- Email: [email protected]
Is idiopathic pancreatitis an autoimmune disease?
circular muscle strip contractions were measured in response to bethanechol (.03-3001*M) Musculans whole mounts of the intestine were stained for myelopemxidase (MPO)-positive leukocytes. Sterile intestinal muscle preparations were incubated for 24h and tissue culture supematants assayed for NO By the Griess reaction (~M/100mg'tissue) Data are mean -+ SEM, N =4-5. Results: Yoh significantly decreased the IM-induced delay in gastrointestinal transit without affecting transit in controls (GC: control-veh 8.9 + 0.3, control-Yoh 9 7 -+ 0.2, IM-veh 6 7 + 0.8, IM-Yoh 8.6 + 0.3). Yoh significantly improved the IM-induced suppression of in vgro contractility, which was not attenuated by vehicle (contractility at 100p.M Bethanechol g/s/ram2: control-veh 4 12 _+0 99, control-Yoh 4.15 -+ 0 79, iM/veh 2.16 _+0 68, IMYoh 2.84 -+0.93). The muscularis leukocyte infiltrate that increased after IM was not affected by Yoh (MPO-posinve cells/ field: control-veh 1.2-+0.2, control-Yoh 1.1 +-0.2, IM-veh 617 -+ 148,1M-Yoh 69.0--+ 10 1). NO production increased 5.6 fold after IM in tissue culture supematants. This increase was completely inhibited by the selective iNOS inhibitor L-NiL Yohimbine attenuated the increase after IM by 35% (NO control-veh = 5.46 _+0.59, controlY o h = 6 . 1 2 +- 1.00, l M - v e h = 3 f l . 3 7 _+ 1.36, IM-veh with L - N I L = 8 9 4 0 + . 1 7 , IMYoh= 19.83 -+ 5.8). Conclusion: This study suggests that the effect of alpha-2 adrenoreceptor stimulation on the development of postoperative ileus is in part mediated through alpha-2 adrenergic regulation of post-surgical NO production within the intestinal muscularis.
705 Protein Kinase C ~ (PKC~) Regulates TNF~-lnduced Expression of Intercellular Adhesion Molecule 1 (ICAM-1) via Transcription Factors Spl and NF-KB p65 in Human Colonic Circular Smooth Muscle Cells (HCCSMC) Konrad Pazdrak, Xuan-Zheng Shi, Sushil K Sarna The inducible expression of ICAM-1 in HCCSMC upon stimulation with TNFe~ suggests that these cells contribute to the overall inflammatory response seen in gut inflammation. The aim of this study was to investigate the regulation of ICAM-1 expression in HCCSMC by PKC~ and transcription factors Spl and NF-KB p65. The essential information for TNFc~induced ICAM-1 expression is contained within the region spanning -187/-178 of the ICAM1 promoter (-187/-178RE). Agarose-DNA pull down assays showed that -187/-178RE binds p65 extracted from the nuclei of TNFoc-stimulated HCCSMC. The binding of p65 peaked within i h and then gradually declined to 35% of maximal binding after 24 h of stimulation. By contrast, ICAM-1 expression was initially detected at 3 h and was on the rise for up to 48 h. The - 187/- 178RE was also able to bind Sp 1 nuclear factor that is known for maintenance of gene expression. The Spl binding to -187/-178RE was detectable after 1 h of stimulation and its increase of binding was maintained for at least 12 h of TNFa stimulation Since both transcription factors have been reported to serve as nuclear substrates tbr PKC~, we investigated whether PKC~ regulates the expression of ICAM.1 through Spl. Stimulation with TNFa resulted ni a two-fold increase of enzymatic activity of nuclear PKC~ for at least up to 12 h. Western blotting confirmed the nuclear presence of Spl, PKC~ and p65 in stimulated HCCSMC. Autoradiography of Sp 1 nuclear immunoprecipitates from 32P-labeled HCCSMC showed TNFa-induced phosphorylation of Spl. Inhibition of PKC~ activity with myristoylated PKC[ pseudosnhstrate inhibitor complete[y blocked TNFa-induced phosphorylatinn of Sp 1 and its binding to - 187/- 178RE, pointing to the essential role of Sp 1 phosphorylation for DNA binding. PKC~inhibition also inhibited the expression of [CAM- 1 in HCCSMC, as assessed by flow cytometry. Our results demonstrate, for the first time, a regulatory role of Spl in TNFa-responsive ICAM-1 gene expression. The activity of PKC~-Spl pathway seems to he mediated by the -187/-178RE, a regmn that binds also NF-/~B p65, but with different kinetics and perhaps in an Spl exclusive manner. Spl may maintain gene expression during diminished NF-KB binding activity.
