Izumi Takeyoshi

Fibrin Glue Sandwich Prevents Pancreatic Fistula following Distal Pancreatectomy

Pancreatic fistula is a major form of morbidity following pancreatic resection. We conducted a nonrandomized clinical trial comparing the sealing and sandwich techniques of spraying fibrin glue to prevent pancreatic fistula following distal pancreatectomy. The pancreas was transected with a scalpel to identify and suture the main pancreatic duct and its small branches. In the sealing group, fibrin glue was sprayed over the closed pancreatic stump and sutures. Alternatively, in the sandwich group fibrin glue was sprayed so as to cover and join the cut surface of the pancreatic remnant, which was then held closed with sutures. Altogether 111 patients were included in the study (90 with gastric cancer, 10 with esophageal cancer, and 11 with pancreatic cancer). Patients were nonrandomly assigned to the sandwich or the sealing group. Morbidity was 21.8% for the patients in the sandwich group versus 33.9% in the sealing group. Pancreatic fistulas occurred in 9.0% of the sandwich group versus 26.8% of the sealing group. The incidence of fistula was thus significantly lower in the sandwich group. The incidence of fistula was also significantly lower in the sandwich group for gastric malignancy patients undergoing extended radical lymphadenectomy down to the paraaortic lymph nodes combined with left adrenalectomy. Of the patients with gastric malignancy, pancreatic fistulas occurred in 9.3% of the sandwich group versus 25.5% of the sealing group. The fibrin glue sandwich technique is simple and reliable and should be valuable for complementing other prophylactic methods of preventing pancreatic fistula.

Expression and Mutations of KCNJ5 mRNA in Japanese Patients with Aldosterone-Producing Adenomas

CONTEXT Mutations of the KCNJ5 gene have recently been identified in patients with aldosterone-producing adenomas (APA). OBJECTIVE Our objective was to investigate the expression and mutations of the KCNJ5 gene in Japanese patients with APA. DESIGN AND PATIENTS We sequenced KCNJ5 cDNA and measured KCNJ5 mRNA levels in 23 patients with APA operated on at Gunma University Hospital. MAIN OUTCOME MEASURES Mutations and mRNA levels of the KCNJ5 gene were examined and compared to those in cortisol-producing adenomas (Cushings syndrome) and pheochromocytomas. RESULTS Of the 23 patients with APA, 15 (65.2%) had two somatic mutations of the KCNJ5 gene: 12 cases of p.G151R (eight with c.451G>A, and four with c.451G>C) and three cases of p.L168R (c.503T>G). Levels of KCNJ5 mRNA were significantly higher in the APA with mutations than those without. Immunohistochemistry also showed a stronger staining of KCNJ5 on the cell membrane in the tumor with a mutation. Furthermore, a PCR-restriction fragment length polymorphism assay with c.503T>G revealed the mutant mRNA to be expressed at a similar level to the wild type. The level of KCNJ5 mRNA in cortisol-producing adenomas was approximately 30% of that in APA, and almost no expression was observed in pheochromocytomas. CONCLUSION We found that: 1) a significant number of patients with APA had somatic mutations of the KCNJ5 gene; 2) KCNJ5 mRNA levels were higher in the APA with KCNJ5 mutations; and 3) the expression of KCNJ5 mRNA was significantly higher in APA than cortisol-producing adenomas and pheochromocytomas.

Stromal Macrophage Expressing CD204 is Associated with Tumor Aggressiveness in Lung Adenocarcinoma

