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Background & Aims: Serine protease inhibitor Kazal type 1 (SPINK1), which is structurally similar to epidermal growth factor, is thought to inhibit trypsin activity and to prevent pancreatitis. Point mutations in the SPINK1 gene seem to predispose humans to pancreatitis; however, the clinical significance of SPINK1 mutations remains controversial. This study aimed to elucidate the role of SPINK1. Methods: We generated Spink3-deficient (Spink3 −/−) mice by gene targeting in mouse embryonic stem cells. Embryonic and neonatal pancreases were analyzed morphologically and molecularly. Specific probes were used to show the typical autophagy that occurs during acinar cell death. Results: In Spink3 −/− mice, the pancreas developed normally up to 15.5 days after coitus. However, autophagic degeneration of acinar cells, but not ductal or islet cells, started from day 16.5 after coitus. Rapid onset of cell death occurred in the pancreas and duodenum within a few days after birth and resulted in death by 14.5 days after birth. There was limited inflammatory cell infiltration and no sign of apoptosis. At 7.5 days after birth, residual ductlike cells in the tubular complexes strongly expressed pancreatic duodenal homeodomain-containing protein 1, a marker of pancreatic stem cells, without any sign of acinar cell regeneration. Conclusions: The progressive disappearance of acinar cells in Spink3 −/− mice was due to autophagic cell death and impaired regeneration. Thus, Spink3 has essential roles in the maintenance of integrity and regeneration of acinar cells. [Copyright &y& Elsevier]

Abstract: In squid jigging operations for neon flying squid (Ommastrephes bartramii), 30–40% of hooked squid are estimated to fall off (i.e., become detached) from the jigs during the drum-hauling process, caused by breakage/severance of the hooked tentacle(s). Reducing fall-off events can lead to higher productivity and more efficient use of the squid resource. In the present study, there were 950 fall-off events either directly observed or assessed from the residue of tentacle(s) left on the jigs compared with the 1720 total captures, which comprises 35.6% of 2670 total hooked squids. The fall-off ratio according to the mantle length (ML) was examined using logistic curve analysis and a higher fall-off ratio for smaller squid was confirmed through size selectivity curve analysis. F 50, the 50% probability of fall off from the jig, was estimated to be 37.4cm ML. The breaking strength of a single tentacle was determined to be similar to the body weight (BW) of squid smaller than 41.4cm ML, indicating a high possibility of tentacle breakage in the case of 1 tentacle grabbing the jig for smaller-sized squid. [Copyright &y& Elsevier]

In order to estimate exactly changes in the murine peristaltic movement by acetylcholine (ACh), we detected the circular muscle activity in the murine small intestine by measuring the changes in intraluminal pressure of the duodenal segment in vitro. When ACh was applied into the tissue baths, the basal tone of the duodenal preparation along the longitudinal axis increased, whereas the intraluminal pressure that reflected circular muscle activity decreased in a dose-dependent manner. Thus, the circular muscle layer of the murine duodenum relaxed by ACh. The circular relaxations by ACh were also observed in the other parts (jejunum and ileum) of the murine small intestine. The ACh-induced fall in the intraluminal pressure of the murine duodenal preparation was antagonized by atropine (10−6 M), not tetrodotoxin (10−6 M). The results indicate ACh acts as not only the excitatory transmitter for the longitudinal muscle but also the inhibitory transmitter for the circular muscle, and suggest that the functional receptors to ACh are different between longitudinal and circular muscle cells in the murine small intestine. [Copyright &y& Elsevier]

Introduction: The pancreatic secretory trypsin inhibitor (PSTI) is thought to be a specific inactivation factor of intrapancreatic trypsin activity. N34S mutation of PSTI was reported in chronic pancreatitis patients from all over the world including us, and is thought to be one of the predisposing factors for pancreatitis. The N34S mutation located close to the reactive lysine–isoleucine site (K41∼I42) might lead to a decreased inhibitory capacity. N34S is also in complete linkage with other intronic sequence variants such as IVS1-37T→C and IVS3-65insTTTT. Materials/Methods: Based on the hypothesis above, we performed biochemical experiments. (I) Activity of recombinant PSTI protein with N34S was compared to that of the wild type. (II) Molecular weight of PSTI from a patient with N34S was analysed by Western blotting to check the possibility of abnormal splicing (e.g. splice-out of exon 4). Results: (I) The function of N34S PSTI remained unchanged compared to wild-type PSTI. (II) The molecular weight of PSTI protein from a patient with N34S was not altered. Conclusion: We could not detect any functional or configurational abnormalities of N34S PSTI protein by the two experiments mentioned above. Other factors, such as translational abnormality by intronic mutation, may be associated with the predisposition to pancreatitis. [Copyright &y& Elsevier]

