 § IELs as well as circulating T-cells but retain functional B-cells, NK cells, and granulocytes. Methods: Male C57B1/6 ]3 / x 8" mice were injected (ip) with either PBS or CD4-CD45RB h~OT-cells (5 x 105 cells per mouse) obtained from splenocytes of wild type donors. At 8-10 wks following injection, mice were sacrificed and pieces of colon were fixed or frozen for blinded histopathologacal analyses and mRNA determinations, respectively. Results: In contrast to the original report describing the generation of ]3 / x 8 ~ mice, we found that PBS-nijected ]3 / x 8 ~ mice did not develop chronic colitis when maintained under specific pathogen Jree (SPF) conditions. These mice did not lose body weight over the course of 8- i 0 wks whereas ]3/ x ~" mice injected with CD4" CD45RB h~g~ T-ceils lost approximately 14% of their body weight during the same time period. In addition, colon weight/length ratios for T-cell injected mutant mice were significantly greater than those for PBS injected animals (6.94 _+0.93 vs. 3.06 -+ 0.21, respectively; p = 0.005) Blinded histopathological sconng of each colon revealed moderate to severe colitis in the T-cell

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Abstracts