Background: Tumor tissue is composed of variable numbers of cancer cells and stromal cells, and tumor-associated macrophages are recruited into cancer-induced stroma and produce a specific microenvironment. Alternatively, activated macrophages (M2 phenotype) are known to be related to tumor progression and outcome, and CD204 has been reported to be expressed in M2 macrophages in some tumors. Methods: To investigate whether CD204-positive macrophages reflect tumor aggressiveness in adenocarcinoma of the lung, we investigated the relationships between the numbers of CD204-positive stromal macrophages and both clinicopathological features and outcome in 170 consecutive resected cases. We also examined the relationships between the numbers of CD204-positive macrophages and the expression levels of cytokines involved in the migration and differentiation of M2 macrophages. Results: The numbers of CD204-positive macrophages were significantly correlated with several prognostic factors. The log-rank test showed a significant association between the numbers of CD204-positive macrophages and a poor outcome (p = 0.0073), whereas the numbers of macrophages expressing CD68, a pan-macrophage/monocyte marker, were of marginal prognostic significance (p = 0.0789). We evaluated associations between the levels of expression of the cytokines IL-6, IL-10, IL-12a, IL-12b, M-colony-stimulating factor, IFN-gamma-., and monocyte chemoattractant protein-1 in cancer tissue and the numbers of CD204-positive macrophages. The expression levels of IL-10 and monocyte chemoattractant protein-1, which are involved in differentiation, accumulation, and migration of M2 macrophages, were significantly correlated with the numbers of CD204-positive macrophages (p = 0.031 and p = 0.031, respectively). Conclusion: These findings demonstrated that CD204-positive macrophages clearly reflect the tumor-promoting phenotype of tumor-associated macrophages in lung adenocarcinoma.

Effects of a dual inhibitor of tumor necrosis factor-α and interleukin-1 on lipopolysaccharide-induced lung injury in rats : Involvement of the p38 mitogen-activated protein kinase pathway

ObjectiveSepsis is a major cause of adult respiratory distress syndrome. In this study, we evaluated the effect of FR167653, which is a potent suppressant of tumor necrosis factor (TNF)-&agr; and interleukin (IL)-1 production, on lipopolysaccharide (LPS)-induced lung injury and lethality in rats, and we examined the involvement of p38 mitogen-activated protein (MAP) kinase in the action of FR167653. DesignProspective, randomized study. SettingAnimal research facility in a university. SubjectsMale Sprague-Dawley rats weighing 200–270 g. InterventionsAll the animals were assigned to one of the following four groups: control group, FR-only group, LPS-only group, and LPS/FR group. Animals in the LPS-only and LPS/FR groups received 6 mg/kg of LPS intravenously. The animals in the FR-only and LPS/FR groups also received an infusion of FR167653 at 0.2 mg·kg−1·hr−1, commencing 30 mins before the LPS (or vehicle) injection and continuing for 5.5 hrs. Measurements and Main Results LPS significantly induced the accumulation of pulmonary neutrophils and lung edema, both of which were significantly attenuated by treatment with FR167653. FR167653 also significantly decreased the LPS-induced lethality. Histologically, tissue damage was milder in the LPS/FR group than in the LPS-only group. Serum concentrations of TNF-&agr; and IL-1&bgr; and plasma concentrations of thromboxane B2 were all suppressed in the LPS/FR group compared with the LPS-only group. Western blot analysis revealed that FR167653 inhibited the phosphorylation of p38 MAP kinase in lung tissues. ConclusionsFR167653 administration decreased serum TNF-&agr; and IL-1&bgr; concentrations, which was associated with decreased lung injury and lethality. The mechanism responsible for the decreased TNF-&agr; and IL-1 may be related to the inhibitory effect of FR167653 on p38 MAP kinase activation.

Significance of epidermal growth factor receptor gene mutations in squamous cell lung carcinoma

Epidermal growth factor receptor (EGFR) gene mutations have been reported to be clinically significant in non-small cell lung cancer (NSCLC). However, because most previous studies focused only on adenocarcinomas, EGFR mutations in other histotypes are poorly investigated. We evaluated the frequency of EGFR gene mutations in squamous cell carcinoma (SCC) and its clinicopathological features. In total, 89 frozen tumor specimens that had been first diagnosed as SCCs, were examined for EGFR mutations in exons 19 and 21 using direct sequencing, PNA-enriched sequencing and SmartAmp2. Additionally, pathological investigation, including immunostaining for p63 and TTF-1, alcian blue staining and EGFR mutation-specific immunohistochemistry in mutation-positive samples was also performed. The frequency of EGFR mutations was 5.6% (5/89); all mutations were deletions in EGFR exon 19. Immunohistological investigation of these samples revealed that two of five were positive for p63 and TTF-1 staining, and showed production of mucin, as evidenced by alcian blue staining. Consequently, three of the samples were considered to be true SCC at final pathological diagnosis, while the remaining two samples were revised to adenosquamous carcinoma and adenocarcinoma. The final frequency of the EGFR mutations in true SCC was 3.4% (3/87). In conclusion, EGFR mutations were found in a small, but significant, number of SCC tumor samples and thus EGFR mutational analysis was useful in the accurate diagnosis of SCC. Our data demonstrate that EGFR mutational analysis should be performed not only in adenocarcinoma, but also in SCC to allow accurate diagnosis and treatment.

