the existence of Helicobacter heilmannii not only in the fundic mucosa but in the lung andliver. Twelve and eighteen months after the infection, approximately 100% of infected micehad liver and pulmonary lesions. Some of the MALT lymphomas were defined as belongingto the high-grade group by the PCNA positivity. Eradication of the bacteria induced shrinkageof the tumor in the liver and the lung with apoptotic cells as well as in the stomach.Conclusion: Long-term infection with Helicobacter heilmannii in C57BL/6 mouse inducedlow- and high-grade hepatic and pulmonary MALT lymphoma as well as gastric lymphoma.Eradication of the bacteria brought about shrinkage of the hepatic and pulmonary lesionsas well as gastric MALT lymphoma.

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MALT Lymphoma Stem Cells in H. Heilmannii-Infected MiceMasahiko Nakamura, Hidenori Matsui, Tetsufumi Takahashi, Somay Y. Murayama,Shin'ichi Takahashi, Toshifumi Hibi, Kanji Tsuchimoto

Background & Aims: Although cancer and leukemia stem cells are indispensable in theinitiation and enlargement of lesions,MALT lymphoma is at first polyclonal with the persistentinfection of Helicobacter species and then changes to monoclonal with genetic aberrations.To clarify the existence of similar cell populations in MALT lymphoma, we performed ahistochemical analysis in Helicobacter heilmannii-induced gastric and hepatic MALTlymphoma. Methods: We used a Helicobacter heilmannii sample isolated from the stomachof a cynomolgus monkey andmaintained in C57BL/6mouse stomachs. Mucosal homogenateswere used to inoculate C57BL/6 mice, which were then examined over 24 months. Macro-scopic observations were carried out, and PCR analysis of the bacteria of the Helicobacterspecies was performed at intervals over the observation period. Histochemical analysis wasperformed using the gastric and hepatic MALT lymphoma with monoclonal and polyclonalantibodies against Doublecortin-like kinase (DCAMKL1), Musashi-1, and proliferating cellnuclear antigen (PCNA). In addition, the effects of eradicating the bacteria were estimatedbased on the 1-week oral administration of amoxicillin, clarithromycin, and lansoprazole.Results: DCAMKL1 and Musashi-1 positivities were recognized in the lymphocytes locatedin the marginal zone of theMALT lymphoma both in the gastric and hepatic MALT lymphomatissues. PCNA-positive cells showed a similar distribution in the lymphoma tissues, whilethe number of positive cells was several times higher. After the eradication of the bacilli,the number of bacteria and the size of the MALT lymphoma tissues significantly decreased,and the DCAMKL1 and musashi-positive cells were still present in the marginal zone of thelymphoma. Conclusions: MALT lymphoma stem cells were found to exist. From the perspect-ive of the histochemical analysis, the MALT lymphoma stem cells were found to exist inthe marginal zone of the lymphoma tissue, and were resistant to the ordinary eradication treat-ment.

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H. pylori Infection Induced Internalization of Duodenal Cellular Iron ExporterFPN1Kenro Hirata, Hidekazu Suzuki, Hitoshi Tsugawa, Yoshimasa Saito, Juntaro Matsuzaki,Seiichiro Fukuhara, Sawako Okada, Toshifumi Hibi

Background Iron is an essential micronutrient indispensable for diverse biological processesincluding erythropoiesis, oxidative metabolism and cellular immune responses. While ironis absorbed only via the duodenal epithelium, it is lost with exfoliation of the alimentarycanal epithelial cells. Therefore, iron metabolism is mainly controlled in the duodenum.Recently, it was reported that H. pylori infection can cause iron deficiency. The presentstudy was designed to examine the effect of H. pylori infection on the iron absorption,focusing on the duodenal iron transporter FPN1 (basolateral) and DMT1 (apical). In addition,hepcidin-25, which is a hormone produced in the liver and plays a role as a central regulatorof iron homeostasis, was also examined. Methods FPN1 and DMT1 were quantified in thecontrol and H. pylori-infected C57/BL6 mice (3 months post-inoculation) by quantitativeRT-PCR and western-blot analysis. Total protein was prepared separately, from fractions ofthe cytoplasm and cell membrane. Serum hepcidin-25 was assessed by ELISA using a specificanti-hepcidin25 IgG. Intracellular iron was evaluated by Berlin blue staining. Results In H.pylori-infected mice, no significant change was found in the mRNA expression level, however,the FPN1 protein from the membrane fraction was significantly decreased and that of thecytoplasmic fraction was significantly increased as compared with the values in the controlmice (p<0.05 and p<0.05 respectively), unlike the case for DMT1. In the H. pylori-infectedmice, 96.5±1.7% of FPN1 existed on the membrane, while in control mice, 83.6±4.4%existed in this fraction. The serum hepcidin-25 levels were similar in the H. pylori-infectedmice and control mice (51.15±1.28ng/ml, 50.58±2.29ng/ml: no significant difference). Exam-ination by Berlin blue staining showed positive staining for only inside the duodenal epithelialcells in the H. pylori-infected mice. Conclusion. Our result suggested that iron absorptionthrough the duodenum might be disrupted by H. pylori infection via FPN1 internalization.In addition, iron might accumulate inside the duodenal epithelial cells in the H. pylori-infected mice, as no significant changes in DMT1 expression were found. These findingswas considered to be one of the mechanisms of iron deficiency or duodenal ulcer inducedby H. pylori infection.

