Keitaro Kakinoki

Pancreas preservation by the 2-layer cold storage method before islet isolation protects isolated islets against apoptosis through the mitochondrial pathway

BACKGROUND Apoptosis in isolated islets has been implicated in primary nonfunction or early graft failure after islet transplantation. Recently, pancreas preservation by the 2-layer method (TLM) before islet isolation has been proved to improve the islet yield, quality, and transplant results not only in experimental models, but also in clinical settings. We examined the influence of TLM on apoptosis of isolated islets. METHOD Rat islets freshly isolated and after pancreas preservation by TLM or conventional cold storage in University of Wisconsin solution (UW) were examined and compared. Islet apoptosis was assessed by TUNEL and annexin V assays. The apoptosis pathways involved were investigated by measurement of caspase 3, 8, and 9 activities and by immunoblotting for total and phosphorylated c-Jun NH2-terminal kinase (JNK) and p38. RESULTS Islet apoptosis in the UW group was significantly increased compared with the fresh and TLM groups. Both caspase 3 and 9 activities in the UW group were higher than in the fresh and TLM groups with an approximate increase of 2- to 3-fold. On the other hand, there was no significant difference in caspase 8 activity among these 3 groups. JNKs were strongly activated both in the TLM and UW groups; although they were not activated in the fresh group, p38 was activated to almost the same levels in these 3 groups. CONCLUSIONS Pancreas preservation by TLM before islet isolation protects isolated islets against apoptosis mainly through the mitochondrial pathway. Pancreas storage before islet isolation even with TLM triggers activation of JNKs in isolated islets.

18F-fluorodeoxyglucose positron emission tomography in the diagnosis of small pancreatic cancer

AIM To investigate the role of (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) in the diagnosis of small pancreatic cancer. METHODS This study involved 31 patients with proven invasive ductal cancer of the pancreas. The patients were divided into 3 groups according to the maximum diameter of the tumor: TS1 (maximum tumor size ≤ 2.0 cm), TS2 (> 2.0 cm and ≤ 4.0 cm) or TS3-4 (> 4.0 cm). The relationships between the TS and various diagnostic tools, including FDG-PET with dual time point evaluation, were analyzed. RESULTS The tumors ranged from 1.3 to 11.0 cm in diameter. Thirty of the 31 patients (97%) had a positive FDG-PET study. There were 5 patients classified as TS1, 15 as TS2 and 11 as TS3-4. The sensitivity of FDG-PET, computed tomography (CT) and magnetic resonance imaging (MRI) were 100%, 40%, 0% in TS1, 93%, 93%, 89% in TS2 and 100%, 100%, 100% in TS3-4. The sensitivity of FDG-PET was significantly higher in comparison to CT and MRI in patients with TS1 (P < 0.032). The mean standardized uptake values (SUVs) did not show a significant difference in relation to the TS (TS1: 5.8 ± 4.5, TS2: 5.7 ± 2.2, TS3-4: 8.2 ± 3.9), respectively. All the TS1 tumors (from 13 to 20 mm) showed higher SUVs in FDG-PET with dual time point evaluation in the delayed phase compared with the early phase, which suggested the lesions were malignant. CONCLUSION These results indicate that FDG-PET with dual time point evaluation is a useful modality for the detection of small pancreatic cancers with a diameter of less than 20 mm.

Protection against experimental small intestinal ischaemia–reperfusion injury with oxygenated perfluorochemical

Intestinal ischaemia–reperfusion (IR) injury frequently occurs in abdominal surgery. Perfluorochemical (PFC) can be used to oxygenate intestinal organs directly and allows adenosine 5′‐triphosphate (ATP) production within the submerged organs during ischaemia. This study was designed to evaluate the protective effect of PFC in IR injury, focusing on cytokine production in rat small intestine.

Hepatic preconditioning using lipopolysaccharide: association with specific negative regulators of the Toll-like receptor 4 signaling pathway.