Calpains form a superfamlly of Ca2+-dependent intracellular cysteine proteases with various isoforms. Two isoforms, µ- and m-calpains, are ubiquitously expressed and known as conventional calpains. It has been previously shown that the mammalian calpains are activated during mitosis by transient increases in cytosolic Ca2+ concentration. However, it is still unknown whether the activation of calpains contributes to particular events in mitosis. With the use of RNA interference (RNAi), we investigated the roles of calpains in mitosis. Cells reduced the levels of m-calpain, but not µ-calpain, arrested at prometaphase and failed to align their chromosomes at the spindle equator. Specific peptidyl calpain inhibitors also induced aberrant mitosis with chromosome misalignment. Although both m-calpain RNAi and caipain inhibitors affected neither the separation of centrosomes nor the assembly of bipolar spindies, Mad2 was detected on the kinetochores of the misaligned chromosomes, indicating that the prometaphase arrest induced by calpain inhibition is due to activation of the spindle assembly checkpoint. Furthermore, when calpain activity was inhibited in cells having monopolar spindles, chromosomes were clustered adjacent to the centrosome, suggesting that calpain activity is involved in a polar ejection force for metaphase alignment of chromosomes. Based on these findings, we propose that activation of m-calpain during mitosis is required for cells to establish the chromosome alignment by regulatlng some molecules that generate polar ejection force. [ABSTRACT FROM AUTHOR]

The immunohistochemical localization of membrane-associated phospholipase A2 (M-PLA2) in normal human pancreases and 30 cases of pancreatic ductal carcinomas was investigated. In pancreatic ductal carcinomas, the immunoreactivity was observed in 25 cases (83%). Among the clinocopathological factors of pancreatic cancer, the incidence of expression of this enzyme is significantly higher in infiltrative type cancers. Furthermore, the expression of M-PLA2 was significantly increased in tumors, which had a larger amount of interstitial tissue. On the other hand, human M-PLA2 added exogenously to the fibroblast cell lines Swiss 3T3 and BALB/3T3 was found to augment their DNA synthesis. The stimulation of DNA synthesis was not affected by treatment with indomethacin. These results suggest that this enzyme could be involved directly in the proliferation of interstitial tissue through its own function. [Copyright &y& Elsevier]

Sahin et al. introduced a new approach, termed serological identification of antigens by recombinant expression cloning (SEREX), for identification of tumor antigens. This approach uses diluted serum from cancer patients to detect prokaryotically expressed cDNA libraries prepared from tumors. Our SEREX study of human pancreatic adenocarcinoma led to the identification of 18 genes. We characterized one of them, heat shock protein 105 (hsp105), which showed over-expression especially in pancreatic ductal and colon adenocarcinoma. Also, our SEREX study of squamous cell carcinoma of head and neck (SCCHN) led to the identification of 19 genes, including three kinds of novel cancer/testis (CT) antigen. These antigens defined by us may be pertinent candidates for cancer-diagnosis and related immunotherapy. [Copyright &y& Elsevier]

The proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin and tryptase. PAR-2 was widely expressed in human pancreatic cancer cell lines and in malignant and nonmalignant human pancreatic tissues. The expression of PAR-2 was found to be higher in the tissues with infiltrative growth pattern than those with expansive growth pattern. Moreover, significantly higher expression of PAR-2 was observed in the tissues that were accompanied with severe fibrosis. Even in the same specimen, the intensity of immunoreactivity tended to be stronger in the part accompanied with severe fibrosis than that accompanied with mild fibrosis. Similarly, the higher expression of PAR-2 was observed in chronic pancreatitis with severe fibrosis than with mild fibrosis. Higher expression of PAR-2 in pancreatic cancer showing infiltrative growth and higher degree of fibrosis may indicate that the activation of PAR-2 is involved in cancer invasion and the induction of fibrosis in human pancreatic cancer. [Copyright &y& Elsevier]