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Helicobacter pyloriRegulates RKIP Function Through cag-DependentPhosphorylation in Gastric Cancer CellsErika Moen, Sicheng Wen, Talha Anwar, Omobola Onikoyi, Devasis Chatterjee, Steven F.Moss, Talha Anwar

Background: H. pylori infection is strongly associated with gastric cancer development. H.pylori activates the transcription factors STAT3 (signal transducer and activators of transcrip-tion) and NF-κB in gastric epithelial cells, through mechanisms involving macrophage-derived IL-6. Raf kinase inhibitory protein (RKIP) inhibits the mitogen activated proteinkinase cascade initiated by Raf-1 and nuclear factor kappa B (NF-κB) and is an activator of

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apoptosis in several human cancers. STAT3 is often constitutively phosphorylated in gastriccancer. RKIP is a negative regulator of STAT3; RKIP expression in tumors has been positivelycorrelated with a higher survival rate in gastric cancer patients. Aim & Methods: To evaluatethe role of RKIP in H. pylori infection by co-culturing AGS gastric cancer cells with H.pyloribacteria. Results: Both H. pylori infection and IL6 induced a dose- and time-dependentincrease in phosphorylation of STAT3 and RKIP. RKIP phosphorylation was dependent uponprotein kinase C and occurred mainly in the nucleus. RKIP transfection enhanced H. pylori-mediated apoptosis. Overexpression of RKIP in which the S153 residue was mutated tocysteine, abolished H. pylori-mediated RKIP phosphorylation and apoptosis and RKIP tran-scriptional activation. Infection of AGS cells with H. pylori in the presence of the proteasomeinhibitor MG132 resulted in an increase of RKIP protein levels, suggesting that RKIP expres-sion is regulated through proteasomal degradation. In the presence of the histone acetyltransferase, CBP, RKIP transcription was enhanced, but RKIP stimulated STAT3 transcriptionwas inhibited. Analysis of a panel of isogenic mutant H. pylori strains revealed that theeffects of H. pylori on RKIP phosphorylation and expression were dependent upon geneswithin H. pylori's cag pathogenicity island, specifically cagL. H. pylori infection also led toa dose- and time-dependent increase in expression of Snail, amediator of epithelial-mesenchy-mal transition that is also a negative regulator of RKIP transcription. Conclusions: Takentogether, these results indicate that RKIP function is regulated by phosphorylation at S153after H. pylori infection, and by acetylation by CBP. Post-translational modification of RKIPfunction by the cag pathogenicity island regulates survival pathways in gastric cancer cells.Our data suggest the existence of a complex feedback loop between H. pylori infection,RKIP, Snail and STAT3 in gastric cancer

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Immunophenotyping Gastric Lymphocytes During H. pylori Infection in MiceUsing Multicolor Flow Cytometric AnalysisVictoria E. Ruiz, Monisha Sachdev, Rebecca Haberman, Travis Brown, Jaqueline Carroll,Songhua Zhang, Sicheng Wen, Joshua Fischer, Steven F. Moss

