Introduction of pseudo-base benzimidazole derivatives into nucleosides via base exchange by a nucleoside metabolic enzyme.

In alternate organic synthesis, biocatalysis using enzymes provides a more stereoselective and cost-effective approach. Synthesis of unnatural nucleosides by nucleoside base exchange reactions using nucleoside-metabolizing enzymes has previously shown that the 5-position recognition of pyrimidine bases on nucleoside substrates is loose and can be used to introduce functional molecules into pyrimidine nucleosides. Here we explored the incorporation of purine pseudo bases into nucleosides by the base exchange reaction of pyrimidine nucleoside phosphorylase (PyNP), demonstrating that an imidazole five-membered ring is an essential structure for the reaction. In the case of benzimidazole, the base exchange proceeded to give the deoxyribose form in 96 % yield, and the ribose form in 23 % yield. The reaction also proceeded with 1H-imidazo[4,5-b]phenazine, a benzimidazole analogue with an additional ring, although the yield of nucleoside was only 31 %. Docking simulations between 1H and imidazo[4,5-b]phenazine nucleoside and the active site of PyNP (PDB 1BRW) supported our observation that 1H-imidazo[4,5-b]phenazine can be used as a substrate by PyNP. Thus, the enzymatic substitution reaction using PyNP can be used to incorporate many purine pseudo bases and benzimidazole derivatives with various functional groups into nucleoside structures, which have potential utility as diagnostic or therapeutic agents.

Role of podoplanin and Kupffer cells in liver injury after ischemia-reperfusion in mice.

AIM: To investigate the relationship between the intrahepatic expression of podoplanin (PDPN) and Kupffer cells (KCs) in ischemia-reperfusion (I/R) liver damage. METHODS: C57Bl/6 mice were injected with 200 µl of clodronate liposomes (macrophage depletion; MDP group) to deplete KCs or control liposomes (control group) via the ophthalmic vein plexus 24 h prior to ischemia. Animals were subjected to 90 min of partial hepatic ischemia (70%), followed by reperfusion, and were then killed at designated time points. Serum and liver tissues were harvested for further analyses. RESULTS: Serum ALT levels, mortality rates, and the percentage of necrotic area in liver sections were significantly higher in the MDP group than in the control group. PDPN was expressed in the lymphatic epithelium, interlobular bile duct epithelium, and in some hepatocytes in each group. Its expression in hepatocytes was down-regulated in the MDP group. The accumulation of platelets in the sinusoid was reduced 6 h after I/R in the MDP group. Tissue HGF and IGF-1 levels decreased in the MDP group. CONCLUSIONS: These results suggest that KCs play a key role in the activation of platelets through direct contact with PDPN-positive hepatocytes in I/R livers.

An extremely rare combination of choledochocele and bile duct duplication escalating severe acute pancreatitis and cholangitis: A case report.

BACKGROUND: Combination of choledochocele and extra-hepatic duct duplication is an extremely rare congenital abnormality. CASE PRESENTATION: The patient was an 81-year-old Japanese man. He visited the emergency room for severe abdominal colic pain. He was diagnosed with severe pancreatitis with cholelithiasis and treated conservatively by percutaneous trans-hepatic gallbladder drainage (PTGBD) for 4 months. Thereafter, he was transferred to our institute and cholangiography was performed via the PTGBD tube, revealing cholecysto- and choledocho-lithiasis. The cystic-duct joined the right hepatic duct with extra-hepatic bile duct duplication and the terminal bile duct flowed into the cystic papilla of Vater. The main pancreatic duct also joined into the cystic papilla. These observations confirmed choledochocele with extra-hepatic bile duct duplication. Surgical exploration was performed, and hepatico-jejunostomy with hepatic-ductplasty and cholecystectomy with choledocholithotomy were carried out. He was discharged and his course was uneventful. CONCLUSION: A very rare combined case of choledochocele with bile duct duplication, which would escalate the pancreatitis and cholangitis, was successfully treated. Their pathogeneses in relation to pancreaticobiliary maljunction is discussed.

Changes in Function and Dynamics in Hepatic and Splenic Macrophages in Non-Alcoholic Fatty Liver Disease.

