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Detection of Helicobacter DNA in the bile from the obstructed bile duct
GASTROENTEROLOGYVol. 114, No. 4
A526 AGA ABSTRACTS • G2144
TRANSITION METALS ARE PREFERENTIALLY PRECIPITATED IN THE CORES OF SOLITARY CHOLESTEROL GALLSTONES. HS Kaufman. KP Murray, JH Shin, P Frasca, P Laraia, PA Lipsett, KD Lillemoe, HA Pitt. Dept. of Surgery, Johns Hopkins, Baltimore, MD, Medical College of Wisconsin, Milwaulkee, WI and EMSL Analytical, Westmont, NJ. Cholesterol gallstones contain precipitated calcium salts and proteins which are often concentrated in stone centers. Up to 15% of patients with cholesterol gallstones possess a large solitary gallstone. Biliary transferrin, a potent pronucleator of cholesterol crystals, binds a variety of transition metals such as Fe, Cu and Zn. The purpose of this study was to compare the relative concentrations of Ca, Fe, Cu and Zn in the centers and peripheries of solitary and multiple gallstones. Therefore, solitary (S, n=13) and multiple (M, n=15) cholesterol stones were collected, washed, dried and stored at room temperature. Representative areas from each core and periphery were dissected. Powdered samples were subjected to acid digestion and prepared for quantitative elemental analysis by inductively coupled plasma analysis (ICP). Ca, Fe, Cu, and Zn concentrations from the core and periphery in each stone type were: Stone Ca Fe Cu Zn Type & Site (mg/kg) (mg/kg) (mg/kg) (mg/kg) S Core 2750 -+900* 22 -+4* 36 -+ 9* 32 ± 8* S Periphery 8200-+3000 12-+2 11-+2 19-+3 M Core 5400 -+ 1800 23 -+ 5 50 ± 14t 42 -+ 15 M Periphery 5900 - 2200 20 ± 4 18 ± 8 19 ± 4 *p < 0.05 vs solitary periphery, t p < 0.05 vs multiple periphery (paired Student's t-test) These data demonstrate that 1) calcium is more concentrated in the peripheries of solitary gallstones while 2) transition metals are more concentrated in the cores of solitary gallstones. Only Cu was significantly more concentrated in the cores than peripheries of multiple gallstones. We conclude that the role of calcium and the transition metals differ between solitary and multiple stones. • G2145 INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) IS THE PRINCIPAL ISOFORM EXPRESSED IN HUMAN GALLBLADDER. A,p, Keaveny, G.D. Offner, N.H. Afdhal. Section of Gastroenterology, Boston University School of Medicine, Boston, MA. Introduction: We have previously shown that nitric oxide (NO) plays an important role in the maintenance of basal inhibitory tone in the prairie dog gallbladder (Salomons et al., Am. J. Physiol. 272:G770-8, 1997) Furthermore, these studies also showed that NO inhibits the contractile response of the gallbladder to cholecystokinin and bethanecol. Immunohistochemical staining of prairie dog gallbladder mucosa suggested that these activities of NO could be correlated with expression of neuronal NOS (bNOS). Little is known, however, about the expression of NOS in the human gallbladder, and the aim of this study was to compare the pattern of NOS isoform expression in normal gallbladder and in gallbladder tissue obtained from patients with gallstone disease. Methods: Normal gallbladder tissue was obtained from livers not suitable for organ transplantation. Diseased gallbladder tissue was obtained at cholecystectomy from patients with cholesterol gallstones. RNA was isolated from homogenates of whole gallbladder and RT-PCR reactions were carried out using primers specific for bNOS, iNOS, and endothelial NOS (eNOS). In addition, proteins in tissue homogenates were subjected to gel electrophoresis (SDS PAGE) and Western blotting. Blots were probed with commercially available polyclonal antibodies against bNOS, iNOS and eNOS. Results: Analysis of PCR products obtained with iNOS-specific primers revealed a DNA fragment of the expected size in RNA isolated from both normal and diseased gallbladder. PCR reactions performed with bNOS- or eNOS-specific primers did not yield detectable NOS DNA fragments in RNA from any tissue sample. Similarly, Western blots probed with anti-iNOS antibodies showed a major 130 kDa immunoreactive band in both normal and diseased gallbladder tissue whereas blots probed with anti-bNOS or anti-eNOS showed no prominent immunoreactive proteins with the correct molecular weight. Parallel RT-PCR experiments carried out with RNA isolated from a well characterized gallbladder cell line as well as RNA isolated gallbladder epithelial cells grown in primary culture also revealed iNOS DNA fragments. This suggests that the iNOS mRNA and protein detected using homogenates of whole gallbladder are derived primarily from mucosal cells. Conclusions: At both the mRNA and protein levels, iNOS appears to be the major NOS isoform expressed in the human gallbladder. Expression of this isoform in gallbladder epithelial cells points to a potential role for iNOS in regulation of mucosal cell function. • G2146 DETECTION OF HELICOBACTER DNA IN THE BILE FROM THE OBSTRUCTED BILE DUCT. J. T. Kim. I. H. Roe, Y. J. Jin, J. H. Shin, J. H. Lee. Division of Gastroenterology, Deprtment of Surgery and Medicine, Research Institute for Gastroenterology, Dankook University College of Medicine, Chun-an, South Korea Objectives Several newly recognized Helicobacter spp. can cause persistent hepatitis, hepatoma, cholangiopancreatitis, and cholecystitis in animals.