It has been reported that a large number of carcinoma cells are able to produce a variety of cytokines. We also reported that many carcinoma cell lines constitutively produce IL-8 in supernatant cultures. To evaluate the in vivo function of IL-8 of carcinoma cells, we investigated the expression of IL-8 and IL-8 receptors (IL-8R) in human carcinoma cells and the role of IL-8 and IL-8R in the growth of carcinoma cells. IL-8 mRNA was detected in 16 out of 20 (80%) carcinoma cell lines and 20 out of 24 (83.3%) carcinoma tissues by Northern blot analysis. IL-8R mRNA was expressed in 7 out of 11 (63.6%) carcinoma cell lines by reverse transcriptase–polymerase chain reaction (RT-PCR). Neutrophil chemotactic activity in supernatant cultures of carcinoma cell lines correlated with immunoreactive IL-8 concentration. Growth of carcinoma cells was significantly inhibited in the presence of anti-IL-8 antibodies or IL-8R antisense oligonucleotides. These results revealed that IL-8 and IL-8R are expressed in the majority of carcinoma cells, and it suggests that they might play a role in the growth of carcinoma cells.On the other hand, the NF-κB/Rel families are crucial transcriptional factors for regulation of IL-8 in immune and inflammatory responses. To determine if IL-8 production in carcinoma cells is associated with activation of nuclear factor κB (NF-κB), we investigated MCF-7 breast carcinoma cells with 12-O-teradecanoyl-phorbol-13-acetate (TPA) treatment. After TPA treatment, ELISA showed IL-8 production was increasing in culture supernatant. Western blotting revealed that in concert with the decline in cytosolic IκB, NF-κB exhibited similar kinetics of translocation to the nucleus. Electrophoric mobility shift assay (EMSA) also revealed that activation of NF-κB. These findings suggest that activation of NF-κB regulates IL-8 expression in carcinoma cells. [Copyright &y& Elsevier]

Recently, differences in histological diagnosis of colonic neoplasms between Japan and Western countries have been noted. We examined the relationship between the histopathology and immunohistochemical localization of six genetically distinct type IV collagen α chains, the major component of basement membrane (BM), in normal and neoplastic colonic tissues. α1/α2(IV) and α5/α6(IV) chains were observed in normal mucosa and tubular adenomas with mild/moderate atypia. In non-invasive cancers, both α1/α2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of α5/α6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The differential expressions of the type IV collagen α5/α6 chains could be one of the diagnostic markers of the invasiveness of colon cancer. [Copyright &y& Elsevier]

To investigate the biological significance of skin-derived anti-leukoproteinase (SKALP)/elafin in squamous cell carcinoma (SqCC), the immunohistochemical expressions of SKALP/elafin in SqCC (lung 46 and esophagus 34) were studied. The results were compared with the immunohistochemical staining of a proliferating cell nuclear antigen (PCNA) and a TDT-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay in serial sections. The expression of SKALP/elafin had a high incidence in SqCC (lung 82.6% and esophagus 73.5%). In contrast, no expression was observed in uninvolved bronchial epithelium and normal esophagus except for hyperplastic cells. SKALP/elafin expression was located in the cell layer just underneath the cornified envelope of each cell nest, and DNA fragmentation was observed in the same cell layer in which SKALP/elafin was expressed. PCNA expression was observed in the basal layer of each cornified envelope, and both expressions were clearly separated. In lung SqCC, immunoreactive score (IRS) of SKALP/elafin expression correlated significantly with the differentiation and it tended to increase in accordance with the degree of differentiation. In esophageal SqCC, there was also an obvious relationship between the expression of SKALP/elafin and its differentiation. There was no correlation between the expression and other clinicopathological factors. These results suggest that SKALP/elafin is possibly involved in the cell differentiation program, finally leading to keratinization in SqCC of human lung and esophagus. SKALP/elafin may be a beneficial molecular target for detection or even the development of a new therapeutic method against cancer. [Copyright &y& Elsevier]

