in this multicenter series. However, PFTG lacks specificity and larger studies will be neededto assess the accuracy of the PFTG for differentiating advanced from indolent pancreaticmucinous lesions.Table 1. Comparison of Test Performance

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Comparison of K-RAS-2 and LOH Mutational Analysis with Pancreas CystFluid CEA Levels and HistologyJayaprakash Sreenarasimhaiah, Saad F. Jazrawi, Luis F. Lara, Shou Jiang Tang

BACKGROUND: The main objective of pancreatic cyst fluid analysis is to differentiate cystsof mucinous or malignant variety from other cysts which have a benign course. Previousinvestigators have reported that K-ras-2 point mutation and at least two mutations of allelicimbalance or loss of heterozygosity (LOH) with good quality DNA is characteristic ofmucinous cystic neoplasm (MCN). OBJECTIVE: Identify the clinical impact of DNA analysisof pancreatic cyst fluid with its correlation to cyst fluid chemistry and histologic analysis.METHODS: This is a retrospective analysis of all consecutive patients with pancreatic cystswho presented for endoscopic ultrasound (EUS) examination with fine-needle aspiration(FNA) biopsy over an 18 month period until October 2007. DNA analysis was performed byRedPath Integrated Pathology (Pittsburg, PA). Fluid analysis included carcinogenic embryonicantigen (CEA) with values >192ng/dL suggestive of MCN. The standard for comparison wassurgical histopathology when available. RESULTS: 27 consecutive patients with cysts andsamples submitted for DNA analysis were examined including 15 men and 12 women (meanage 62.8 and 61.3 years, respectively). In 20 patients, all parameters including cyst fluid,DNA analysis, and histology were available for comparison. Consistent findings were seenin 7/20 (35%) in which all parameters suggested negative benign findings. 3/20 (15%) hadpositive K-ras-2 mutations of which only 2/3 had true histologic MCN suggesting a 33%false positivity. 7/20 (35%) of patients had at least 2 LOH mutations; however only 2/7were actually positive on histology for MCN suggesting 71% false positivity. In the samegroup, CEA levels >192ng/dL were seen in 7/20 of which 4/7 had histologic malignancydemonstrating 42.9% false positivity. In a group of 9 malignant cysts (MCN, side branchintraductal papillary mucinous neoplasm, and cystic degeneration of adenocarcinoma), 7(78%) had CEA levels >192ng/dL. In this group, 7 had DNA analysis of which 4/7 (57.1%)were falsely negative for K-ras-2 and 3/7 (42.9%) falsely negative for LOH. CONCLUSIONS:Consistency in histology, CEA levels, and K-ras-2 and LOH mutations was seen in only35% of cases, all of which were benign cysts. In the detection of malignant cysts, elevatedCEA levels were more predictive of histology in comparison to K-ras-2 or LOH mutations.Additionally, false positivity of LOH mutations was noted to be considerably higher thanK-ras-2 mutations or even fluid CEA levels. These findings suggest that DNA mutation analysisshould not be used routinely but rather selectively in the evaluation of pancreatic cysts.

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TNF-α Inhibits Human Intestinal NHE8 Gene Expression in Caco2 Cells ViaTyrosine Kinase PathwayHua Xu, Huacong Chen, Jiali Dong, Fayez K. Ghishan