Background. Preconditioning using lipopolysaccharide (LPS), a Toll-like receptor (TLR)-4 ligand, has been demonstrated to attenuate ischemia-reperfusion injury (IRI) in several organs but has not been sufficiently elucidated in the liver. We investigated the molecular mechanism of protection induced by LPS preconditioning against hepatic IRI. Methods. BALB/c mice underwent 70% hepatic ischemia for 90 min. LPS was injected intraperitoneally 20 hr before ischemia at a range of 1 to 1000 &mgr;g/kg. Hepatic injury was evaluated based on serum alanine aminotransferase levels and histopathology. Inflammatory cytokine expression, nuclear factor-&kgr;B activation, and c-Jun N-terminal kinase phosphorylation were investigated after reperfusion. Additionally, preischemic expression of negative feedback inhibitors of the TLR4 cascade was examined. Results. Only the 100 &mgr;g/kg LPS pretreatment significantly reduced serum alanine aminotransferase levels and histopathologic damage 6 hr after reperfusion; there was no difference among other LPS concentrations. In mice pretreated with LPS, intrahepatic expression of tumor necrosis factor-&agr; and interleukin (IL)-6 as well as activation of nuclear factor-&kgr;B and c-Jun N-terminal kinase were inhibited 1 hr after reperfusion, whereas expression of IL-10, an anti-inflammatory cytokine, was induced. Suppressor of cytokine signaling (SOCS)-1, SOCS-3 and IL-1 receptor-associated kinase-M were upregulated by LPS exposure in the preischemic period. Conclusions. Hepatic LPS preconditioning elicited the upregulation of specific negative regulators in the TLR4 signaling pathway. Preischemic induction of these regulators plays an important role as immunologic preparation for the subsequent ischemia-reperfusion and produces resistance to liver injury. Preoperative modulation of the TLR4 pathway might become an alternative therapeutic strategy against hepatic IRI.

Successful 24-hour preservation of ischemically damaged canine small intestine by the cavitary two-layer method.

Background. The purpose of this study is to examine the possibility of a long-term preservation of the ischemically damaged intestine by the cavitary two-layer method (TLM) in canine small intestinal transplantation. Methods. The grafts were allotransplanted without preservation immediately (group 1) or after 30 minutes of warm ischemia (group 2). The ischemically damaged grafts were also allotransplanted after cold preservation for 24 hours in University of Wisconsin (UW) solution (group 3) or the cavitary TLM (group 4). Seven-day survivals, tissue adenosine triphosphate (ATP) concentrations, absorption tests, and histopathology were examined. Results. seven-day survivals in groups 1, 2, 3, and 4 were 8 of 8, 6 of 8, 0 of 8, and 6 of 8, respectively. In group 4, significant recovery of ATP tissue level was seen after preservation compared with group 3, and absorption function and regeneration of the graft mucosa recovered at day 14. Conclusions. Ischemically damaged canine small intestine could be preserved for 24 hours by the cavitary TLM.

Protective effect of lipopolysaccharide preconditioning in hepatic ischaemia reperfusion injury

BACKGROUND Preconditioning using lipopolysaccharide (LPS), a toll-like receptor 4 (TLR4) ligand, has been demonstrated to reduce ischaemia/reperfusion injury (IRI) in some organs, but its effect in the liver has not been elucidated. We examined the liver protective mechanism and correlated signalling pathway of LPS preconditioning in mice. METHODS BALB/c and TLR4 mutant mice underwent 90 min of 70% hepatic ischaemia. Lipopolysaccharide (100 µg/kg) was injected intraperitoneally 20 h or 30 min before ischaemia. Liver damage after reperfusion was examined using serum samples and liver specimens. To analyse the mechanism of preconditioning in detail, phosphorylation of representative signalling mediators to nuclear factor-κB (NF-κB) activation, Akt and interleukin-1 receptor-associated kinase-1 (IRAK-1), and expression of a negative feedback inhibitor, suppressor of cytokine signalling-1 (SOCS-1), were evaluated by Western blotting. RESULTS Pretreatment with LPS only 20 h before ischaemia elicited a preconditioning effect; however, preconditioning was absent in TLR4 mutant mice. Lipopolysaccharide significantly decreased serum alanine aminotransferase, tumour necrosis factor-α, hepatocyte necrosis and NF-κB activity after reperfusion. Phosphorylated IRAK-1 was suppressed by LPS, whereas no difference was observed in phosphorylated Akt. Pre-ischaemic LPS provided early induction of SOCS-1. DISCUSSION Late-phase LPS preconditioning provided liver protection against IRI through the downregulation of the TLR4 cascade derived from early induction of SOCS-1 during ischaemia/reperfusion.

Detailed analysis of mucosal restoration of the small intestine after the cavitary two-layer cold storage method.

Small bowel transplantation (SBT) is associated with a high incidence of infectious complications because of ischemia/reperfusion (I/R) mucosal injury concomitant with potent immunosuppression. In this study, we evaluated whether the cavitary two‐layer method (cTLM) could reduce I/R injury and allow early mucosal restoration, particularly after prolonged preservation and transplantation.

Surgical approach for extrahepatic metastasis of HCC in the abdominal cavity.