Background: Helicobacter pylori infection is associated with severe chronic inflammation, yetthe host immune response is rarely able to clear the bacterium. Thymus derived lymphocytepopulations such as T-helper 1, T-helper 2, T-helper 17, and T-regulatory cells are knownto play a significant role in the chronicity of H. pylori infection as well as contributing toongoing gastric pathology. It is yet to be established, how these immune cells interact inthe gastric environment during H. pylori infection and how changes in the gastric T- lympho-cyte cell repertoire may lead to gastric carcinogenesis. Murine studies have been limited byan emphasis on splenic lymphocyte populations as surrogates of their gastric mucosalcounterparts. Aim: To characterize T-helper and T-regulatory populations and functionalparameters during chronic H. pylori infection in a gastric cancer susceptible mouse modelusing eight color flow cytometric analysis. Methods: p27-deficient mice and wild-typecontrols on an identical C57BL/6 background were infected with 109 cfu of H. pylori strainSS1 and euthanized 30 weeks post-infection. Stomachs were digested in a DTT/EDTA mixfollowed by collagenase treatment. Cell isolates were passed through a sucrose gradient toisolate gastric lymphocytes. Cell viability was assessed via trypan blue exclusion. Gastriclymphocytes were re-stimulated ex-vivo with CD3/CD28 antibodies for 4 hours. Cells werethen cultured with monensin for an additional 2 hours. After culture, lymphocytes werestained using markers specific for Th1 (TCRβ+CD4+,IFNγ+), Th2 (TCRβ+CD4+,IL-4), Th-17 (TCRβ+, CD4+ IL-17A+) and Treg subsets (TCRβ, CD4+, CD25+, Foxp3+), as well asfor a rare subset of NKT cells (TCRβ+, NK1.1). Cell populations were enumerated by flowcytometric analysis. Results: Yields of 1-2 million viable lymphocytes per stomach wereroutinely obtained. Flow cytometric analysis of gastric lymphocytes isolated from wild typeand p27-deficient mice revealed the presence of several discrete CD4+ T- helper lymphocytepopulations expressing IL-17A, IL-4, Foxp3 and CD25. Interestingly CD4 negative NK1.1positive cells were also identified in infected wild-type and p27-deficient mice. There wasa significant increase in CD4+ IFNγ expressing cells in infected p27-deficient mice comparedto uninfected and wild-type infected mice, #p < 0.05 versus p27+HP group; *p < 0.01versus p27+HP group. Conclusion: Discrete populations of T-helper, T-regulatory and NKTcells can be isolated from the gastric mucosa of H. pylori-infected mice p27-deficient andwild-type mice, and immunophenotyped for cytokine and transcription factor expression.H. pylori infection markedly increased the population of CD4+ IFNγ secreting lymphocytes,suggesting the involvement of this T helper subset in the pathogenesis of gastric cancer inthis model.

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Molecular Mechanism of Gastric MALT Lymphoma Formation by Helicobacterheilmannii InfectionTetsufumi Takahashi, Hidenori Matsui, Asako Takizawa, Yoko Komatsu, Keiko Shinagawa,Shin'ichi Takahashi, Toshifumi Hibi, Masahiko Nakamura, Kanji Tsuchimoto

We have developed a C57BL/6 mouse model of gastric mucosa-associated lymphoid tissue(MALT) lymphoma induced by a single intragastric infection with Helicobacter heilmanniistrain TKY isolated from a cynomolgus monkey. We observed an accumulation of B lympho-cytes along with destruction of glandular elements and the presence of lymphoepitheliallesions consistent with low-gradeMALT lymphomas with almost 100% probability of infectedC57BL/6J mice 6 months after infection (Nakamura et al., Infect. Immun., 75, 1214-1222,2007). However, the mechanism of gastric MALT lymphoma formation by infection withH. heilmannii is still unclear. In this connection, it has been reported that the expression ofInterleukine-10 (IL-10) is upregulated in the stomach of H. heilmannii infected mice. There-fore, we tried to identify the molecular mechanism of gastric MALT lymphoma formationby infection with H. heilmannii in IL-10 knockout mice. Materials and Methods: Thehomogenates of gastric mucosa of infected mice were orally administrated to 6-week oldfemale C57BL/6J and IL-10 knockout mice. Total RNA prepared from the gastric mucosasix months after infection, was subjected to the microarray and quantitative RT-PCR analyses.Genes with a paired t-test, p value (with Benjamini and Hochberg multiple testing correction)of <0.05 inmicroarray analysis were considered that the expression was significantly different.KEGG pathway analysis was performed using these listed genes. The viable number of

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