703 Up-regulation of PARd and PAR-2 Expression Contributes to Nematode-induced l~lypercontractility of Murine Intestinal Smooth muscle A/ping Zhao, Harry Dawson, Joseph F. Urban, Fred D. Finkdman, Terez Shea-Dnnohue Nematode induces a hypercontractility of intestinal smooth muscle that contributes to worm expulsion. Protease-activated receptors (PARs) are a subclass of the seven transmembranespanning, G protein-coupled receptors and are expressed throughout the gut. Activation of PARs is involved also in inflammation suggesting an immunological control of the PAR response. Aim: To determine N. brasiliensis {Nb.)-induced changes in 1) smooth muscle function and 2) PAR-1 and PAR-2 expression in murine small intestine. Methods: Balb/c mice were inoculated subcutaneously with 500 Nb. at L3 and studied 9 days later. For functional studies, segmep.ts of jejunum were suspended longuudinally in organ baths and stretched to 9.9 mN (optimal length). Responses to acetylchoIine (ACH, lnM-lmM), PAR1 activating peptide SFLLRN (1-100p,M), and PAR-2 agonist trypsin (lnM-lg,M) were determined. Frequency-dependent responses to EPS (1-20 Hz, 80V, 0.1ms) were also constructed. For receptor expression, total RNA was isolated using TRhol and then reversetranslated to cDNA using First Strand cDNA Sypthase Kit (MBI Fermentas). Real-time quantitative PCR was performed by using ABI Prism 7700; Primers and probes were designed by using Pnmer Express; PCR amplifications were as follows: 50~ for 2 rain, 95~ for 10 min, 40 cycles of 95~ for 15s and 60~ for 1 rain Results: In control mice, ACH and EPS induced concentration- and frequency-dependent contraction in intestinal smooth muscle, respectively Trypsin and SFLLRN induced similar responses consisting of a small relaxation and followed by a concentration-dependent contraction. Nb. infection significantly increased responses to ACH and EFS as well as to trypsin and SFLLRN. Correspondingly, there was an up-regulation of PAR-1 and PAR-2 mRNA expression in No. infected mice. Conclusions: Activation of PAR-1 and PAR-2 evoked a contraction of routine intestinal smooth muscle that was enhanced by Nb. infection. These data indicate that nematode-induced hypercontracttlity of routine intestinal smooth muscle, which contributes to the worm expulsion, may be attnbuted, in part, to the up-regulation of PAR-1 and PAR-2 expression. Supported by NIH AI/DK 49316 (TSD)
706 Acid Infusion Induced Esophageal Hypersensitivity is Associated with a Hypersensitivity of Esophageal Contractile Activity Vikas Bhalla, Jianmin Liu, Ravinder Mittal Background and Aims; We recently found a close temporal correlation between the onset of heartburn induced by acid infusion into the esophagus (positive 8emstein test) and a sustained esophageal contraction (SEC). A number of investigators have observed that repeated acid infusion induces stronger symptoms (symptom hypersensitivity). The goal of our study was to determine if symptom hypersensitivity was associated with esophageal contractile hypersensitivity. Methods:Subjects with chronic symptoms of reflux (heartburn & chest pain) underwent simultaneous manometric pressure recording and ultrasound imaging of the esophagus. Nom',al saline and 0.1 N HCL was sequentially infused into the esophagus and subjects scored symptoms of heartburn on a scale of 1-10. Saline and HCL infusions were repeated in 10 subjects with a positive Bemstein test. Data Analysis: Ultrasound images were digitized and analyzed every three seconds during the entire study period and the mnacularis propria (circular and longitudinal muscle) thickness was measured using computerized image analysis software Results: Acid infusion in esophagus induced heartbum. Esophageal contractions were of higher amplitudes and longer in duration during acid as compared to saline period. SECs were also identified during acid infusion period. Second acid infusion induced heartburn ~ t h shorter latency and stronger severity than first one. Contractions amplitudes and duration were higher dunng 2 "~ acid infusion than 1~ acid infusion. In addition, the number and the amplitude of SECs were greater during 2 "d as compared to 1~ acid infusion Conclusion:l) Our data, for the first time, shows that acid induced symptom hypersensitivity is associated with hypersensitMty of esophageal contractile activity, 2) It is possible that acid induced motor events are the cause of heartburn.
Tendon (N/cmz) mRNA EFS(20Hz, TRYPSIN SFLLRN 80V) (lpM) (100pU) PAR-I PAR-2 Cmtrol 7.3q,1 8,6~1.0 2.6q.0 11.5~.0,9 . . . . Nb. 28.0,2.9~ 23.4='2.5" 5.7~1.'2" 21.0~2.5" 320~ 270" Tension=rr~aos*8EM; mRNA=%of Control;*p<0,05;"p<0.01 vs Control;rP4 AGH(1raM)
704
Effect of acid infusionon symptomsand sustained esophagealcontraction
Differential Effects of Inflammation on Agonist-lnduced Contractility of Circular Smooth Muscle Cells Run W Wells, Gerald P. Morns, Michael G Blennerhassett
Time period
Alteredmotility ts characteristic of intestinal inflammation, but despite its subsequent resolution, some individuals have persistent motility problems as part of a post-inflammatory imtable bowel syndrome (IBS). To increase basic knowledge of both the acute and lasting effectsof inflammation on smooth muscle, we studied the contractile responses of individual circularsmooth muscle cells (SMC) isolated during rat colitis (TNBS) throughout the course of inflammation to recovery. Video microscopy was used to measure the percent contraction of cells exposed to the contractile agonists carhachol (CCh), 5-HT, histamine or KC1 (30 cells per animal per concentration of agonist; n = 3-9 animals/group). Maximal contraction to CCh, 5-HT, histamine or KC1 caused an overall shortening of 29 +/-1, 23 +/-2, 25 +/2 and 29 +/-2%, respectively (mean +/-sere). Inflammation caused decreased responsiveness Io CCh and 5-HT (Days 2-16), with a maximal decrease of 22% for CCh on Day 2 and 29% for 5-HT on Day 4 (p<0.05). By Day 36, CCh-induced contraction had returned to control levels, while 5-HT-induced contraction remained impaired (-18%). In contrast, there were no changes observed for histamine at any time. Although KCl-induced contraction became impaired by Day 4 (p<0.05), the mammal response was still substantially greater than that induced by either CCh or 5-HT, suggesting that both receptor and non-receptor mediated changes to contraction occurred during peak inflammation. We conclude that the impaired contractility observed at the single cell level contributes to impaired colonic motility during inflammation. However, persistent changes in sensitivity to excitatory agunists are detectable post inflammation, when the tissue function appears to have returned to normal. The prolonged attenuation in the 5-HT response detected in this model of colitis suggests that a similar event may contribute to the development or exacerba4ion of post-inflammatory [BS Supported by the CIHR and CDHF.
