Usefulness of supernatant of pancreatic juice for genetic analysis of K-ras in diagnosis of pancreatic carcinoma.

AIMS: To ascertain whether analysis of K-ras mutations at codon 12 (KRM) in the supernatant of pure pancreatic juice (PPJ) is more useful for the diagnosis of pancreatic carcinoma (PCa) than that in sediment, the authors analyzed KRM in DNA extract from not only the sediment but also the supernatant of PPJ and compared the results. METHODOLOGY: PPJ was collected endoscopically from 19 patients with PCa and 25 patients with chronic pancreatitis (CP). DNA was extracted from the supernatant and the sediment of PPJ. Mutant allele-specific amplification (MASA) was performed for KRM analysis with the DNA extracts from these samples. RESULTS: The incidence of KRM in the supernatant of PPJ was 89% (17 of 19) in patients with PCa and 28% (7 of 25) in patients with CP, whereas that in the sediment was 79% (15 of 19) in patients with PCa and 20% (5 of 25) in patients with CP. Although there was no significant difference in KRM incidence between supernatant and sediment, the positive rate of KRM was higher in the former. Additionally, with regard to the PCa cases, KRM were found in the supernatant alone in four cases and in the sediment alone in two cases. Consequently, by a combination assay, all of the patients with PCa showed KRM in either the supernatant or sediment of PPJ. Although there was no relation between the incidence of KRM in PPJ and the location and size of tumor, and clinical stage of carcinoma in the patients with PCa, two patients with clinical stage I disease showed KRM in the supernatant. CONCLUSION: These results suggest that the positive rate of KRM in the supernatant is not lower than that in the sediment, and simultaneous analysis of KRM in the supernatant and sediment of PPJ enhances the genetic diagnosis of PCa.

P8 expression is induced in acinar cells during chronic pancreatitis.

The p8 gene is barely expressed in the normal pancreas, but is overexpressed in acute pancreatitis. To elucidate the dynamic expression of p8 mRNA and its significance in the course of chronic pancreatitis, we investigated the p8 expression in spontaneous chronic pancreatitis in the WBN/Kob rat as well as in humans and arginine-treated rat pancreatic acinar AR4-2J cells. p8 mRNA was significantly increased at 12 weeks when chronic pancreatitis first appeared in the WBN/Kob rats. p8 was immunolocalized in the acinar cell nuclei. Acinar cell apoptosis was significantly increased at 12 and 20 weeks in the WBN/Kob rats. In AR4-2J cells, p8 mRNA was significantly induced at 4 hr after arginine addition. Apoptosis of AR4-2J cells was not increased during the strong expression of p8 mRNA. These results suggest that p8 is induced in the acinar cells during chronic pancreatitis as the self-defence mechanism against proapoptotic insults.

Effect of camostat mesilate on the expression of pancreatitis-associated protein (PAP), p8, and cytokines in rat spontaneous chronic pancreatitis.

An oral protease inhibitor, camostat mesilate (CM) has been used clinically for chronic pancreatitis (CP) in Japan, but it lacks enough scientific evidence of its effectiveness. The aim of this study was to analyze the effect of CM on the gene expressions of pancreatitis-associated protein (PAP), p8, and cytokines such as interleukin-6 and transforming growth factor-beta1 in spontaneous CP model (WBN/Kob rats). CM (10 mg/100 g body weight), mixed in MB-3 diet, was administered orally and gene expressions were analyzed by reverse transcription-polymerase chain reaction. In untreated WBN/Kob rats, the gene expressions of all the four factors peaked at 12 weeks, whereas they were significantly suppressed in the CM-treated rats. CM significantly increased the body weight and pancreatic wet weight, and it significantly inhibited inflammatory changes and fibrosis of the pancreas. These results suggest that CM inhibits pancreatic inflammation and fibrosis through the suppression of gene expressions of PAP, p8, and cytokines in CP.