Our recent study has demonstrated that brain (fetal)-type glycogen phosphorylase (BGP) positive foci (BGP foci) have a vital role in the de novo colorectal carcinogenesis. This paper reviewed the investigation on the genetic alternations in the BGP foci and the clarification of the mechanisms of de novo colorectal carcinogenesis. De novo colorectal carcinomas with invasion into submucosa or superficial muscularis propria were selected from resected specimens. Investigations of the p53, K-ras and APC mutations were performed in the BGP foci, BGP negative colorectal mucosa and de novo colorectal carcinoma. No K-ras mutation was observed in all of the cases. Mutations of p53 and APC were 14 (50.0%) and 9 (32.1%) in de novo colorectal carcinomas, and 11 (39.3%) and 1 (3.6%) in BGP foci, respectively. Both p53 and APC mutations were detected in 8 and 1, p53 mutation alone in 6 and 10, APC mutation alone in 1 and 0 out of 28 de novo colorectal carcinomas and BGP foci, respectively. Our sequential studies propose that the two major pathways, i.e., the p53–APC pathway and the p53 alone pathway in the de novo colorectal carcinogenesis. [Copyright &y& Elsevier]

GC-associated DNA primase (GANP) contains two functionally important regions, the N-terminal RNA/DNA primase and C-terminal MCM3-associated/import domains, which might be involved in DNA replication. Here, we investigated the functions of these regions by transfection studies. In addition to the C-terminal MCM3-binding (Gmap80) region, the RNA/DNA primase region (Gp) possesses an additional MCM3-interaction activity that mediates its translocation between the nucleus and the cytoplasm. Gp with an N-terminal nuclear localization signal sequence (NLS) is present in the nucleus, but Gmap80 with a C-terminal NLS appeared in the cytoplasm. The whole GANP (Ganp) protein is present in both nuclear and cytoplasmic compartments, in a Crm1-dependent manner. Gp associated with MCM3 through the NLS, and MCM3 induced nuclear localization of Ganp. NLS-negative MCM3 does not have such activity. These results suggest that the NLS of MCM3 is involved in nuclear translocation of Ganp. The nuclear-cytoplasm MCM3 translocation was demonstrated in mammalian cells, and over-expression of Ganp, but not of Gp or Gmap80, facilitated this translocation of MCM3 in co-transfectants. Moreover, introduction of Ganp induced DNA synthesis in the transfectants but dominant-negative mutants did not. It is concluded that GANP is translocated from the nucleus to the cytoplasm in association with MCM3 and is involved in DNA synthesis. [Copyright &y& Elsevier]

The presence of RON and its variant isoform in malignant and non-malignant human colonic tissues was investigated by immunohistochemistry using paraffin-embedded sections and RT-PCR analysis followed by direct sequencing of PCR product using RNAs isolated from frozen tissues. In normal colonic mucosa of both human fetus and adult, RON was uniformly expressed in cript cells, especially in the bottom of cripta. On the other hand, the expression was distributed heterogeneously in adenomas and in colon cancer. The expression of RON was significantly related to the degree of differentiation of colon cancer. The RT-PCR analysis of RNA isolated from malignant and non-malignant colonic tissue revealed the presence of two RON mRNA isoforms. Direct sequencing of two major products was revealed to be identical to that of human wild-type RON and a splicing variant of RON transcript which has been reported in human gastric cancer cell line, KATO-III. It is indicated that both wild type RON and its valiant isoform play an important role in regulating the normal function of colonic mucosa such as differentiation and motile activity, and the expression of both wild-type RON and its valiant isoform could be considered to be reduced during malignancy of human colonic mucosa. [Copyright &y& Elsevier]