l . ~ c y to Heartburn (Seconds)
HuIlbum Severity
Musclethick. Pressure Pressure hess during 10 amplitudeof durationabove mlnutes of Acld swallows(%) 15 mmHg during score(1-10) perfuldon(%) t t (%}swallow t
Acid1 317!137 5.3'!2.4 1'0"1!6 115 23 117 ~15 Add2 93~47' 8.5 '1.5" 118,10' 143 ~43" 151 ~17" 1": The vaFuesshownhere are percentagechangein referencewith the salineinfusionperiod, whichis 100%. * : p < 0.05 comparedwith acid 1
707 Is Idiopathic Pancreatitis an Autoimraune Disease? Karine Nahon-Uzan, Philippe Levy, Dermot O'foole, Nadia Belmatoug, Philippe Ponsot, Laurent Palazzo, Pascal Hammel, Valerie Vilgrain, Philippe Ruszniewski Three forms of idiopathic chronie pancreatitis (following exclusion of alcoholic, hereditary and metabolic causes) have been described on imaging: a) pseudotumora[; b) duct-destructive c) chronic pancreatitis (CP) with duct dilatation Aim: to identify autotmmune stigmata in the three forms of idiopathic CP. Methods: all patients who underwent exploration of idiopathic CP between 1996 and 2001 were included. The following were performed for each patient: detailed history and clinical examination by an internal medicine specialist in autoimmune disorders; auto-annhodies and immunogloublin screening; pancreatic duct imaging (ERCP or MR-pancreatography); and EUS. The different forms were thus defined: a) pseudotumoral: nodule presence on imaging; b) duct-destructive as described by Ectors
A-89
AGA
Abstracts
et al (1); c) dilated ducts according to Cambridge classification Patients with an obvious cause (alcohol, metabolic, hereditary, ductat obstruction) were excluded. Results: 60 patients were included (pseudotumora[ : n = 11 ; duct-destructive : n = 28 ; dilated ducts : n = 21 ). Duct-destructive types commenced at an earlier age than the dilated ducts type (median : 28 vs 39 years : P< 0,05). A tendency towards more frequent episodes of acute pancreatitis was observed in the duct-destructive type (86%) compared to dilated ducts (71%) and pseudotumoral (55%) types. Cholestasis was more frequent in pseudotumoral forms (46%) vs duct-destructive (11%) vs dilated ducts (6% : P< 0,007) Pancreatic calcifications were unique to the dilated duct type (81% ; P
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'I08 Progression of Endocrine Insufficiency in Tropical Caicific Pancreatis: A Longitudinal Follow-up Study of 53 Non-Diabetic Subjects K. K. Barman, V. S. Rajah, R. Deepa, Suresh T. Churl, V. Mohan Background/Aims: Tropical calcific pancreatitis (TCP), a form of idiopathic, early-onset calcific chronic pancreatitis is the most prevalent form of chronic pancreatitis in South India. Diabetes (DM) in TCP is also called fibrocalculons pancreatic diabetes (FCPD) and is often seen at diagnosis of TCP. The aim of this longitudinal follow-up study was to delineate the natural history of pancreauc endocnne insufficiency in TCP patients who presented without DM. Methods: Seventy-s/x TCP patients, referred to MDRF for evaluation of pancreatic endocrine function, who did not have DM by WHO criteria at baseline, were included in this study. The 53/76 subjects (70%) available for follow-up underwent yearly oral glucose tolerance tests for evaluation of pancreatic endocrine function until they developed DM. Pancreatic exocrine function was measured using fecal chymotrypsin. Baseline demographic (age, gender) and clinical (age at onset of pain, body mass index (BMI)) characteristics were noted. Time to development of DM was calculated nsmg Kaplan Meir method. Data are expressed as mean _+standard deviation. Results: Baseline characteristics (age, gender, fasting plasma glucose) of the 53 study subjects were similar to those of the 23 subjects not available for follow-up. The 53 subjects were followed for a mean of 6.8 years (range: 1- 21). Their mean age at baseline was 27 _+ 12 years, 68% were males, and the mean BM1 was 19.5 +_ 4.3 kg/m ~. On fofiow-up, 24/53 (45%) developed DM; the incidence of DM in this cohort was 6 6 per 100 person years of follow up. Compared to subjects who remained non-diabetic on follow-up, the subjects who developed DM were older (24 -+ 11 vs 31 _+12 years, p<0.03), had higher BMI (18.4 +- 3.8 vs 21.4 kg/m 2 +- 4.6, p<0.03) and Bad lower fecal chymotrypsin (3.9 +- 2.9 vs 1.4-+ l. 1, p < 0 001) at baseline. Of the subjects who developed DM, 63% had a family history of DM compared to 34% of non-diabetic subjects (p =us). Median time to development of DM after diagnosis of TCP was 12.5 years and the diagnosis of TCP was made a median of 5.2 years after onset of pain Conclusion: In TCP there is progressive deterioration in pancreatic endocrine function, with development of FCPD in nearly half the patients on foflow-up. FCPD is merely the later stage in the natural history of TCP.