BACKGROUND/AIMS Despite recent development of therapeutic strategies for intrahepatic lesions, standard guidelines for treatment of extrahepatic metastases of hepatocellular carcinoma have not been established. METHODOLOGY Surgical resection for intra-abdominal extrahepatic metastases of hepatocellular carcinoma was performed on 10 patients at our institution between 1992 and 2008. We retrospectively examined the clinicopathologic features and significance of a surgical approach in these patients. RESULTS Nine of the 10 patients received treatment for primary hepatocellular carcinoma before surgery for intra-abdominal extrahepatic metastasis. A simultaneous intrahepatic lesion was detected in half of the patients when the extrahepatic metastasis was resected. Extrahepatic recurrent organs included adrenal glands, lymph nodes, abdominal wall, stomach and diaphragm. The mean survival period after resection was 36.1 months. Two patients are still alive without further recurrence. One patient died of retroperitoneal recurrence and 7 died of intrahepatic recurrence or liver failure after resection. CONCLUSIONS With careful case selection, considering that not all extrahepatic metastases suggest systemic spread of hepatocellular carcinoma, surgical treatment for metastatic lesions in the abdominal cavity can provide a relatively good prognosis.

Clinicopathological comparison between intrahepatic cholangiocarcinoma arising in livers positive and negative for hepatitis B or C virus

To the Editor: An increasing incidence of intrahepatic cholangiocarcinoma (ICC) has been recognized worldwide (1, 2). Although there are a few well-established risk factors for ICC, including primary sclerosing cholangitis, liver fluke infection, and hepatolithiasis, none of these risk factors can explain the recent trend for the increase of ICC (1). Recent reports have pointed to the potential role of chronic liver disease, hepatitis C, and possibly hepatitis B infections in the development of ICC (1–8). However, it remains to be clarified regarding how the hepatitis virus induces the carcinogenesis of ICC, or how the virus affects the treatment and prognosis of ICC. We compared the clinicopathological features and the prognosis after the surgical treatment of ICCs arising from livers with and without hepatitis B virus (HBV) or hepatitis C virus (HCV), with an aim of clarifying the impact of HBV or HCV infection on the development of ICC and on the prognosis of patients. Forty-one patients who had undergone a primary attempt of a curative resection for ICC were involved. The following clinicopathological features were compared for patients both with and without HBV or HCV: patient’s age, gender, liver function tests including the serum level of aspartate aminotransferase, prothrombin time, indocyanin green dye retention rate, the serum levels of carcinoembryonic antigen and a-fetoprotein (AFP), the size of the tumor, the staging of the cancer according to the TNM classification, and the histopathological findings of the cancer and liver parenchyma. The differences were examined statistically using either Mann–Whitney’s U-test or the w test. The survival rates after surgery were obtained using the Kaplan–Meier method. To identify the prognostic

Improved quantity and in vivo function of islets isolated by reduced pressure-controlled injection of collagenase in a rat model.

In islet transplantation, insufficient yield is a major obstacle to one-donor/one-recipient transplant. Collagenase, which is injected via a pancreatic duct to separate islets from acini, can so easily distribute into the islet core that it may result in disruption of islets. The purpose of this study was to evaluate the superiority of reduced pressure-controlled collagenase injection (RPCI) at 80 mmHg on islet isolation to injection at 180 mmHg by examining in vivo transplant experiments besides the yield and the glucose stimulation test in a rat model. Lewis rat pancreases were distended with collagenase solution at 80 mmHg pressure as the RPCI group (group 1) and at 180 mmHg (group 2), followed by isolation. The yield in group 1 (1100 ± 160 islets with 2750 ± 530 IEQ) was significantly higher than that in group 2 (900 ± 130 islets with 1570 ± 350 IEQ, p < 0.01) due to the significant difference of the number of islets sized >150 μm in diameter, although the purity was not significantly different between the two groups. Stimulation indices in the glucose stimulation tests were 2.88 ± 1.12 in group 1 and 1.93 ± 0.62 in group 2 (p < 0.05). The cure rate by transplantation of 100 islets to diabetic nude mice in group 1 (8/10) was significantly higher than that in group 2 (3/10, p < 0.05). In a syngenic transplant model of 90% of islets isolated from one donor, the cure rates were 100% and 67% in groups 1 and 2, respectively (NS). The area under the curve on the graph of IPGTT on postoperative day 28 in group 1 was significantly smaller than that in group 2 (p < 0.05). In conclusion, our data show that RPCI at 80 mmHg could contribute to consistently high islet yield and in vivo function in a rat model. It was suggested that the current human protocol should be reviewed from this viewpoint.