Recently some studies have been reported that Helicobacter DNA can be found in the bile from the patients with diseased bile duct, although its clinical significance is still unclear. We also found that Helicobacter DNA might be detected in the bile from the intrahepatic duct stones, bile duct cancers, and pancreatic head cancers. We investigated the existence, and character of Helicobacter in the bile from the obstructed bile duct, the relationship with pH and the other bacteria found in the bile. Methods Twenty-eight bile samples (15 from bile duct cancer, 6 from pancreatic head cancer, 7 from bile duct stones) were obtained from the PTBD route. Bile pH measurement, and Helicobacter culture in microaerophillic and unaerobic conditions were performed. The primers chosen for PCR amplification for detection and characterization were ureA (411 bp) and cagA gene (298 bp), respectively. Primer of 16srRNA for all known bacteria including Helicobacter was used, and the kinds of bacteria were identified by RFLP. Results Helicobacter DNA was detected in 39.3%. The bile pH was not related with presence of Helicobacter (7.83-+ 0.41 vs 7.78 ± 0.48). The prevalence of cagA was 35.7% and 16s rRNA was found in 46.4%. The specific 16s rRNA band for Helicobacter was observed in 14.3%. All the cultures were not successful. Conclusions Although the Helicobacter spp. were not cultured, Helicobacter exists in the bile from the diseased bile duct, and coexists with other bacteria. These results should stimulate studies to ascertain whether these Helicobacter play a role in the pathogenesis of bile duct diseases in humans. G2147
PAPILLARY STRUCTURE IS NOT AFFECTED BY ENDOSCOPIC PAPILLARY BALLOON DILATION, Y. Komats~, H. Isayama, T. Takemura, K. Tateishi, N. Toda, M. Ohhashi, M. Yamagata, M. Tada, H. Yoshida, T. Kawabe, Y. Shiratori and M. Omata. Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Department of Pathology, Japanese Red Cross Medical Center, Japan Background and Aim: Endoscopic papillary balloon dilation (EPBD) is a
safe and effective procedure in the management of bile duct stones and it seems to be a less traumatic approach than sphincterotomy. However, little is known about architectural change of papilla after EPBD. We investigate the effects of balloon dilation on papillary structure. Patients and Methods: Between May 1994, and September 1997, EPBD was performed in 315 patients (293 patients for removal of bile duct stone, 22 patients for insertion of stent or baby cholangioscope). Of those, histological examinations were undertaken in 8 patients. The indications of EPBD were bile duct stone removal in 2 patients, stent insertion in 5 patients and cholangioscopy in 1 patient. The specimens were obtained by autopsy in 5 patients and by surgical operation in 3 patients (15 days to 9 months after EPBD). Serial sections through the papilla were stained by H & E and Masson's trichrome. The specimens were also obtained from 4 patients without biliary disease as control. Results: No smooth muscle disruption nor architectural distortion was observed in all of 8 patients, although mild inflammation were seen in 5 patients. No fibrosis nor stenosis was detected in the patient examined 9 month after EPBD. Conclusion: The results show that papillary architecture is not affected by EPBD. It may imply that EPBD preserves papillary function. G2148 PERIPAPILLARY DIVERTICULA: ASSOCIATION WITH AN INCREASED INCIDENCE OF CHOLEDOCHOLITHIASIS. DIFFICULTIES IN ERCP? SPHINCTEROTOMY AND EXTRACTION OF