Pneumatosis intestinalis and hepatic portal venous gas caused by mesenteric ischemia in an aged person.

An 82-year-old woman complaining of abdominal pain and vomiting was admitted to our emergency department. Abdominal X-ray, ultrasonography, and computed tomography showed hepatic portal venous gas, as well as pneumatosis intestinalis. We first suspected superior mesenteric arterial thrombosis. However, her physical findings, including computed tomography scanning and laboratory data, did not support the presence of bowel necrosis. The gas disappeared after 1 day. After the 12th day, she had recovered with conservative therapy, and she was discharged on the 41st day. Many reports indicate that hepatic portal venous gas is often associated with bowel necrosis, and urgent operation is recommended in such instances. In this patient, total colonoscopy on the 7th day revealed longitudinal redness, suggesting mesenteric ischemia. Thus, we speculate that this is a rare case of mesenteric ischemia without bowel necrosis associated with both pneumatosis intestinalis and hepatic portal venous gas.

Overexpression of p8 is inversely correlated with apoptosis in pancreatic cancer.

A recently identified gene, p8, has cell growth-promoting activity and is strongly induced in acute pancreatitis. In this study, we detected p8 and single-stranded DNA (ssDNA) for apoptosis by immunohistochemistry in human pancreatic cancer. The p8 was overexpressed (>30% per 1000 cancer cells) in 26 of 44 (59%) pancreatic cancers, and apoptosis (ssDNA-positive cells >10% per 1000 cancer cells) was recognized in 18 of 44 (41%) pancreatic cancers. There was a significant inverse correlation between the p8 overexpression and apoptosis (P < 0.05). Moreover, the expression pattern of high p8 and low ssDNA was seen significantly more often in lower age (<65 years), in moderately or poorly differentiated cancers, and in node-positive cases (P < 0.05). The p8 expression and apoptosis were not significantly correlated with survival. These results suggest that p8 overexpression is involved in antiapoptotic activity and the biological characteristics of pancreatic cancer.

Expression of tumor necrosis factor-alpha, interleukin-6, and interferon-gamma in spontaneous chronic pancreatitis in the WBN/Kob rat.

To clarify the pathophysiological significance of cytokines in chronic pancreatitis (CP), we analyzed tissue expressions of various cytokines in the onset and progression of spontaneous CP in the WBN/Kob rat. Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3) for 20 weeks, and 6 rats were killed every 4 weeks. Pathologically, CP occurred at 12 weeks and progressed thereafter. The inflammation and fibrosis peaked at 12 and 16 weeks, respectively. By semiquantitative reverse transcription-polymerase chain reaction, the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and interferon (IFN)-gamma mRNAs peaked at 8, 12, and 16 weeks, respectively. Immunohistochemistry showed IL-6 expression in infiltrating inflammatory cells and vascular endothelial cells, whereas TNF-alpha was expressed in both acinar and infiltrating cells. IFN-gamma was localized to acinar, infiltrating and ductal cells, and its expression intensity showed significant correlation with those of fibrosis, type III collagen and alpha-smooth muscle actin. The in situ hybridization results were consistent with the RT-PCR data. These results suggest that tissue expressions of TNF-alpha and IL-6 are involved in the onset of pancreatitis and that IFN-gamma expression is related to the progression of CP.

Hepatic peribiliary cysts diagnosed by magnetic resonance cholangiography.

We report a 76-year-old man with hepatic peribiliary cysts diagnosed by magnetic resonance cholangiopancreatography (MRCP). On his first admission, in 1991, the patient was misidentified as having localized dilatation of the left intrahepatic bile ducts, from the ultrasound (US) and computed tomographic (CT) findings, or primary sclerosing cholangitis from endoscopic retrograde cholangiopancreatography (ERCP). However, US and CT in 1998 suggested worsening of the lesions. MRCP was performed for the first time, revealing hepatic peribiliary cysts in the hepatic hilum and along the left hepatic ducts. Drip infusion cholangiography (DIC)-CT confirmed the extraluminal compression of the bile ducts, caused by the cysts, without an influx of contrast medium into the hepatic peribiliary cysts. Retrospective evaluation showed increases in the size and number of the cysts in 1998 compared with the findings in 1996.