710
Endoscopic Ultrasound-Guided Pancreatic Trucut Needle Biopsies in Patients with Suspicious Pancreatic Masses Alberto Larghi, Elizabeth C. Verna, Stawos S. Stavropoulos, Heidrun Rotterdam, Jennifer J. Koehnken, Charles J. Lightdale, Peter D. Stevens Background: A trucut needle biopsy able to obtain specimens from the pancreas and other perigastric organs under EUS guidance has recently been developed and successfully tested in animals. The purpose of our study was to prospectively assess the clinical utility of this new diagnostic tool in patients with suspicious pancreatic masses. Methods: Patients with a pancreatic mass detected by CT were studied. EUS-guided trucut needle biopsy (EUSTNB) was performed with a linear echoendoscope using a disposable 19-gauge needle (Quick Core Biopsy, Wilson-Cook Medical Inc.). In a subset of patients, EUS-guided fine needle aspiration (EUS-FNA) with a 22-gauge needle was performed during the same procedure. Patients were assessed for complications throughout the following 48-hour period. Results: Sixteen patients (mean age 62; M/F 8/8) underwent EUS-TNB. Fragmentation of the biopsy specimens was frequently observed with fragment length ranging from 0.1 to 1.1 cm Histological examination revealed pancreatic cancer in 11 patients (69%). Four of the 5 patients with negative EUS-TNB underwent CT-guided biopsy, two of which were positive for adenocarcinoma. EUS-FNA was also performed in 11 patients. In four of the seven patients with positive EUS-TNB, EUS-FNA results were negative, while in all other cases results of both procedures were equiva/ent. No acute or long-term complications were observed. Conclusions: Our preliminary data show that pancreatic tissue for histological examination can be safely obtained with EUS-TNB. Studies comparing EUS-TNB and EUSFNA in large patient populations are needed to determine if this technique will increase the diagnostic yield and accuracy of EUS in the evaluation of patients with pancreatic
709
711
A Shortened Endoscopic Pancreatic Function Test (ePFT) May Be Used as Screening Test for Chronic Pancreatitis (CP) in Patients with Chronic Abdominal Pain (CAP) Darwin Conwell, Gregory Zuccaro, John Vargo, Frederick Vanlente, John Dumot, Particia Trolfi, Jufie Tsiramhidis
Clinical and Nutritional Effects of Anti-oxidant Supplementation: a Prospective Randomized Study in Patients with Chronic Pancreatitis Pierre H. Deprez, Sebastien Delazzer, Laurence Galanti, John Lebrun, Andre Geubel, Yves Horsmans The aim of our study was to compare the nutritional, metabolic, functional and clinical effects of a diet adapted to chronic pancreatitis versus anti-oxidant supplementation. Patients: Thirty patiems with chronic pancreatitis were given an adapted diet plus or minus antioxidant supplementation (Quatral | rid) in a prospective, randomized study with a crossover after 3 months. The dietary counseling was aimed to correct all errors detected during a preliminary dietary evaluation. The latter showed an insufficient intake of vii A (33% of patients), vit D (93%), vii E (86.7%), vit C (76.7%), copper (83.3%), zinc (30%), selenium (56.7%). During the study, dietary assessment, clinical pain evaluation 0/AS), nutritional and metabolic assessment (BMI, fat mass, basal metabolism), determination of vitamins and anti-oxidants blood levels and measurement of exocrine (fecal elastase and steatorrhea) and endocrine function (HOMA test) were performed at start point, after 3 and 6 months. Results: In basal state, a significant percentage of patients was shown to have a hypermetabolic status (46.7%), an excessive BET (60%) with a correct nutrient repartition (lipid 35%, protein 16% and glucids 46%), a low BMI (33.3%), and low levels of albumin and prealbumin (10.7%, 22.2%, respectively), vii D (60.7%), vit C (64.3%), and zinc (33.3%). The anti-oxidant system was impaired m half our patients (total anti-oxidant level) with decreased plasma glutathion peroxidase levels in 25% and superoxyde dismutase in 43.5% of patients. Exocrine function was impaired in 66.7% of our patients and diabetes was present in 267%. Mean pain score was 31.7%. No significant changes were seen with the adapted diet. The oral antioxidant supplements significantly increased levels of vit C, vit E, carotene, zinc, selenium, glutathion peroxydase and superoxyde dismutase (P
BACKGROUND. We have developed a 1 hour endoscopic collection method (ePFT) using synthetic porcine secretin to evaluate CAP pts with and without risk factors (+RF, -RF) for CP. A shorter test would be desirable and more time efficient. AIM: 1) Examine the ePFT in pts with CAP and CP 2) Determine accuracy of specific timed aspiration periods. METHODS. 3 groups of pts studied: 1) CAP without risk factors (CAP-RF), 2) CAP with risk factors (CAP + RF), and 3) chronic pancreatitis (CP). ERCP confirmed CP dLagnosis. ePFT protocol: 1) upper endoscopy, 2) IVP secretin (0.2 mcg/kg) over 1 min, 3) gastric fluid discard, 4) duodenal fluid aspirations via endoscope in trap at 0, 15, 30, 45, and 60 rain after secretin injection, 5) analysis for bicarb conc( Normal > 80 meq/L). Data analysis: p-values calculated using one-sided t-test; szgnificance 0.05. RESULTS. 55 pts studied: 23 CAP-RF, 15 CAP+RE, and 17 CP Peak bicarb conc for all groups in graphic. The CAPRF group is significantly greater than 80 meq/L (p=0.008; 95% CI 83.34-92.48 meq/L). The CAP+ RF group is significantly less than 80 meq/L (p=0.0127; 95% CI 64.77-78.83 meq/L). The CP group is significantly less than 80 meq/L (p<0.001, 95% CI 37.55-58.56 meq/L) The diagnostic accuracy of specific timed aspirations dunng peak pancreatic secretion (30-45 rain) are shown tn table. CONCLUSIONS: i) The ePFT using synthetic porcine secretin can distinguish pts with CP from CAP. 2) Duodenal aspiration obtained at 30 minutes during EGD represents peak pancreatic secretion and is accurate for the diagnosis of CP. CLINICAL IMPLICATION: A brief and simple screening ePFT may be performed by IV injection of secretin followed by endoscopy and simple aspiration 30 minutes following injecnon.