Introduction: Sodium Hydrogen Exchanger isoform 8 (NHE8) is the newest member of theNHE family. This transporter is located on the apical membrane of the intestinal epithelialcells and is highly expressed in the early stages of development. Reports have shown thatintestinal NaCl absorption is reduced in the gastrointestinal tract of patients with inflammat-ory bowel diseases and in animal models of chronic enteritis, but the involvement of NHEisoforms in the pathogenesis of inflammatory disorders and the mechanism of inflammation-associated diarrhea remains unclear. We have shown that NHE8 function is reduced byTNF-α in the gastrointestinal tract, and this inhibition involves down-regulation of NHE8transcription. To further understand the underlining mechanism, the current study investig-ates the promoter response region and the signaling transduction pathway activated by TNF-α in regulating NHE8 gene expression in inflammatory conditions. Methods: A series of 5'deletion constructs of the human NHE8 gene promoter were made by PCR and restrictionenzyme digestion and subcloned into pGL3 basic reporter vector. These promoter constructswere then transfected into Caco2 cells by Effectene method and treated with TNF-α for 20hours before promoter reporter assay. The signaling transduction pathways after TNF-αtreatment were performed by treating cells with inhibitors AG1478, H7, and PD98059. Todetermine the involvement of EGF receptor (EGFR) in TNF-α mediated human NHE8expression down-regulation, anti-EGFR antibody was used to block EGFR activation. Results:Promoter reporter assays demonstrated that the TNF-α response region is located in theproximal promoter region of the human NHE8 gene. The effect of TNF-α on human NHE8gene expression could be completely blocked by receptor tyrosine kinase inhibitor AG1478and also partially blocked by MAP kinase inhibitor PD98059. Further study indicated thatneutralization EGFR by EGFR antibody could not abolish the effect of TNF-α on humanNHE8 gene expression. Conclusion: The TNF-α response region is located in the proximalpromoter region of the human NHE8 gene. Inhibition of the human NHE8 gene expressionby TNF-α is completely prevented by blocking receptor tyrosine kinase activation or partiallyprevented by blocking the MAP kinase activation. EGFR activation is not involved in thisregulation. This investigation was supported by NIH grant R01-DK073638.

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Regulation of the Human Anion Exchanger SLC26A3 Gene Expression ByIFNγSeema Saksena, Amika Singla, Shivani Katyal, Nikhil Bansal, Waddah A. Alrefai,Krishnamurthy Ramaswamy, Pradeep K. Dudeja

Two members of the SLC26 gene family, SLC26A3 or DRA (Down Regulated in Adenoma)and SLC26A6 or PAT-1 (Putative Anion Transporter-1) are known to play a major role inapical Cl-/OH- (HCO3

-) exchange process in the human intestine. We have previously shownthe inhibitory effects of IFNγ (30 ng/ml, 24h) on both SLC26A3 & A6 expression andpromoter activity. We also demonstrated that the effects of IFNγ on SLC26A6 gene expressionwere mediated via IRF-1 transcription factor. However, the molecular mechanisms underlyingthe transcriptional regulation of SLC26A3 gene expression by IFNγ in the intestine are notyet known. Current studies were, therefore, aimed at delineating the molecular mechanismsinvolved in IFNγ induced inhibition of SLC26A3 promoter activity. Human intestinal Caco2cells were transiently transfected with DRA promoter constructs and promoter activity wasdetermined by luciferase assay. DNA-protein binding was assessed by electrophoretic mobilityshift assay (EMSA). IFNγ (30 ng/ml, 24h) significantly reduced DRA promoter activity(p1183/+114) by ~40%. To identify the region of the SLC26A3 promoter responsive toIFNγ, the effects of IFNγ on 5′-deletion constructs of SLC26A3 promoter in Caco2 cellswere examined. Deletions from p-1183/+114 to p-790/+114 abolished the inhibitory effectsof IFNγ indicating that IFNγ response element is located in the -1183/-790 region. Sequenceanalysis of the -1183/-790 region revealed the presence of a potential binding site (-939/-918) for STAT1 transcription factor. Earlier studies have shown that the effects of IFNγcould be mediated through STAT1 (Signal Transducer and Activator of Transcription 1).EMSA demonstrated an increase in protein binding to the oligonucleotide spanning thepotential STAT site (-939/-918) in IFNγ-treated nuclear extracts. The binding was abolishedin the presence of excess of cold consensus STAT1 oligo or STAT1 antibody. Conclusion:Our studies provide evidence for the involvement of STAT1 in the inhibition of SLC26A3gene expression by IFNγ in the human intestine. We speculate that binding of STAT1 toDRA promoter in response to IFNγmay contribute to the down-regulation of DRA expressionassociated with diarrheal disorders such as IBD. (Supported by NIH and Dept of Vet Affairs).