The effect of FR167653 on pulmonary ischemia-reperfusion injury in rats

BACKGROUND A novel synthesized organic compound, FR167653, has been characterized as a potent suppressant of interleukin-1 and tumor necrosis factor-alpha. We designed this experimental study to evaluate the effect of FR167653 on ischemia-reperfusion injury of the rat lung. METHODS Following general anesthesia, the left bronchus, pulmonary artery and vein were clamped for 1 hour. FR167653 was administered continuously beginning 30 minutes before the onset of ischemia and extending for 2 hours after reperfusion. Thirty-eight Wistar rats were divided into 4 groups according to the dose of FR167653 at the rate of 0.1, 0.05 and 0.025 mg/kg/hr in each group. After the optimal dose was obtained from the result of 1-week survival rate, the group with the optimal dose was compared with a control group by using such parameters as arterial oxygen saturation (SaO(2)), arterial oxygen tension (PaO(2)), cytokines, the expression of p38 MAP kinase and histologic study. RESULTS Survival rate of the group received FR at the rate of 0.1 mg/kg/hr (FR0.1 group) was best among the 4 groups. SaO(2) levels and PaO(2) levels after 2-hour of reperfusion were significantly (p < 0.05, respectively) higher in the FR0.1 group than in the control group. After 2-hour reperfusion, IL-1 beta was lower in the FR0.1 group than in the control group, and the expression of p38 MAP kinase was reduced in the FR0.1 group compared with the control group. In histologic study after 2-hour of reperfusion, alveolar damage with edema and interstitial thickening localized along the alveolar duct were observed in the control group, whereas these findings were remarkably less evident in the FR0.1 group. CONCLUSIONS We concluded that FR167653 ameliorates ischemia-reperfusion injury of the lung and may inhibit the production of proinflammatory cytokines by means of the inhibition of p38 MAP kinase.

Prognostic significance of L-type amino-acid transporter 1 expression in surgically resected pancreatic cancer

Background:The expression of L-type amino-acid transporter 1 (LAT1) is tumour-specific and has been shown to have essential roles in cell growth and survival. However, little is known regarding the clinical significance of LAT1 expression in pancreatic cancer. This study was conducted to determine the prognostic significance of LAT1 expression.Methods:A total of 97 consecutive patients with surgically resected pathological stage I–IV pancreatic ductal adenocarcinoma were retrospectively reviewed. Tumour sections were stained by immunohistochemistry for LAT1, CD98, Ki-67 and vascular endothelial growth factor (VEGF), and microvessel density was determined by CD34 and p53.Results:L-type amino-acid transporter 1 and CD98 were highly expressed in 52.6% (51/97) and 56.7% (55/97) of cases, respectively (P=0.568). The expression of LAT1 within pancreatic cancer cells was significantly associated with disease stage, tumour size, Ki-67, VEGF, CD34, p53 and CD98. L-type amino-acid transporter 1 expression was confirmed to be a significant prognostic factor for predicting poor outcome by multivariate analysis.Conclusion:L-type amino-acid transporter 1 expression is a promising pathological marker for the prediction of outcome in patients with pancreatic cancer.