STONES. A. Konstantinldis, A.Germanopoulos, E.Kanellopoulou, V.Delis, V.Balatsos, A. Pantes, V.Vamvakousis, V.Ekonomou, Ph.Georgopoulos, N.Skandalls. GastroenterologistUnit,General Hospital "G.Gennimatas",ATHENS. Aim: To clarify if peripaplliary diverticula are associated with an increased incidence of choledocholitiasis. To what extent their presence confer difficulties during ERCP, sphincterotomy and extraction of choledochal stones. Patients: The patients had problems from the biliary tree and / or pancreas and unterwent ERCP. Method: Duodenescopy, assesment of perlpaplliary diverticula, ERCP, Sphincterotomy and extraction of choledochal stones. Results: Choledocholithiasis was found in 338 ( 23.8% ) among 1422 patients ( aged 17-93 years ) who unterwent ERCP. In 96 ( 6.8% ) patients ( male 35, female 61 with a mean age 65.6 years ) a diverticulum was found in the area of papllia and for all them there was a strong indication of choledocholithiasis. In 89 p. ( 92.7% ) cannulation and opacification of CBD was achieved. All of them had choledochal stones. Sphincterotomy was done in 87 p. ( In 2 the endoscopist had reservations). Ductal clearance was not achieved during the lrst ERCP in 12 p. The reasons were: sphincterotomy was not done in 2, in 4 there were multiple stones, in 3 the stone could not be collected by a basket catheter, 2 couldn't endure the stress of the procedure, in 1 bleeding during sphincterotomy complicated the ERCP. The percentage of successful ERCP was similar in patients without peripapillary diverticulum. Conclusion: 1) Peripapillary diverticula was seen in 6,8 % of the patients who underwent ERCP and in 1/4 of them who had choledocholithiasis. 2) Their presence didn't confer major difficulties in the cannulation-opacification of CBD and extraction of stones. 3) A very strong correlation of peripapillary diverticula to choledochal stones was found.
A526 AGA ABSTRACTS • G2144
TRANSITION METALS ARE PREFERENTIALLY PRECIPITATED IN THE CORES OF SOLITARY CHOLESTEROL GALLSTONES. HS Kaufman. KP Murray, JH Shin, P Frasca, P Laraia, PA Lipsett, KD Lillemoe, HA Pitt. Dept. of Surgery, Johns Hopkins, Baltimore, MD, Medical College of Wisconsin, Milwaulkee, WI and EMSL Analytical, Westmont, NJ. Cholesterol gallstones contain precipitated calcium salts and proteins which are often concentrated in stone centers. Up to 15% of patients with cholesterol gallstones possess a large solitary gallstone. Biliary transferrin, a potent pronucleator of cholesterol crystals, binds a variety of transition metals such as Fe, Cu and Zn. The purpose of this study was to compare the relative concentrations of Ca, Fe, Cu and Zn in the centers and peripheries of solitary and multiple gallstones. Therefore, solitary (S, n=13) and multiple (M, n=15) cholesterol stones were collected, washed, dried and stored at room temperature. Representative areas from each core and periphery were dissected. Powdered samples were subjected to acid digestion and prepared for quantitative elemental analysis by inductively coupled plasma analysis (ICP). Ca, Fe, Cu, and Zn concentrations from the core and periphery in each stone type were: Stone Ca Fe Cu Zn Type & Site (mg/kg) (mg/kg) (mg/kg) (mg/kg) S Core 2750 -+900* 22 -+4* 36 -+ 9* 32 ± 8* S Periphery 8200-+3000 12-+2 11-+2 19-+3 M Core 5400 -+ 1800 23 -+ 5 50 ± 14t 42 -+ 15 M Periphery 5900 - 2200 20 ± 4 18 ± 8 19 ± 4 *p < 0.05 vs solitary periphery, t p < 0.05 vs multiple periphery (paired Student's t-test) These data demonstrate that 1) calcium is more concentrated in the