Expression of clusterin in pancreatic acinar cell injuries in vivo and in vitro.

Clusterin is a secretory glycoprotein that is highly induced in several tissues in response to injury. The pathophysiologic significance of clusterin in the pancreas remains largely unknown. The aim of this work was to examine whether clusterin is expressed in spontaneous chronic pancreatitis in the WBN/Kob rat and to investigate the relationship between clusterin and apoptosis in pancreatic acinar AR4-2J cells. In the in vivo study, 4-week-old male WBN/Kob rats developed chronic pancreatitis at 12 weeks. Clusterin mRNA was expressed after 12 weeks and then decreased. Immunohistochemistry showed clusterin expression in the acinar cells. In the in vitro study, clusterin mRNA and protein were strongly induced in AR4-2J cells treated either with arginine, menadione, tumor necrosis factor-alpha or transforming growth factor-beta1. In the time course study with arginine or menadione, clusterin mRNA was expressed after 4 hours and peaked at 8 and 24 hours, whereas DNA fragmentation peaked at 72 hours. Our results show that clusterin is overexpressed in the pancreas at the onset of chronic pancreatitis in vivo and in cultured acinar cells in response to various stimuli in vitro, suggesting that clusterin is a defense mechanism of the exocrine pancreas.

Expression of p8 in human pancreatic cancer.

The p8 gene is a recently identified gene with mitogenic activity. p8 expression is induced in acute pancreatitis, pancreatic development, and regeneration. However, the expression of p8 in pancreatic cancer is not reported. We investigated p8 expression in 72 human pancreatic tissues, including 38 pancreatic cancers (PCs), by immunohistochemistry. p8 was overexpressed (positive cells >25% in 1,000 cells) in 71% (27 of 38) of PCs, but in only 17% (3 of 18) of chronic pancreatitis cases. There was no overexpression in mucinous cystadenoma or in normal pancreas. The p8 overexpression rate in PC was significantly higher than that in other conditions (P < 0.05). Reverse transcription-PCR analysis confirmed p8 mRNA overexpression (tumor/nontumor ratio >2) in 75% (3 of 4) of PCs. p8 was overexpressed also in human pancreatic cancer cell lines (MIA PaCa-2 and PANC-1). These results suggest that p8 is involved in the development of pancreatic cancer, reflecting its mitogenic activity.

Apoptosis in rat spontaneous chronic pancreatis: role of the Fas and Fas ligand system.

The Fas/Fas ligand (FasL) system is suggested to be correlated to the onset of inflammation and apoptosis in various diseases. However, whether Fas and FasL are expressed in chronic pancreatitis is unknown. The aim of this study was to examine the expression of the Fas/FasL system and to analyze its correlation with apoptosis in a spontaneous chronic pancreatitis model (the WBN/Kob rat). Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3). Different groups of rats were killed every four weeks, and pancreata were histopathologically examined. Fas and FasL mRNAs in the pancreas were detected with a reverse transcription-polymerase chain reaction method. The cellular localization of Fas and FasL mRNA and protein was determined with in situ hybridization (ISH) and immunohistochemistry (IHC). Apoptosis was detected with a terminal deoxynucleotidyltransferase-mediated method. Fas and FasL mRNA were expressed when the pancreas was still pathologically normal, and showed a biphasic peak at 12 and 20 weeks. ISH and IHC confirmed that Fas and FasL are expressed in the cytoplasm of acinar cells, ductal cells, and lymphocytes. An apoptotic index in acinar cells correlated to the expression of Fas and FasL mRNAs. These results suggest that the expression of the Fas/FasL system is involved in acinar cell apoptosis and the onset and progression of chronic pancreatitis in the WBN/Kob rat.