LPA1 receptors mediate stimulation, whereas LPA2 receptors mediate inhibition, of migration of pancreatic cancer cells in response to lysophosphatidic acid and malignant ascites

Malignant ascites from pancreatic cancer patients has been reported to stimulate migration of pancreatic cancer cells through lysophosphatidic acid (LPA) and LPA(1) receptors. Indeed, ascites- and LPA-induced migration was inhibited by Ki16425, an LPA(1) and LPA(3) antagonist, in Panc-1 cells. Unexpectedly, however, in the presence of Ki16425, ascites and LPA inhibited cell migration in response to epidermal growth factor (EGF). The inhibitory migratory response to ascites and LPA was also observed in the cells treated with pertussis toxin (PTX), a G(i) protein inhibitor, and attenuated by a small interfering RNA (siRNA) specific to the LPA(2) receptor. The inhibitory LPA action was reversed by the regulators of G-protein signaling domain of p115RhoGEF, dominant-negative RhoA or C3 toxin. Indeed, LPA activated RhoA, which was attenuated by the siRNA against the LPA(2) receptor. Moreover, LP-105, an LPA(2) agonist, also inhibited EGF-induced migration in the PTX-treated cells. A similar inhibitory migration response through LPA(2) receptors was also observed in YAPC-PD, BxPC-3, CFPAC-1 and PK-1 pancreatic cancer cell lines. LPA also inhibited the invasion of Panc-1 cells in the PTX-treated cells in the in vitro Matrigel invasion assay. We conclude that LPA(2) receptors are coupled to the G(12/13) protein/Rho-signaling pathway, leading to the inhibition of EGF-induced migration and invasion of pancreatic cancer cells.

Living-related liver transplantation for type II citrullinemia using a graft from heterozygote donor.

BACKGROUND Type II citrullinemia (CTLN2) characterized by a liver-specific argininosuccinate synthetase deficiency is an adult onset genetical disorder caused by the mutation of SLC25A13 gene, which results in fulminant hyperammonemia often with poor prognosis. METHODS A 16-year-old Japanese boy presented fulminant hyperammonemia and encephalopathy and recovered after aggressive medical treatment. The patient was diagnosed as CTLN2 by plasma amino acid pattern and detection of the mutated SLC25A13 gene. We performed living-related liver transplantation (LRLT) using a graft from the genetically proven heterozygote father. RESULTS Serum amino acid concentration was normalized within a day after transplantation without protein restriction and medication. The patients postoperative course was natural. The patient is back in school 6 months after surgery. CONCLUSIONS Living-related liver transplantation using a graft from genetically proven heterozygote donors might be a permissible treatment modality for CTLN2. Long-term observation may be necessary to make a definite conclusion possible.

Effects of a p38 mitogen-activated protein kinase inhibitor as an additive to university of wisconsin solution on reperfusion injury in liver transplantation.

BACKGROUND Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in the development of ischemia/reperfusion injury in nonhepatic organs, such as the heart. However, the role of p38 MAPK activation in the liver is unclear. We examined the effects of FR167653, a novel p38 MAPK inhibitor, as an additive to University of Wisconsin (UW) solution in rat liver transplantation. METHODS Rat orthotopic liver transplantation was performed after 30 hr of cold storage using UW solution with or without FR167653. Ten-day survival rates, serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels, liver tissue blood flow, histological findings, and activities of p38 MAPK and p46/p54 c-Jun N-terminal kinase (JNK) in liver grafts were evaluated. RESULTS The addition of FR167653 significantly increased animal survival rates. FR167653 significantly suppressed serum ALT and LDH levels and improved liver tissue blood flow after transplantation. FR167653 also ameliorated histological damage to the liver graft. Neither p38 MAPK nor p46/p54 JNKs was activated during cold storage, whereas both were markedly activated within 30 min of reperfusion and remained activated until 60 min after reperfusion. FR167653 inhibited the activation of p38 MAPK both 30 and 60 min after reperfusion, but it did not affect the activation of p46/p54 JNKs. CONCLUSIONS The addition of FR167653 to UW solution improved liver graft viability and animal survival rates associated with the inhibition of p38 MAPK activation. These results suggest that inhibiting the activation of p38 MAPK may attenuate ischemia/reperfusion injury in liver transplantation.