peripheries of solitary gallstones while 2) transition metals are more concentrated in the cores of solitary gallstones. Only Cu was significantly more concentrated in the cores than peripheries of multiple gallstones. We conclude that the role of calcium and the transition metals differ between solitary and multiple stones. • G2145 INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) IS THE PRINCIPAL ISOFORM EXPRESSED IN HUMAN GALLBLADDER. A,p, Keaveny, G.D. Offner, N.H. Afdhal. Section of Gastroenterology, Boston University School of Medicine, Boston, MA. Introduction: We have previously shown that nitric oxide (NO) plays an important role in the maintenance of basal inhibitory tone in the prairie dog gallbladder (Salomons et al., Am. J. Physiol. 272:G770-8, 1997) Furthermore, these studies also showed that NO inhibits the contractile response of the gallbladder to cholecystokinin and bethanecol. Immunohistochemical staining of prairie dog gallbladder mucosa suggested that these activities of NO could be correlated with expression of neuronal NOS (bNOS). Little is known, however, about the expression of NOS in the human gallbladder, and the aim of this study was to compare the pattern of NOS isoform expression in normal gallbladder and in gallbladder tissue obtained from patients with gallstone disease. Methods: Normal gallbladder tissue was obtained from livers not suitable for organ transplantation. Diseased gallbladder tissue was obtained at cholecystectomy from patients with cholesterol gallstones. RNA was isolated from homogenates of whole gallbladder and RT-PCR reactions were carried out using primers specific for bNOS, iNOS, and endothelial NOS (eNOS). In addition, proteins in tissue homogenates were subjected to gel electrophoresis (SDS PAGE) and Western blotting. Blots were probed with commercially available polyclonal antibodies against bNOS, iNOS and eNOS. Results: Analysis of PCR products obtained with iNOS-specific primers revealed a DNA fragment of the expected size in RNA isolated from both normal and diseased gallbladder. PCR reactions performed with bNOS- or eNOS-specific primers did not yield detectable NOS DNA fragments in RNA from any tissue sample. Similarly, Western blots probed with anti-iNOS antibodies showed a major 130 kDa immunoreactive band in both normal and diseased gallbladder tissue whereas blots probed with anti-bNOS or anti-eNOS showed no prominent immunoreactive proteins with the correct molecular weight. Parallel RT-PCR experiments carried out with RNA isolated from a well characterized gallbladder cell line as well as RNA isolated gallbladder epithelial cells grown in primary culture also revealed iNOS DNA fragments. This suggests that the iNOS mRNA and protein detected using homogenates of whole gallbladder are derived primarily from mucosal cells. Conclusions: At both the mRNA and protein levels, iNOS appears to be the major NOS isoform expressed in the human gallbladder. Expression of this isoform in gallbladder epithelial cells points to a potential role for iNOS in regulation of mucosal cell function. • G2146 DETECTION OF HELICOBACTER DNA IN THE BILE FROM THE OBSTRUCTED BILE DUCT. J. T. Kim. I. H. Roe, Y. J. Jin, J. H. Shin, J. H. Lee. Division of Gastroenterology, Deprtment of Surgery and Medicine, Research Institute for Gastroenterology, Dankook University College of Medicine, Chun-an, South Korea Objectives Several newly recognized Helicobacter spp. can cause persistent hepatitis, hepatoma, cholangiopancreatitis, and cholecystitis in animals.