It is well known that heme oxygenase-1 (HO-1) as well as nitric oxide (NO) synthase expressed in tumor cell line and solid tumor. NO may be beneficial to the tumor growth, not only because enhanced vascular permeability in solid tumor is mediated by NO, but because the l-arginine-dependent NO pathway mediates angiogenic activity and vascular endothelial growth factor (VEGF) induces angiogenesis via formation of NO. However, NO has a powerful cytostatic action on tumor cells by inhibiting DNA synthesis and causing oxidative injury. While it has been suggested that induction of HO-1 may provide an important protective response by cells against oxidative damage, HO-1 expression in tumor cells is strongly enhanced by NO donor. Strong induction of HO-1 is observed in solid tumors after occlusion or embolization of the tumor-feeding artery, indicating that ischemic stress which may involve oxidative stress triggers HO-1 induction. In addition, it is of great importance that an HO inhibitor, zinc protoporphyrin IX, suppressed the tumor growth to a great extent. In conclusion, HO-1 expression in the solid tumor may confer resistance of tumor cells to hypoxic stress as well as to NO-mediated cytotoxicity. [Copyright &y& Elsevier]

Similarities between cancer cells and neutrophil, such as circulating by single cells, attachment to the vascular system of the target and invasion into the target, lead to a hypothesis that cancer cells might produce neutrophil elastase or a very similar protease. Although the production of immunoreactive polymorphonuclear leukocyte elastase (ir-PMN-E) can be measured by enzyme immunoassay (EIA) in human cancer and cancer cell lines, we were having difficulty to detect this at mRNA level.TNF-α stimulation induces neutrophil elastase to a detectable level by RT-PCR in human cancer cell lines. In SUIT2, neutrophil elastase mRNA was detectable from 0.1 ng/ml of TNF-α stimulation which in EBC1 and MCF7 requires 1.0 ng/ml of TNF-α. For PC-3, there was no induction. The RT-PCR direct sequencing method revealed that the induced neutrophil elastase has no mutation.Although the amount of this molecule is low, as neutrophil elastase is a protease, the function in living systems does not depend on their amount but mainly on the activity they display. The result of this study indicates that some of the cancer cells could produce neutrophil elastase by stimulation of TNF-α and also suggests that neutrophil elastase produced by cancer cells is involved in clinical aspects of malignancy under inflammatory conditions. [Copyright &y& Elsevier]

We found that EBC-1, a human non-small cell lung cancer (NSCLC) cell line, formed multiple metastases in the lung after subcutaneous inoculation into severe combined immunodeficiency (SCID) mice. SCID mice were injected in the flank with 106 EBC-1 cells. Metastases in the lung were counted in histologic sections and stained immunohistochemically for neutrophil elastase (NE). Solid tumors were palpable in the flank at 3 weeks and grew steadily until 10 weeks. EBC-1 cells formed multiple metastases in the lung at 7 weeks; their numbers increased steadily until 12 weeks in all mice. Immunoreactivity for NE was intense in the metastatic tumor cells. As a marker for circulating tumor cells, human β-actin mRNA was detected in blood by reverse transcriptase–polymerase chain reaction (RT-PCR). Blood samples obtained at 3 weeks after tumor inoculation contained human β-actin mRNA. Our NSCLC EBC-1 pulmonary metastasis model is reliable, technically simple, and predictably results in pulmonary metastasis from early hematogenous spread. This model may be useful for screening potential new drugs, including specific NE inhibitors, for effectiveness against pulmonary metastasis of NSCLC. [Copyright &y& Elsevier]

We demonstrated the dynamic aspects of granulocyte activation in rat severe acute pancreatitis which was induced by cerulein and aggravated following lipopolysaccharide (LPS) injection. Pancreatitis induced by cerulein increased intracellular elastase activity of granulocytes in the blood. However, significant systemic cytokinemia was not provoked under such conditions. After induction of severe pancreatitis by LPS, intracellular elastase activity of circulating granulocytes decreased markedly and immediately. The timing of this decrease was concomitant with induction of systemic hypercytokinemia and granulocytes migration into the lung. Overall results provide the following implications: (1) circulating granulocytes are activated by the induction of mild pancreatitis; (2) the activation of granulocytes is mediated by other factors than systemic cytokinemia, such as locally produced cytokine; (3) those priming granulocytes immediately and significantly migrate from the circulation into extravascular space by the induction of endotoxemia; (4) the migration of granulocytes, in turn, may be mediated by systemic cytokinemia. [Copyright &y& Elsevier]