Antifibrotic effect of the herbal medicine Saiko-keishi-to (TJ-10) on chronic pancreatitis in the WBN/Kob rat.

Chronic pancreatitis is characterized by fibrosis. We reported an anti-inflammatory effect of the herbal medicine Saiko-keishi-to (TJ-10) on chronic pancreatitis. This study aimed to elucidate the antifibrotic effect of TJ-10. Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3) with or without TJ-10 (80 mg/100 g body weight) for 20 weeks. Pancreata were histopathologically examined at every 4 weeks, and the expression of fibrosis-related factors such as transforming growth factor beta1 (TGF-beta1), fibronectin (FN), alpha-smooth muscle actin (alpha-SMA), and type III collagen was analyzed. In untreated WBN/Kob rats, chronic pancreatitis developed at 12 weeks and progressed with marked fibrosis at 16 weeks, and the expression of TGF-beta1 and FN peaked at 12 weeks. However, in the TJ-10-treated rats, the rate of pancreatic fibrosis and the expression of TGF-beta1, FN, alpha-SMA, and type III collagen at 12 and 16 weeks decreased significantly compared to those in the untreated rats. These results suggest that TJ-10 inhibits the pancreatic fibrosis by the suppression of TGF-beta1 expression.

Effect of herbal medicine keishi-to (TJ-45) and its components on rat pancreatic acinar cell injuries in vivo and in vitro.

BACKGROUND: In an attempt to clarify the mechanism of the effect of a herbal medicine, Saiko-keishi-to (TJ-10), which is a combination of Keishi-to (TJ-45) and Sho-saiko-to (TJ-9), we investigated the effects of these two herbal medicines and their components on pancreatic acinar cell injury models in vivo and in vitro. METHODS: Four-week-old male WBN/Kob rats were fed an MB-3 pellet diet containing herbal medicine (TJ-9, TJ-10 and TJ-45). Expressions of pancreatitis-associated protein (PAP) and manganese superoxide dismutase (Mn-SOD) were analyzed with a reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The herbal medicines and two of their components, Keihi (Cinnamomi cortex) and Shakuyaku (Paeoniae radix alba), were tested in vitro using an arginine-treated rat pancreatic acinar AR4-2J cell injury model. The inducible nitric oxide synthase (iNOS) was assayed in in vitro experiments. RESULTS: TJ-45-treated WBN/Kob rats showed no evidence of pancreatitis whereas there were pathological changes of chronic pancreatitis in TJ-9-treated WBN/Kob rats. PAP was not expressed and Mn-SOD expression was increased in the TJ-10-, and TJ-45-treated rats. The herbal medicines and two components suppressed PAP mRNA expression and enhanced Mn-SOD and iNOS mRNA expression in arginine-treated AR4-2J cells. CONCLUSION: These results suggest that the herbal medicine TJ-45 is effective for chronic pancreatitis caused by pancreatic ischemia.

Pancreatitis-associated protein levels in pancreatic juice from patients with pancreatic diseases.

BACKGROUND: Pancreatitis-associated protein (PAP), the acute-phase protein of the pancreas, is overexpressed in acute pancreatitis. Serum PAP levels were reported to be useful as an indicator of the severity, prognosis and healing of acute pancreatitis. Although PAP was originally identified in pancreatic juice, there has been no clinical report on PAP levels in pancreatic juice. This study was conducted to determine levels of PAP in pancreatic juice (PJ-PAP) in various human pancreatic diseases. METHODS: PAP levels in endoscopically aspirated PJ were measured by enzyme-linked immunosorbent assay in 86 patients with pancreatic diseases. RESULTS: 55% of 22 patients with pancreatic cancer (PC) and 25% of 49 patients with chronic pancreatitis (CP) were positive (> 350 ng/ml) for PJ-PAP. PJ-PAP levels were significantly higher in PC than in CP, in which PJ-PAP was also significantly higher than in 15 control subjects. There was no significant correlation between PJ-PAP and serum PAP, and combination assay of serum PAP and/or PJ-PAP detected 80% of PC cases and 44% of CP cases. CONCLUSIONS: We have demonstrated that human PAP could be detected in pancreatic juice from patients with pancreatic diseases. Determination of PAP in pancreatic juice might be helpful for early detection of pancreatic injury.