Recently some studies have been reported that Helicobacter DNA can be found in the bile from the patients with diseased bile duct, although its clinical significance is still unclear. We also found that Helicobacter DNA might be detected in the bile from the intrahepatic duct stones, bile duct cancers, and pancreatic head cancers. We investigated the existence, and character of Helicobacter in the bile from the obstructed bile duct, the relationship with pH and the other bacteria found in the bile. Methods Twenty-eight bile samples (15 from bile duct cancer, 6 from pancreatic head cancer, 7 from bile duct stones) were obtained from the PTBD route. Bile pH measurement, and Helicobacter culture in microaerophillic and unaerobic conditions were performed. The primers chosen for PCR amplification for detection and characterization were ureA (411 bp) and cagA gene (298 bp), respectively. Primer of 16srRNA for all known bacteria including Helicobacter was used, and the kinds of bacteria were identified by RFLP. Results Helicobacter DNA was detected in 39.3%. The bile pH was not related with presence of Helicobacter (7.83-+ 0.41 vs 7.78 ± 0.48). The prevalence of cagA was 35.7% and 16s rRNA was found in 46.4%. The specific 16s rRNA band for Helicobacter was observed in 14.3%. All the cultures were not successful. Conclusions Although the Helicobacter spp. were not cultured, Helicobacter exists in the bile from the diseased bile duct, and coexists with other bacteria. These results should stimulate studies to ascertain whether these Helicobacter play a role in the pathogenesis of bile duct diseases in humans. G2147
PAPILLARY STRUCTURE IS NOT AFFECTED BY ENDOSCOPIC PAPILLARY BALLOON DILATION, Y. Komats~, H. Isayama, T. Takemura, K. Tateishi, N. Toda, M. Ohhashi, M. Yamagata, M. Tada, H. Yoshida, T. Kawabe, Y. Shiratori and M. Omata. Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Department of Pathology, Japanese Red Cross Medical Center, Japan Background and Aim: Endoscopic papillary balloon dilation (EPBD) is a
safe and effective procedure in the management of bile duct stones and it seems to be a less traumatic approach than sphincterotomy. However, little is known about architectural change of papilla after EPBD. We investigate the effects of balloon dilation on papillary structure. Patients and Methods: Between May 1994, and September 1997, EPBD was performed in 315 patients (293 patients for removal of bile duct stone, 22 patients for insertion of stent or baby cholangioscope). Of those, histological examinations were undertaken in 8 patients. The indications of EPBD were bile duct stone removal in 2 patients, stent insertion in 5 patients and cholangioscopy in 1 patient. The specimens were obtained by autopsy in 5 patients and by surgical operation in 3 patients (15 days to 9 months after EPBD). Serial sections through the papilla were stained by H & E and Masson's trichrome. The specimens were also obtained from 4 patients without biliary disease as control. Results: No smooth muscle disruption nor architectural distortion was observed in all of 8 patients, although mild inflammation were seen in 5 patients. No fibrosis nor stenosis was detected in the patient examined 9 month after EPBD. Conclusion: The results show that papillary architecture is not affected by EPBD. It may imply that EPBD preserves papillary function. G2148 PERIPAPILLARY DIVERTICULA: ASSOCIATION WITH AN INCREASED INCIDENCE OF CHOLEDOCHOLITHIASIS. DIFFICULTIES IN ERCP? SPHINCTEROTOMY AND EXTRACTION OF STONES. A. Konstantinldis, A.Germanopoulos, E.Kanellopoulou, V.Delis, V.Balatsos, A. Pantes, V.Vamvakousis, V.Ekonomou, Ph.Georgopoulos, N.Skandalls. GastroenterologistUnit,General Hospital "G.Gennimatas",ATHENS. Aim: To clarify if peripaplliary diverticula are associated with an increased incidence of choledocholitiasis. To what extent their presence confer difficulties during ERCP, sphincterotomy and extraction of choledochal stones. Patients: The patients had problems from the biliary tree and / or pancreas and unterwent ERCP. Method: Duodenescopy, assesment of perlpaplliary diverticula, ERCP, Sphincterotomy and extraction of choledochal stones. Results: Choledocholithiasis was found in 338 ( 23.8% ) among 1422 patients ( aged 17-93 years ) who unterwent ERCP. In 96 ( 6.8% ) patients ( male 35, female 61 with a mean age 65.6 years ) a diverticulum was found in the area of papllia and for all them there was a strong indication of choledocholithiasis. In 89 p. ( 92.7% ) cannulation and opacification of CBD was achieved. All of them had choledochal stones. Sphincterotomy was done in 87 p. ( In 2 the endoscopist had reservations). Ductal clearance was not achieved during the lrst ERCP in 12 p. The reasons were: sphincterotomy was not done in 2, in 4 there were multiple stones, in 3 the stone could not be collected by a basket catheter, 2 couldn't endure the stress of the procedure, in 1 bleeding during sphincterotomy complicated the ERCP. The percentage of successful ERCP was similar in patients without peripapillary diverticulum. Conclusion: 1) Peripapillary diverticula was seen in 6,8 % of the patients who underwent ERCP and in 1/4 of them who had choledocholithiasis. 2) Their presence didn't confer major difficulties in the cannulation-opacification of CBD and extraction of stones. 3) A very strong correlation of peripapillary diverticula to choledochal stones was found.