Background: Interleukin-18 (IL-18) production is the main mechanism of hepatic injury in several animal models via further gamma interferon (IFN-γ) release. IL-18 also functions as an inducer of proinflammatory cytokines as TNF-α, which are also shown to induce organ injuries in many severe inflammatory conditions. To clarify the mechanism of hepatic injury developed in acute pancreatitis, plasma concentrations of IL-18 and its related cytokines were analyzed. Patients and methods: Plasma levels of IL-18, IL-12, IFN-γ, TNF-α, soluble TNF receptor (sTNF-R) and soluble Fas ligand (sFas-L) were measured by ELISA. Results: Plasma concentrations of IL-18 in patients with hepatic injury increased during the first few days apparently and remained so to some extent thereafter. However, IFN-γ showed no apparent increase in plasma concentrations. Those of TNF-α and sTNF-R remained increased in the hepatic injury group. Conclusion: These results suggest that IL-18 may act as a pro-inflammatory cytokine, which provokes the cytokine storm and eventually hepatic injury. [Copyright &y& Elsevier]

Objectives: We clarified a distinctive feature of response to surgical stress in elderly patients compared to nonelderly patients. Subjects: Two hundred and ninety-two consecutive patients received elective gastrointestinal surgery. Patients were analyzed for physiological status, cytokine levels, surgical stress parameters and postoperative status of SIRS and complications. Cytokine responses according to patient''s age: Plasma levels of interleukin 6 (IL-6) fluctuated in higher concentrations in elderly patients. Peak plasma levels of IL-6 were significantly high in patients aged over 75 years in total gastrectomy, over 65 years in pancreaticoduodenectomy and over 60 years in major hepatectomy. Mechanisms of hypercytokinemia in elderly patients: Renal dysfunction caused hypercytokinemia in elderly patients. Hypercytokinemia and SIRS: SIRS was more frequently encountered and continued for a long time in elderly patients, giving us an early sign of postoperative complications. Conclusions: In order to prevent postoperative complications in elderly patients, we must shorten status of SIRS due to excessive hypercytokinemia. [Copyright &y& Elsevier]

Purpose: We aimed to clarify the possibility of hematogenous spreading of cancer cells by microwave coagulation therapy (MCT) and radio-frequency ablation (RFA) for liver cancers. Molecular marker: Carcinoembryonic antigen (CEA) mRNA and melanoma antigen-3 (MAGE-3) mRNA were selected as a genetic marker. PCR method: Blood samples were collected before, during and just after the treatments. Genetic markers were examined using LightCycler reverse transcriptase–polymerase chain reaction (RT–PCR) assay with SYBR Green I. The samples with both positive markers were defined as genetic positive. Molecular metastasis and recurrence: We evaluated peripheral blood samples of 70 patients with liver tumors; 64 hepatocellular carcinomas (HCCs), 1 cholangiocellular carcinoma (CCC), and 5 metastatic liver tumors (MLTs). Genetic positive rates in RT–PCR assay before treatment were 42.2% in HCC, 0% in CCC, and 20% in MLT. Among genetic negative patients before operation, 45% of patients in MCT and 29% in RFA changed to genetic positive as a result of the procedures. Recurrence rates were 55% and 50% in MCT and 43% and 88% in RFA of the patients assessed negative to negative and negative to positive, respectively. Conclusion: Local ablation therapies may promote hematogenous seeding of cancer cells. [Copyright &y& Elsevier]

Reverse transcription-polymerase chain reaction (RT-PCR) technique is extremely useful for sensitive detection of cancer cells, but it requires at least 8 h. If the precise information on the cancer spread based on RT-PCR is available during surgery, we can give the optimal therapy to each patient during operation. We aimed to establish the ultra-rapid procedure of RT-PCR for application to the intra-operative diagnosis. We have established a one-step real-time RT-PCR using LightCycler with hybridization probes, in combination with automated mRNA extraction on MagNA Pure LC. The method can be completed in only 70 min after sampling and has a comparable sensitivity to conventional RT-PCR and/or two-step real-time RT-PCR with a wide (10–106) dynamic range. When this method was applied to the intra-operative detection of free cancer cells in peritoneal lavage, the results were available during operation in all cases. We could detect cancer cells more effectively compared to conventional cytology and RT-PCR. This protocol is easily accomplished, delivers rapid, sensitive and reproducible results, and is therefore applicable to intra-operative diagnosis. [Copyright &y& Elsevier]