Myxedema accompanied by huge portal-systemic shunt without portal hypertension.

A 43-year-old woman with a huge portal-systemic shunt accompanied by myxedema showed slow speech and behavior. Several imaging studies revealed a bold portal-systemic shunt from the splenic vein to the left renal vein. In addition, hypothyroidism caused by chronic thyroiditis was diagnosed, and synthesized thyroxine replacement was effective for the symptoms. However, the serum ammonia and indocyanin green retention remained in the abnormal range, nevertheless the portal vein pressure was normal and findings of liver cirrohsis were not recognized histologically. Surgical shunt closure was performed, resulting in normalized serum ammonia levels and serum branched chain amino acids /aromatic amino acids ratio, and improvement of the ammonia tolerance test.

Does the depth of gastric ulceration influence a modified dual therapy with amoxicillin and lansoprazole for Helicobacter pylori-associated gastric ulcer?

PURPOSE: To clarify whether the depth of ulceration evaluated by endoscopic ultrasonography (EUS) influences a modified dual therapy with amoxicillin and lansoprazole for the treatment of Helicobacter pylori-positive patients with gastric ulcer. PATIENTS AND METHODS: Twenty-two consecutive cases of gastric ulcer (nine superficial ulcers and 13 deep ulcers) in H pylori-positive patients were studied. Ten of 22 patients received a two-week eradication therapy with amoxicillin 1500 mg/day, lansoprazole 30 mg/day and a new antiulcer agent with features in common with sucralfate, ecabet sodium, 2.0 g/day. They continued to receive the same doses of lansoprazole and ecabet sodium for the next six weeks. The other 12 patients received the same therapy except for those who underwent the four-week amoxicillin treatment. All patients underwent EUS both at the start of the study and eight weeks later. They then received ecabet sodium alone for the next six months as a maintenance therapy, followed by a six-month interval with no treatment. The final endoscopy was done one year after H pylori eradication therapy was completed to evaluate H pylori status and ulcer recurrence. RESULTS: The rates of endoscopic healing and H pylori eradication in the nine patients with superficial ulcer were 100%, irrespective of the period of amoxicillin treatment. In contrast, the rates of endoscopic evidence of healing and H pylori eradication in the 13 patients with deep ulcer were different for each period of amoxicillin treatment; that is, the rates of reduction in ulcer determined by echo and H pylori eradication in the four patients treated with the two-week amoxicillin course were significantly lower (P=0.03) than those in the nine patients treated with the four-week course. CONCLUSION: Ulcer depth is likely to influence the success of amoxicillin treatment for H pylori-positive patients with gastric ulcer.

Effect of herbal medicine Saiko-keishi-to (TJ-10) on rat spontaneous chronic pancreatitis: comparison with other herbal medicines.

BACKGROUND: In an attempt to obtain evidence of the beneficial effects of TJ-10, we investigated the gene expression of PAP, an acute phase protein specific for pancreatitis in rat spontaneous chronic pancreatitis. METHODS: Four-wk-old male WBN/Kob rats were fed with MB-3 pellet diet containing herbal medicine. There were two administration groups for each drug: the prophylactic group administered from 4-12 wk, and the therapeutic group administered from 12-20 wk. Untreated control rats were fed with MB-3 alone. Histopathologic changes and PAP gene expressions were analyzed at 12 and 20 wk. RESULTS: In the prophylactic group, TJ-10-treated WBN/Kob rats showed no evidence of pancreatitis, and there was the amelioration of pancreatitis in the pancreata of the rats treated with other herbal medicines except TJ-24 at 12 wk. PAP mRNA was not expressed in the TJ-10-treated rats, and PAP gene expression was suppressed in rats treated with other drugs except TJ-107. In the therapeutic group, the amelioration of pancreatitis was seen only in TJ-10-treated rats, but PAP gene expression was significantly suppressed in the rats treated with all herbal medicines tested, compared with that in untreated control rats. CONCLUSION: An herbal medicine Saiko-keishi-to (TJ-10) delayed the onset of chronic pancreatitis in the WBN/Kob rat, and suppressed the pancreatitis-associated protein (PAP) gene expression more significantly than other herbal medicines.