Dlagne~c Accuracy ePFT Aspirins
CAP-RF CAP~RF CP Overall Accuracy
AGA Abstracts
N 23 15 17
30 mln (%) O111,/ 45 mln (~) Only 3e, 4~ r ~ ComNnee ~.) 19 (82) 13 (56) 21 (91,3) 15 (100) 12 (80) 15 (100) 17 llCO) 17(100) 17(100) 51 (96.2) 42 (76) 53 (96.3)
A-90
705 Protein Kinase C ~ (PKC~) Regulates TNF~-lnduced Expression of Intercellular Adhesion Molecule 1 (ICAM-1) via Transcription Factors Spl and NF-KB p65 in Human Colonic Circular Smooth Muscle Cells (HCCSMC) Konrad Pazdrak, Xuan-Zheng Shi, Sushil K Sarna The inducible expression of ICAM-1 in HCCSMC upon stimulation with TNFe~ suggests that these cells contribute to the overall inflammatory response seen in gut inflammation. The aim of this study was to investigate the regulation of ICAM-1 expression in HCCSMC by PKC~ and transcription factors Spl and NF-KB p65. The essential information for TNFc~induced ICAM-1 expression is contained within the region spanning -187/-178 of the ICAM1 promoter (-187/-178RE). Agarose-DNA pull down assays showed that -187/-178RE binds p65 extracted from the nuclei of TNFoc-stimulated HCCSMC. The binding of p65 peaked within i h and then gradually declined to 35% of maximal binding after 24 h of stimulation. By contrast, ICAM-1 expression was initially detected at 3 h and was on the rise for up to 48 h. The - 187/- 178RE was also able to bind Sp 1 nuclear factor that is known for maintenance of gene expression. The Spl binding to -187/-178RE was detectable after 1 h of stimulation and its increase of binding was maintained for at least 12 h of TNFa stimulation Since both transcription factors have been reported to serve as nuclear substrates tbr PKC~, we investigated whether PKC~ regulates the expression of ICAM.1 through Spl. Stimulation with TNFa resulted ni a two-fold increase of enzymatic activity of nuclear PKC~ for at least up to 12 h. Western blotting confirmed the nuclear presence of Spl, PKC~ and p65 in stimulated HCCSMC. Autoradiography of Sp 1 nuclear immunoprecipitates from 32P-labeled HCCSMC showed TNFa-induced phosphorylation of Spl. Inhibition of PKC~ activity with myristoylated PKC[ pseudosnhstrate inhibitor complete[y blocked TNFa-induced phosphorylatinn of Sp 1 and its binding to - 187/- 178RE, pointing to the essential role of Sp 1 phosphorylation for DNA binding. PKC~inhibition also inhibited the expression of [CAM- 1 in HCCSMC, as assessed by flow cytometry. Our results demonstrate, for the first time, a regulatory role of Spl in TNFa-responsive ICAM-1 gene expression. The activity of PKC~-Spl pathway seems to he mediated by the -187/-178RE, a regmn that binds also NF-/~B p65, but with different kinetics and perhaps in an Spl exclusive manner. Spl may maintain gene expression during diminished NF-KB binding activity.
703 Up-regulation of PARd and PAR-2 Expression Contributes to Nematode-induced l~lypercontractility of Murine Intestinal Smooth muscle A/ping Zhao, Harry Dawson, Joseph F. Urban, Fred D. Finkdman, Terez Shea-Dnnohue Nematode induces a hypercontractility of intestinal smooth muscle that contributes to worm expulsion. Protease-activated receptors (PARs) are a subclass of the seven transmembranespanning, G protein-coupled receptors and are expressed throughout the gut. Activation of PARs is involved also in inflammation suggesting an immunological control of the PAR response. Aim: To determine N. brasiliensis {Nb.)-induced changes in 1) smooth muscle function and 2) PAR-1 and PAR-2 expression in murine small intestine. Methods: Balb/c mice were inoculated subcutaneously with 500 Nb. at L3 and studied 9 days later. For functional studies, segmep.ts of jejunum were suspended longuudinally in organ baths and stretched to 9.9 mN (optimal length). Responses to acetylchoIine (ACH, lnM-lmM), PAR1 activating peptide SFLLRN (1-100p,M), and PAR-2 agonist trypsin (lnM-lg,M) were determined. Frequency-dependent responses to EPS (1-20 Hz, 80V, 0.1ms) were also constructed. For receptor expression, total RNA was isolated using TRhol and then reversetranslated to cDNA using First Strand cDNA Sypthase Kit (MBI Fermentas). Real-time quantitative PCR was performed by using ABI Prism 7700; Primers and probes were designed by using Pnmer Express; PCR amplifications were as follows: 50~ for 2 rain, 95~ for 10 min, 40 cycles of 95~ for 15s and 60~ for 1 rain Results: In control mice, ACH and EPS induced concentration- and frequency-dependent contraction in intestinal smooth muscle, respectively Trypsin and SFLLRN induced similar responses consisting of a small relaxation and followed by a concentration-dependent contraction. Nb. infection significantly increased responses to ACH and EFS as well as to trypsin and SFLLRN. Correspondingly, there was an up-regulation of PAR-1 and PAR-2 mRNA expression in No. infected mice. Conclusions: Activation of PAR-1 and PAR-2 evoked a contraction of routine intestinal smooth muscle that was enhanced by Nb. infection. These data indicate that nematode-induced hypercontracttlity of routine intestinal smooth muscle, which contributes to the worm expulsion, may be attnbuted, in part, to the up-regulation of PAR-1 and PAR-2 expression. Supported by NIH AI/DK 49316 (TSD)