The established ultra-rapid quantitative RT-PCR system for intraoperative detection of intraperitoneal free cancer cells, the elucidated cause of peritoneal recurrence after curative operation for patients with non-serosa-invasive gastric cancer, and the extensive intraperitoneal lavage (EIPL) therapy for the prophylaxis of peritoneal recurrence from the results of evaluation using the ultra-rapid quantitative RT-PCR system were reviewed. We established ultra-rapid RT-PCR protocol that enables diagnosis of intraperitoneal cancer spread about 70 min, using LightCycler method in combination with automated mRNA extractor. Both the carcinoembryonic antigen (CEA) and cytokeratin (CK)20 messenger RNA (mRNA) in intraperitoneal lavages after lymph node dissection were identified in three (14.2%) and four (26.7%) patients with submucosal (SM) and muscularis propria (MP) tumors, respectively. Although peritoneal metastasis occurs in some patients with early gastric carcinoma, the reasons have not been fully established. These results clarified that peritoneal metastasis after operation for non-serosal-invasive gastric cancer was caused by opening lymphatic channels during lymph node dissection allowing the spread of viable cancer cells into the peritoneal cavity. Furthermore, EIPL method was performed in five cases with serosa-invasive (SE) gastric carcinoma, and its efficacy was evaluated by the quantitative RT-PCR. The quantitative RT-PCR demonstrated that EIPL reduced free cancer cells from 3.8×105±1.4×105 to 2.8±1.5 cells/100 ml of lavage by from six to eight washes, and they disappeared after the seventh to ninth wash. In conclusion, an ultra-rapid quantitative RT-PCR system for intraoperative detection of intraperitoneal free cancer cells was established for clinical use. It is suggested that the combination with the novel rapid detection system with the intraoperative therapy of EIPL can be a useful prophylactic strategy for peritoneal metastasis from gastric carcinoma. [Copyright &y& Elsevier]

Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor of trypsin. We proposed a hypothesis: if the function of PSTI is impaired by its genetic mutation, trypsin may easily promote autodigestion causing pancreatitis, and performed mutational analysis of PSTI gene in patients with pancreatitis. Two exonic mutations (N34S and R67C) were suggested to be associated with the predisposition to pancreatitis. N34S mutation was co-segregated with two intronic mutations, IVS1-37T>C and IVS3-69insTTTT. Although we analyzed the function of recombinant N34S protein, we could not demonstrate the loss of function of this protein. Intronic mutations rather than N34S itself (IVS1-37T>C+N34S+IVS3-69insTTTT complex) may be associated with the decreased function of PSTI. Alternatively, increased digestion of N34S in vivo may be applicable. As for R67C, the conformational alteration of the protein by forming intramolecular or intermolecular disulfide bonds with 67Cys was strongly suggested. These results, along with the brand new findings in PSTI knockout mice, suggest that genetic mutation of PSTI is one of the important mechanisms for predisposition to pancreatitis by lowering the trypsin inhibitory function. [Copyright &y& Elsevier]

In our previous studies, the cancer dissociation factor (DF), which can induce cell dissociation of weakly invasive pancreatic cancer cells (PC-1), was isolated from a highly invasive cell line (PC-10). In further study, mitogen-activated protein kinase 2 (MEK2) was discovered to be an invasion-metastasis related factor in this model. Extracellular signal-regulated kinase 2 (ERK2) is known to be the downstream kinase of MEK2 in the mitogen-activated protein kinase (MAPK) signaling pathway. Demonstration of the role of ERK2 in DF induced cell dissociation may contribute to elucidation of the signaling regulatory mechanism of tumor invasion-metastasis. The two cell lines mentioned above and the immunofluorescent method were used. In untreated cells, both total ERK2 and phosphorylated ERK (p-ERK) presented constitutively strong expressions in PC-1.0 cells, whereas the relevant expressions were quite weak in PC-1 cells. Nevertheless, addition of the conditioned medium containing DF to the PC-1 cells, significantly induced total ERK2 and p-ERK expressions and dissociation of islandlike cell colonies. Furthermore, the total ERK2 and p-ERK expressions, either the induced expressions in PC-1 cells or constitutive expressions in PC-1.0 cells, were seriously suppressed after treatment with the MEK2 phosphorylation inhibitor, U0126. Correspondingly, obvious cell colonies formation was observed in these two cell lines. In conclusion, ERK2 activation is closely correlated with the DF induced cell dissociation in pancreatic cancer cells. [Copyright &y& Elsevier]