Nonfunctioning islet cell carcinoma of the pancreas with high serum CEA & CA19-9, K-ras codon 12 mutation, and microsatellite instability.

A 55-year-old man with nonfunctioning islet cell carcinoma showing elevation of serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) levels is described with genetic analyses. Pathological examination of the resected specimen revealed two independent islet cell carcinomas, one in the body and the other in the tail of the pancreas. It was proved immunohistochemically that the tumor cells, particularly those in the tail, were immunoreactive to CEA and CA 19-9 and had the property of duct cells, as well as endocrine cells. Gastrin was demonstrated immunohistochemically in these tumor cells, although its level in serum was not elevated. Genetic analyses of the fresh specimens from the tumor in the body revealed K-ras codon 12 mutation and microsatellite instability. These findings are consistent with its progressive clinical course and strongly suggest that these tumors originate, not from the islet cells of Langerhans, but from protodifferentiated cells, capable of giving rise to all the pancreatic cell types.

Expression of transforming growth factor-beta in spontaneous chronic pancreatitis in the WBN/Kob rat.

Transforming growth factor-beta1 (TGF-beta1) is suggested to be a mediator of fibrosis in chronic pancreatitis, but the serial change of TGF-beta1 expression in the onset and progression of chronic pancreatitis is still unclear. We investigated the TGF-beta1 expression in the spontaneous chronic pancreatitis model. Four-week-old male WBN/Kob rats were fed with special pellet diet (MB-3) for 20 weeks. TGF-beta1 mRNA in the pancreas was detected by reverse transcription-polymerase chain reaction assay from four weeks, and its expression peaked at 12 weeks when the pancreatic fibrosis first appeared. The localizations of TGF-beta1 mRNA and protein were confirmed in the cytoplasm of pancreatic acinar and ductal cells by in situ hybridization and immunohistochemistry, respectively. Although fibronectin expression peaked at 12 weeks and correlated with that of TGF-beta1, its elevated expression tended to be prolonged. Pancreatic fibrosis peaked at 16 weeks after the peak of TGF-beta1 expression. These results suggest that TGF-beta1 expression may be a trigger of the fibrotic process of chronic pancreatitis in the WBN/Kob rat.

Arginine induces apoptosis and gene expression of pancreatitis-associated protein (PAP) in rat pancreatic acinar AR4-2J cells.

Arginine-induced pancreatic acinar cell injury has been reported in vivo, but the mechanism involved is unknown. In this study we investigated the effects of arginine on the cell morphology and pancreatitis-associated protein (PAP) gene expression in rat pancreatic acinar AR4-2J cells in vitro. Arginine inhibited the proliferation of AR4-2J cells in a dose-dependent manner. This decrease in proliferation was due to an increase in apoptosis, as assessed by cell morphology and DNA fragmentation. PAP messenger RNA (mRNA) was expressed at doses of 2.5 and 5.0 mg/ml of arginine, and a time-course study showed that the expression started 2 h after arginine addition and peaked at 6 h. Apoptosis was rarely seen when PAP mRNA was highly expressed, but occurred when PAP mRNA expression was decreased. These results suggest that arginine induces apoptosis and PAP gene expression in pancreatic acinar cells and that PAP might inhibit the induction of apoptosis.