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PI3K, AKT and MAPK Differentially Mediate Mitogenic and Motogenic Effectsof Repetitive Deformation On Intestinal Epithelial CellsChristopher Gayer, Lakshmi S. Chaturvedi, Marc D. Basson

Gut mucosa In Vivo experiences repetitive strain during peristalsis or villous motility whichmay be trophic for the mucosa. In Vitro, cyclic strain stimulates intestinal epithelial prolifera-tion on most matrix proteins but stimulates cell migration across fibronectin, consistentwith the deposition of tissue fibronectin into the matrix in chronic inflammation and mucosalinjury. ERK mediates both effects. The mitogenic effect is p38-independent but the relevanceof p38 to the motogenic effect has not been studied. Furthermore, the upstream effectorsof strain activation of the MAPK are not clear. Since PI3K, AKT, and the MAPK influenceproliferation and migration in cells without strain, we evaluated the role of these signals inthe mitogenic and motogenic effects of strain. Human Caco-2 intestinal epithelial cells werecultured on collagen IV or fibronectin and subjected to an average 10% cyclic strain at 10cycles/min. Proliferation was assessed by cell counting and migration by circular woundclosure. We assayed PI3K by immunoprecipitating its p85 subunit and Western blottingfor its phosphorylated form, and AKT, p38 and ERK by Western blot for their phosphorylatedforms. PI3K, AKT, ERK, and p38 were activated 30-60 min after cyclic strain initiation onboth collagen and fibronectin. Strain stimulation of proliferation on collagen and of motilityon fibronectin was each blocked by either the PI3K inhibitor LY294002 or AKT inhibitor IV.The p38 inhibitor SB203580 blocked motility on fibronectin without affecting proliferation oncollagen. We next examined the effects of PI3K and AKT blockade on ERK and p38. BlockingPI3K prevented strain-induced ERK and p38 phosphorylation, but blocking AKT did not,suggesting that strain activation of PI3K activates ERK and p38 by an AKT-independentpathway. We further investigated whether the role of AKT was isozyme-specific using specificsiRNA to selectively reduce each AKT isoform by ≈70%. The motogenic effect of strainwas blocked by reducing either AKT1 or AKT2. However, only AKT2 reduction blockedproliferation in cells subjected to strain on a collagen substrate; AKT1 reduction had noeffect. These results trace a matrix-dependent pathway involving PI3K, ERK, p38 and AKTthat mediates the mitogenic and motogenic effects of cyclic strain on intestinal epithelialcells in a manner varying in AKT isozyme specificity. PI3K may be a potentially importantupstream target to maintain the gut mucosa in settings of ileus or sepsis when peristalsisand villous motility are aberrant and the mucosa atrophies, while the AKT isozyme specificityoffers a target to selectively modulate different strain-induced effects.

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Pathogenic Roles of COX-2/PGs and iNOS/NO in a Rat Model of IschemicEnteritisTohru Kotani, Yoshino Komatsu, Yuka Nakamori, Koji Takeuchi

Background/Aim: Ischemic enteritis, the disease of which onset is rapid, followed by extensionprogressively and resulting in a high mortality rate, is caused by interruption or profounddecrease of the arterial inflow to the small intestine. We recently established an animalmodel of ischemic enteritis that has a high clinical predictability, yet the pathogenic mechan-ism remains fully explored. In the present study, we investigated the roles of COX-2/PGsand iNOS/NO in the mucosal defense against ischemia-induced intestinal lesions in rats.Methods: Male SD rats were used after 18 h fasting. Ischemic enteritis was induced bypartial ligation of the superior mesenteric artery (SMA). Under ether anesthesia, SMA wasisolated, and a stenosis was produced by placing a needle (23 guage) on the vessel and thenligature of both the vessel and needle, and then a needle was removed from the ligature,resulting in a calibrated constriction of SMA. After the operation, the animals were fednormally and sacrificed various days later up to 3 days. Various COX and NOS inhibitors

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