Yoshifumi Takeyama

Peripheral lymphocyte reduction in severe acute pancreatitis is caused by apoptotic cell death

To investigate impairment of cellular immunity in severe acute pancreatitis, alterations of peripheral lymphocytes in acute pancreatitis were examined. In 48 patients with severe acute pancreatitis, the mean peripheral lymphocyte count on admission was 959 +-105/mm 3, and it was significantly decreased in the patients with subsequent infection (623 +-90/mm 3) in comparison to those without infection (1084 +- 135/ram3). According to an analysis of lymphocyte subsets, although both B and T lymphocytes were decreased in peripheral circulation in the patients with infection, it was primarily CD8-positive lymphocytes that decreased in these subsets. Cell cycle analysis of lymphocytes collected from these patients indicated that apoptotic changes occurred after 24 hours’ incubation in lymphocytes from patients with severe pancreatitis but not in lymphocytes from healthy control subjects. In a rat model of experimental necrotizing pancreatitis, total peripheral lymphocytes and T lymphocytes were significantly decreased 5 hours after induction of pancreatitis. In severe pancreatitis, peripheral lymphocytes are eliminated from systemic circulation possibly as a result of apoptosis. It has been suggested that impairment of cellular immunity due to peripheral lymphocyte apoptosis is linked to the development of subsequent infectious complications in acute pancreatitis.

Carcinosarcoma of the gallbladder with chondroid differentiation

Carcinosarcoma of the gallbladder is an uncommon neoplasm. We herein report the case of a patient with carcinosarcoma of the gallbladder with chondroid differentiation, treated by cholecystectomy with liver segmentectomy and lymph node dissection for a tumor which occupied the entire gallbladder and spread to the liver. Histologically, the tumor contained two distinct components: a mixture of both well and poorly differentiated tubular adenocarcinoma and sarcomatoid tissue with chondroid differentiation. From a review of the literature, it was seen that carcinosarcomas of the gallbladder could be divided into two groups: one group with apparent sarcomatous differentiation, such as chondroid, osteoid, and rhabdomyosarcomatous differentiation, and the other group, of carcinosarcomas with a sarcomatous portion composed of anaplastic spindle cells. Each group had a poor prognosis in spite of surgical resection of tumors. Our patient died of peritoneal dissemination 7 months after surgery.

Apoptotic cell death of renal tubules in experimental severe acute pancreatitis.

BACKGROUNDnRecently renal cell apoptosis has been reported in various disorders that result in renal failure. Thus we hypothesized that renal cell injury resulting from apoptosis is involved in renal failure with severe acute pancreatitis.nnnMETHODSnRenal cell apoptosis in kidneys harvested from rats with necrotizing pancreatitis was evaluated by in situ nick-end labeling. Ascitic fluid that had been collected 6 hours after development of pancreatitis was injected into the peritoneal cavities of healthy rats, and renal apoptosis was also evaluated. The apoptosis-inducing activity of the ascitic fluid was estimated in vitro with use of isolated rat renal tubules and the normal rat kidney cell line NRK52E by nuclear staining, cell cycle analysis, and DNA electrophoresis.nnnRESULTSnApoptosis was detected by in situ nick-end labeling on the renal tubules 6 hours after induction of pancreatitis in vivo. Similar tubular apoptosis was detected in the rats that had intraperitoneal injection of the ascitic fluid. In in vitro analyses the ascitic fluid induced nuclear and DNA fragmentation on the isolated renal tubules and promoted apoptosis on NRK52E cells in a time-dependent manner.nnnCONCLUSIONSnApoptotic cell death of renal tubules occurs in severe acute pancreatitis within several hours and is possibly involved in the mechanism of renal failure through undefined substance(s) in the ascitic fluid associated with pancreatitis.

Hepatocyte growth factor in assessment of acute pancreatitis: Comparison with C-reactive protein and interleukin-6

Serum levels of hepatocyte growth factor (HGF), C-reactive protein (CRP), and interleukin-6 (IL-6) were determined at the time of admission in 38 patients with acute pancreatitis. The clinical utility of HGF for the detection of severe pancreatitis and for predicting prognosis, bacterial infection (infected pancreatic necrosis or sepsis), and organ dysfunction (liver, kidney, and lung) during the clinical course of acute pancreatitis was compared with the clinical utility of CRP and IL-6 by analysis of receiver operator characteristic (ROC) curves. The optimum cutoff levels of HGF for severity, prognosis, infection, hepatic dysfunction, renal dysfunction, and respiratory dysfunction were 0.9, 1.1, 1.0, 1.1, 1.1, and 1.0ng/ml, respectively. HGF was as useful as CRP and more useful than IL-6 for detection of severe pancreatitis and for predicting hepatic dysfunction. Moreover, HGF was more useful than CRP or IL-6 for predicting prognosis, renal dysfunction, and respiratory dysfunction. However, for predicting infection, CRP was more useful than HGF. These results suggest that serum HGF levels on admission may be a useful new clinical parameter for determining the prognosis of acute pancreatitis and that HGF may be closely related to the organ dysfunction of acute pancreatitis.

Molecular cloning and characterization of a ras p21-like GTP-binding protein (24KG) from rat liver

We have isolated cDNA clones from a rat liver cDNA library that encode a ras p21-like small GTP-binding protein (24KG) which was purified from the microsomes-Golgi complex fraction of the rat liver. The cloning was accomplished using polymerase chain reaction amplified with a set of oligonucleotide primers which were designed from the partial amino acid sequences for 24KG. The cDNA contained an open reading frame encoding a 216 amino acid protein with a calculated Mr weight of 24,397. This Mr weight was similar to that of the purified 24KG estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The sequence analysis of 24KG revealed that a 24KG cDNA is the rat counterpart of a rab11 cDNA cloned from a Madin-Darby canine kidney cell cDNA library. The 1.0-kilobase 24KG mRNA corresponding to the isolated cDNA was also detected in various rat tissues, such as brain, testis, spleen, and heart.