706 Acid Infusion Induced Esophageal Hypersensitivity is Associated with a Hypersensitivity of Esophageal Contractile Activity Vikas Bhalla, Jianmin Liu, Ravinder Mittal Background and Aims; We recently found a close temporal correlation between the onset of heartburn induced by acid infusion into the esophagus (positive 8emstein test) and a sustained esophageal contraction (SEC). A number of investigators have observed that repeated acid infusion induces stronger symptoms (symptom hypersensitivity). The goal of our study was to determine if symptom hypersensitivity was associated with esophageal contractile hypersensitivity. Methods:Subjects with chronic symptoms of reflux (heartburn & chest pain) underwent simultaneous manometric pressure recording and ultrasound imaging of the esophagus. Nom',al saline and 0.1 N HCL was sequentially infused into the esophagus and subjects scored symptoms of heartburn on a scale of 1-10. Saline and HCL infusions were repeated in 10 subjects with a positive Bemstein test. Data Analysis: Ultrasound images were digitized and analyzed every three seconds during the entire study period and the mnacularis propria (circular and longitudinal muscle) thickness was measured using computerized image analysis software Results: Acid infusion in esophagus induced heartbum. Esophageal contractions were of higher amplitudes and longer in duration during acid as compared to saline period. SECs were also identified during acid infusion period. Second acid infusion induced heartburn ~ t h shorter latency and stronger severity than first one. Contractions amplitudes and duration were higher dunng 2 "~ acid infusion than 1~ acid infusion. In addition, the number and the amplitude of SECs were greater during 2 "d as compared to 1~ acid infusion Conclusion:l) Our data, for the first time, shows that acid induced symptom hypersensitivity is associated with hypersensitMty of esophageal contractile activity, 2) It is possible that acid induced motor events are the cause of heartburn.
Tendon (N/cmz) mRNA EFS(20Hz, TRYPSIN SFLLRN 80V) (lpM) (100pU) PAR-I PAR-2 Cmtrol 7.3q,1 8,6~1.0 2.6q.0 11.5~.0,9 . . . . Nb. 28.0,2.9~ 23.4='2.5" 5.7~1.'2" 21.0~2.5" 320~ 270" Tension=rr~aos*8EM; mRNA=%of Control;*p<0,05;"p<0.01 vs Control;rP4 AGH(1raM)
704
Effect of acid infusionon symptomsand sustained esophagealcontraction
Differential Effects of Inflammation on Agonist-lnduced Contractility of Circular Smooth Muscle Cells Run W Wells, Gerald P. Morns, Michael G Blennerhassett
Time period
Alteredmotility ts characteristic of intestinal inflammation, but despite its subsequent resolution, some individuals have persistent motility problems as part of a post-inflammatory imtable bowel syndrome (IBS). To increase basic knowledge of both the acute and lasting effectsof inflammation on smooth muscle, we studied the contractile responses of individual circularsmooth muscle cells (SMC) isolated during rat colitis (TNBS) throughout the course of inflammation to recovery. Video microscopy was used to measure the percent contraction of cells exposed to the contractile agonists carhachol (CCh), 5-HT, histamine or KC1 (30 cells per animal per concentration of agonist; n = 3-9 animals/group). Maximal contraction to CCh, 5-HT, histamine or KC1 caused an overall shortening of 29 +/-1, 23 +/-2, 25 +/2 and 29 +/-2%, respectively (mean +/-sere). Inflammation caused decreased responsiveness Io CCh and 5-HT (Days 2-16), with a maximal decrease of 22% for CCh on Day 2 and 29% for 5-HT on Day 4 (p<0.05). By Day 36, CCh-induced contraction had returned to control levels, while 5-HT-induced contraction remained impaired (-18%). In contrast, there were no changes observed for histamine at any time. Although KCl-induced contraction became impaired by Day 4 (p<0.05), the mammal response was still substantially greater than that induced by either CCh or 5-HT, suggesting that both receptor and non-receptor mediated changes to contraction occurred during peak inflammation. We conclude that the impaired contractility observed at the single cell level contributes to impaired colonic motility during inflammation. However, persistent changes in sensitivity to excitatory agunists are detectable post inflammation, when the tissue function appears to have returned to normal. The prolonged attenuation in the 5-HT response detected in this model of colitis suggests that a similar event may contribute to the development or exacerba4ion of post-inflammatory [BS Supported by the CIHR and CDHF.