Background: We investigated the effect of the pretransplant administration of recipient''s blood to donor on rat hepatic allograft survival. Methods: Serum levels of interferon (IFN)-γ were determined after transplantation. Immunocompetent cells from the donor were identified with anti-donor class II MHC (RT1Ba) (OX-76) antibodies. The number of donor-derived CD4+ and CD8+ T cells in hepatic allografts or recipient spleen was determined. Results: LEW rats transplanted with hepatic allografts from ACI rats survived a mean of 11.1±2.1 days. Pretreatment intravenous administration of recipient LEW blood did not prolong the survival of hepatic allografts (10.5±1.7 days). In contrast, pretreatment intraportal administration of recipient LEW blood significantly increased the survival of LEW recipients with ACI livers to 31.7±7.6 days (Table 1). The ratio of ox76+CD4+ or ox76+CD8+ T cell to CD4+ or CD8+ cell was significantly greater (25.6% versus 5.7%, 26.6% versus 6.8%) in hepatic allografts pretreated with pretransplant intraportal administration of LEW recipient blood, rather than those cells in recipient spleen (46.2% versus 34.4%, 63.7% versus 71.4%). Conclusions: Pretransplant intraportal administration of recipient LEW blood to ACI donor rats prolonged the survival of hepatic allografts. This may be associated with the regional graft-versus-host reaction. [Copyright &y& Elsevier]

Tumor necrosis factor-α (TNF-α) is known to play a central part in the pathogenesis of acute pancreatitis. TNF-α and its two receptors (p55 and p75 TNF receptors) have shown to be expressed at various sites in the body. TNF-α is initially expressed as a 26-kDa membrane-associated perform, which is proteolytically processed to a 17-kDa secreted mature form. Recently, TNF-α converting enzyme (TACE) has been identified as a membrane-bound TNF-α activating enzyme. Hence, it is expected that the newly devised TACE inhibitor, Y39083, will be a useful candidate for the treatment of inflammatory disorders provoked by TNF-α, including acute pancreatitis. Y39083 suppressed TNF-α production in vitro and in vivo significantly. However, the production of other cytokines, such as IL-1β, IFNγ and IL-6, in vivo were not. The blockade of secreted TNF-α production by Y39083 could not improve the organ injury.These results suggest that the blockade of activities of secreted TNF-α production is not sufficient to suppress systemic cytokine reaction and distant organ injury. [Copyright &y& Elsevier]

Hepatectomy is one of the most effective means of managing hepatic neoplasms. However, some patients are still susceptible to hepatic failure after the procedure. In patients with hepatic failure after surgery, plasma concentrations of IL-18 increased gradually after 1 week. Interestingly, the increase in the plasma IL-18 correlated with that in serum bilirubin. Because IL-18 is metabolized in the liver and excreted in bile, the increase in plasma IL-18 in patients with hepatic failure reflects the decreased metabolism in the liver. [Copyright &y& Elsevier]

Pancreatic secretory trypsin inhibitor (PSTI) is synthesized in the acinar cells of the pancreas and acts as a potent protease inhibitor that inhibits intrapancreatic activation of trypsin to prevent the occurrence of pancreatitis. Recently, we and other groups reported that some patients with chronic pancreatitis had a point mutation in the PSTI gene. Genetic mutations in the PSTI gene seem to promote a predisposition to pancreatitis, possibly by lowering the threshold of pancreatitis caused by other factors. In order to prove this hypothesis, we generated PSTI knockout mice using cre/mutant lox system. Heterozygous offsprings of chimeras appeared entirely normal. [Copyright &y& Elsevier]