Spontaneous rupture of an intrahepatic bile duct with biloma treated by percutaneous drainage and endoscopic sphincterotomy

A case of spontaneous rupture of an intrahepatic bile duct with biloma formation treated by percutaneous drainage and endoscopic sphincterotomy is reported. A 73-yr-old woman was admitted with fever and abdominal pain. There was no past history of abdominal surgery, instrumentation, or trauma. Ultrasound and computed tomography revealed a massive fluid collection in the abdominal cavity. Endoscopic retrograde cholangiography demonstrated extravasation of contrast medium from a distal biliary radicle in the left lobe of the liver. After successful treatment by percutaneous drainage and endoscopic sphincterotomy, the patient did well. Ultrasound and computed tomography showed resolution of the biloma. Nontraumatic bilomas are very rare: in fact, only 24 cases of spontaneous biloma have been reported. Endoscopic treatment for patients with spontaneous bilomas can be safe and effective, and should be considered.

Hepatocyte growth factor increases in injured organs and functions as an organotrophic factor in rats with experimental acute pancreatitis.

We previously reported that serum hepatocyte growth factor (HGF) levels are elevated in patients with acute pancreatitis and that pancreatitis-associated ascitic fluid (PAAF) contains cytotoxic factor(s) inducing apoptosis on Madin-Darby canine kidney (MDCK) cells. In this study, plasma HGF levels and HGF tissue distribution were investigated in rats with experimental acute pancreatitis, and the effects of HGF on the cytotoxic activity and apoptosis-inducing activity of PAAF also were examined. Plasma HGF levels were elevated in rats with two experimental pancreatitis models of different grades of severity. The degree of its elevation was correlated with the severity and the organ dysfunctions. In rats with severe pancreatitis, HGF protein and messenger RNA (mRNA) levels significantly increased in liver, kidney, and lung, which were injured organs. When anti-HGF neutralizing antibody was administered in severe pancreatitis, liver dysfunction worsened, and apoptotic cells increased in kidney. Recombinant HGF inhibited the cytocidal activity of PAAF on MDCK cells in a dose-dependent manner. Moreover, recombinant HGF prevented the apoptotic cell death (DNA fragmentation, nuclear fragmentation, and caspase-3 activation) induced by PAAF. These results suggest that HGF is produced in injured organs and may function as an organotrophic and antiapoptotic factor against the organ injuries in acute pancreatitis.

Enhancement of fibroblast growth factor-induced diacylglycerol formation and protein kinase C activation by colon tumor-promoting bile acid in Swiss 3T3 cells: Different modes of action between bile acid and phorbol ester

A small amount (50–200 μM) of deoxycholate (DOC), a colon tumor‐promoting bile acid, did not show a direct effect on protein kinase C activity in a cell‐free system, but enhanced fibroblast growth factor (FGF)‐induced diacylglycerol formation and protein kinase C activation in Swiss 3T3 cells. DOC potentiated both reactions induced by submaximal doses of FGF but showed little effect on the maximal levels of the reactions. DOC alone was inactive in eliciting both reactions in the absence of FGF. DOC did not affect the binding of FGF to the cells. Since it has been described that diacylglycerol serves as a messenger for the activation of protein kinase C in the action of FGF in Swiss 3T3 cells [(1985) FEBS Lett. 191, 205‐210], these results suggest that a small amount of DOC increases the sensitivity to FGF of diacylglycerol formation and thereby potentiates protein kinase C activation in this cell line. This action of DOC was in marked contrast to that of 12‐O‐tetradecanoylphorbol‐13‐acetate, a potent tumor‐promoting phorbol ester, which directly activated protein kinase C in cell‐free and intact cell systems.

Induction of apoptotic cell death by pancreatitis‐associated ascitic fluid in Madin‐Darby canine kidney cells

We investigated the cytotoxicity on Madin‐Darby canine kidney (MDCK) cells of pancreatitis‐associated ascitic fluid (PAAF) collected from rats with experimental necrotizing pancreatitis. PAAF reduced viability of MDCK cells in a time‐ and dose‐dependent manner. We detected DNA fragmentation on the PAAF‐treated MDCK cells, indicating that the cytocidal action of PAAF is via apoptosis. From the results obtained, we conclude that PAAF contains factor(s) inducing apoptosis on MDCK cells, and we assume that apoptotic cell death is involved in the mechanism of organ failure in acute pancreatitis.

Purification and characterization of a novel GTP-binding protein with a Mr value of 24,000 from rat liver

About 15% of the total GTP-binding proteins (G proteins) of rat liver homogenate was found in the microsomes-Golgi complex fraction. From this fraction, we purified to near homogeneity and characterized a G protein with a Mr value of 24,000 (24K G). 24K G specifically bound guanosine 5-(3-Q-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 30 nM. 24K G bound maximally about 0.7 mol of GTP gamma S/mol of protein. 24K G hydrolyzed GTP to liberate Pi with a turnover number of about 0.008 min-1. 24K G was not copurified with the beta gamma subunit of heterotrimeric G proteins. The partial amino acid sequences of 24K G revealed that this protein was a novel small G protein.