l . ~ c y to Heartburn (Seconds)
HuIlbum Severity
Musclethick. Pressure Pressure hess during 10 amplitudeof durationabove mlnutes of Acld swallows(%) 15 mmHg during score(1-10) perfuldon(%) t t (%}swallow t
Acid1 317!137 5.3'!2.4 1'0"1!6 115 23 117 ~15 Add2 93~47' 8.5 '1.5" 118,10' 143 ~43" 151 ~17" 1": The vaFuesshownhere are percentagechangein referencewith the salineinfusionperiod, whichis 100%. * : p < 0.05 comparedwith acid 1
707 Is Idiopathic Pancreatitis an Autoimraune Disease? Karine Nahon-Uzan, Philippe Levy, Dermot O'foole, Nadia Belmatoug, Philippe Ponsot, Laurent Palazzo, Pascal Hammel, Valerie Vilgrain, Philippe Ruszniewski Three forms of idiopathic chronie pancreatitis (following exclusion of alcoholic, hereditary and metabolic causes) have been described on imaging: a) pseudotumora[; b) duct-destructive c) chronic pancreatitis (CP) with duct dilatation Aim: to identify autotmmune stigmata in the three forms of idiopathic CP. Methods: all patients who underwent exploration of idiopathic CP between 1996 and 2001 were included. The following were performed for each patient: detailed history and clinical examination by an internal medicine specialist in autoimmune disorders; auto-annhodies and immunogloublin screening; pancreatic duct imaging (ERCP or MR-pancreatography); and EUS. The different forms were thus defined: a) pseudotumoral: nodule presence on imaging; b) duct-destructive as described by Ectors
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et al (1); c) dilated ducts according to Cambridge classification Patients with an obvious cause (alcohol, metabolic, hereditary, ductat obstruction) were excluded. Results: 60 patients were included (pseudotumora[ : n = 11 ; duct-destructive : n = 28 ; dilated ducts : n = 21 ). Duct-destructive types commenced at an earlier age than the dilated ducts type (median : 28 vs 39 years : P< 0,05). A tendency towards more frequent episodes of acute pancreatitis was observed in the duct-destructive type (86%) compared to dilated ducts (71%) and pseudotumoral (55%) types. Cholestasis was more frequent in pseudotumoral forms (46%) vs duct-destructive (11%) vs dilated ducts (6% : P< 0,007) Pancreatic calcifications were unique to the dilated duct type (81% ; P
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'I08 Progression of Endocrine Insufficiency in Tropical Caicific Pancreatis: A Longitudinal Follow-up Study of 53 Non-Diabetic Subjects K. K. Barman, V. S. Rajah, R. Deepa, Suresh T. Churl, V. Mohan Background/Aims: Tropical calcific pancreatitis (TCP), a form of idiopathic, early-onset calcific chronic pancreatitis is the most prevalent form of chronic pancreatitis in South India. Diabetes (DM) in TCP is also called fibrocalculons pancreatic diabetes (FCPD) and is often seen at diagnosis of TCP. The aim of this longitudinal follow-up study was to delineate the natural history of pancreauc endocnne insufficiency in TCP patients who presented without DM. Methods: Seventy-s/x TCP patients, referred to MDRF for evaluation of pancreatic endocrine function, who did not have DM by WHO criteria at baseline, were included in this study. The 53/76 subjects (70%) available for follow-up underwent yearly oral glucose tolerance tests for evaluation of pancreatic endocrine function until they developed DM. Pancreatic exocrine function was measured using fecal chymotrypsin. Baseline demographic (age, gender) and clinical (age at onset of pain, body mass index (BMI)) characteristics were noted. Time to development of DM was calculated nsmg Kaplan Meir method. Data are expressed as mean _+standard deviation. Results: Baseline characteristics (age, gender, fasting plasma glucose) of the 53 study subjects were similar to those of the 23 subjects not available for follow-up. The 53 subjects were followed for a mean of 6.8 years (range: 1- 21). Their mean age at baseline was 27 _+ 12 years, 68% were males, and the mean BM1 was 19.5 +_ 4.3 kg/m ~. On fofiow-up, 24/53 (45%) developed DM; the incidence of DM in this cohort was 6 6 per 100 person years of follow up. Compared to subjects who remained non-diabetic on follow-up, the subjects who developed DM were older (24 -+ 11 vs 31 _+12 years, p<0.03), had higher BMI (18.4 +- 3.8 vs 21.4 kg/m 2 +- 4.6, p<0.03) and Bad lower fecal chymotrypsin (3.9 +- 2.9 vs 1.4-+ l. 1, p < 0 001) at baseline. Of the subjects who developed DM, 63% had a family history of DM compared to 34% of non-diabetic subjects (p =us). Median time to development of DM after diagnosis of TCP was 12.5 years and the diagnosis of TCP was made a median of 5.2 years after onset of pain Conclusion: In TCP there is progressive deterioration in pancreatic endocrine function, with development of FCPD in nearly half the patients on foflow-up. FCPD is merely the later stage in the natural history of TCP.
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Endoscopic Ultrasound-Guided Pancreatic Trucut Needle Biopsies in Patients with Suspicious Pancreatic Masses Alberto Larghi, Elizabeth C. Verna, Stawos S. Stavropoulos, Heidrun Rotterdam, Jennifer J. Koehnken, Charles J. Lightdale, Peter D. Stevens Background: A trucut needle biopsy able to obtain specimens from the pancreas and other perigastric organs under EUS guidance has recently been developed and successfully tested in animals. The purpose of our study was to prospectively assess the clinical utility of this new diagnostic tool in patients with suspicious pancreatic masses. Methods: Patients with a pancreatic mass detected by CT were studied. EUS-guided trucut needle biopsy (EUSTNB) was performed with a linear echoendoscope using a disposable 19-gauge needle (Quick Core Biopsy, Wilson-Cook Medical Inc.). In a subset of patients, EUS-guided fine needle aspiration (EUS-FNA) with a 22-gauge needle was performed during the same procedure. Patients were assessed for complications throughout the following 48-hour period. Results: Sixteen patients (mean age 62; M/F 8/8) underwent EUS-TNB. Fragmentation of the biopsy specimens was frequently observed with fragment length ranging from 0.1 to 1.1 cm Histological examination revealed pancreatic cancer in 11 patients (69%). Four of the 5 patients with negative EUS-TNB underwent CT-guided biopsy, two of which were positive for adenocarcinoma. EUS-FNA was also performed in 11 patients. In four of the seven patients with positive EUS-TNB, EUS-FNA results were negative, while in all other cases results of both procedures were equiva/ent. No acute or long-term complications were observed. Conclusions: Our preliminary data show that pancreatic tissue for histological examination can be safely obtained with EUS-TNB. Studies comparing EUS-TNB and EUSFNA in large patient populations are needed to determine if this technique will increase the diagnostic yield and accuracy of EUS in the evaluation of patients with pancreatic
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A Shortened Endoscopic Pancreatic Function Test (ePFT) May Be Used as Screening Test for Chronic Pancreatitis (CP) in Patients with Chronic Abdominal Pain (CAP) Darwin Conwell, Gregory Zuccaro, John Vargo, Frederick Vanlente, John Dumot, Particia Trolfi, Jufie Tsiramhidis
Clinical and Nutritional Effects of Anti-oxidant Supplementation: a Prospective Randomized Study in Patients with Chronic Pancreatitis Pierre H. Deprez, Sebastien Delazzer, Laurence Galanti, John Lebrun, Andre Geubel, Yves Horsmans The aim of our study was to compare the nutritional, metabolic, functional and clinical effects of a diet adapted to chronic pancreatitis versus anti-oxidant supplementation. Patients: Thirty patiems with chronic pancreatitis were given an adapted diet plus or minus antioxidant supplementation (Quatral | rid) in a prospective, randomized study with a crossover after 3 months. The dietary counseling was aimed to correct all errors detected during a preliminary dietary evaluation. The latter showed an insufficient intake of vii A (33% of patients), vit D (93%), vii E (86.7%), vit C (76.7%), copper (83.3%), zinc (30%), selenium (56.7%). During the study, dietary assessment, clinical pain evaluation 0/AS), nutritional and metabolic assessment (BMI, fat mass, basal metabolism), determination of vitamins and anti-oxidants blood levels and measurement of exocrine (fecal elastase and steatorrhea) and endocrine function (HOMA test) were performed at start point, after 3 and 6 months. Results: In basal state, a significant percentage of patients was shown to have a hypermetabolic status (46.7%), an excessive BET (60%) with a correct nutrient repartition (lipid 35%, protein 16% and glucids 46%), a low BMI (33.3%), and low levels of albumin and prealbumin (10.7%, 22.2%, respectively), vii D (60.7%), vit C (64.3%), and zinc (33.3%). The anti-oxidant system was impaired m half our patients (total anti-oxidant level) with decreased plasma glutathion peroxidase levels in 25% and superoxyde dismutase in 43.5% of patients. Exocrine function was impaired in 66.7% of our patients and diabetes was present in 267%. Mean pain score was 31.7%. No significant changes were seen with the adapted diet. The oral antioxidant supplements significantly increased levels of vit C, vit E, carotene, zinc, selenium, glutathion peroxydase and superoxyde dismutase (P
BACKGROUND. We have developed a 1 hour endoscopic collection method (ePFT) using synthetic porcine secretin to evaluate CAP pts with and without risk factors (+RF, -RF) for CP. A shorter test would be desirable and more time efficient. AIM: 1) Examine the ePFT in pts with CAP and CP 2) Determine accuracy of specific timed aspiration periods. METHODS. 3 groups of pts studied: 1) CAP without risk factors (CAP-RF), 2) CAP with risk factors (CAP + RF), and 3) chronic pancreatitis (CP). ERCP confirmed CP dLagnosis. ePFT protocol: 1) upper endoscopy, 2) IVP secretin (0.2 mcg/kg) over 1 min, 3) gastric fluid discard, 4) duodenal fluid aspirations via endoscope in trap at 0, 15, 30, 45, and 60 rain after secretin injection, 5) analysis for bicarb conc( Normal > 80 meq/L). Data analysis: p-values calculated using one-sided t-test; szgnificance 0.05. RESULTS. 55 pts studied: 23 CAP-RF, 15 CAP+RE, and 17 CP Peak bicarb conc for all groups in graphic. The CAPRF group is significantly greater than 80 meq/L (p=0.008; 95% CI 83.34-92.48 meq/L). The CAP+ RF group is significantly less than 80 meq/L (p=0.0127; 95% CI 64.77-78.83 meq/L). The CP group is significantly less than 80 meq/L (p<0.001, 95% CI 37.55-58.56 meq/L) The diagnostic accuracy of specific timed aspirations dunng peak pancreatic secretion (30-45 rain) are shown tn table. CONCLUSIONS: i) The ePFT using synthetic porcine secretin can distinguish pts with CP from CAP. 2) Duodenal aspiration obtained at 30 minutes during EGD represents peak pancreatic secretion and is accurate for the diagnosis of CP. CLINICAL IMPLICATION: A brief and simple screening ePFT may be performed by IV injection of secretin followed by endoscopy and simple aspiration 30 minutes following injecnon.
Dlagne~c Accuracy ePFT Aspirins
CAP-RF CAP~RF CP Overall Accuracy
AGA Abstracts
N 23 15 17
30 mln (%) O111,/ 45 mln (~) Only 3e, 4~ r ~ ComNnee ~.) 19 (82) 13 (56) 21 (91,3) 15 (100) 12 (80) 15 (100) 17 llCO) 17(100) 17(100) 51 (96.2) 42 (76) 53 (96.3)
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