BACKGROUND: The aim of this study was to investigate the populations and functions of hepatic and splenic macrophages (Mfs) in non-alcoholic fatty liver disease (NAFLD). MATERIALS AND METHODS: Experiment 1: Wild-type and STAM® mice were given chow or high-fat diets for designated periods. In isolated Mfs, phagocytosis and cytokine production were assessed. Immunohistochemistry for CD68 and F4/80 and expression of CD14 and CD16 were assessed. Experiment 2: Bone marrow cells harvested from enhanced green fluorescent protein (EGFP) mice were transplanted into wild-type mice with or without splenectomy after total body irradiation that was kept on methionine- and choline-deficient diets. RESULTS: Experiment 1: The number of CD68-positive cells and the percentage of F4/80-positive/CD68-positive cells increased with the progression of NAFLD. Production of TNF-α and IL-6 by hepatic Mfs was greater than that by splenic Mfs in mice with NASH. The number of CD14+CD16- Mfs increased in the spleen and decreased in the liver in animals that had progressed to NASH. Furthermore, the number of CD14+CD16+ hepatic Mfs was increased in animals that had progressed to NASH with fibrosis. Experiment 2: EGFP-positive cells were observed in the liver after transplantation. In the splenectomy group, EGFP-positive Mfs were also observed; however, the number was significantly less than that in the sham operation group. CONCLUSION: The populations and functions of hepatic and splenic Mfs are altered during the progression of NAFLD. In addition, increased hepatic Mfs during the progression of NAFLD may migrate from bone marrow to the liver via the spleen.

Pituitary Adenylate Cyclase-Activating Polypeptide Prevents Mortality Caused by Septic Peritonitis in Mice.

The purpose of this study was to investigate whether pituitary adenylate cyclase-activating polypeptide (PACAP) prevents mortality due to sepsis in mice. Mice were given PACAP at designated time points before or after cecal ligation and puncture (CLP), and organ injury and mortality were investigated. Serum inflammatory and anti-inflammatory cytokine levels were assessed after CLP. Plasma corticosterone and adrenocorticotropic hormone levels were also measured. Isolated tissue macrophages (Mfs) were incubated with or without PACAP, and production of cytokines was measured. Activation of NF-κB was investigated in tissue Mfs isolated from CLP animal in the presence and absence PACAP in vitro. PACAP treatment significantly prevented acute lung injury and mortality after CLP. Plasma endotoxin levels and bacterial load were not different between PACAP-treated and nontreated groups. Increased serum TNF-α and HMGB1 levels in animals treated with vehicle were significantly blunted in PACAP-treated animals after CLP. Furthermore, serum IL-10 levels were significantly greater in the PACAP-treated group compared with the vehicle group. Production of HMGB1 and TNF-α by isolated hepatic Mfs was significantly inhibited in the presence of PACAP, whereas production of IL-10 by isolated hepatic Mfs and interstitial lung Mfs was significantly increased. Plasma corticosterone and adrenocorticotropic hormone levels were significantly greater in the animals treated with PACAP compared with vehicle after CLP. Activation of NF-κB was significantly inhibited by PACAP in the hepatic Mfs compared with other tissue Mfs. PACAP prevents mortality due to septic peritonitis by inhibiting inflammation via NF-κB activation and possible effects on the brain.

Effects of Medium-chain Triglycerides Administration in Chemically-induced Carcinogenesis in Mice.

AIM: The aim of this study was to investigate the effects of medium-chain triglycerides (MCTs) on chemically-induced hepatic carcinogenesis (HCC) in mice. MATERIALS AND METHODS: In a first set of experiments, mice were treated with diethylnitrosoamine intraperitoneally at two weeks of age. They were fed chow containing MCT or a normal chow diet and sacrificed after 28 weeks. Incidence of hepatic tumor was compared between the two groups. Expression of oxidative stress, and inflammatory cytokines and chemokines in liver tissues were examined. In a second set of experiments, the histopathological findings of the intraperitoneal adipose tissue were assessed, and expression of adipocytokines in the fat tissue was measured. In a third set of experiments, plasma ß-hydroxybutyrate (HB) concentration was measured in both animals fed chow containing MCT and a normal chow diet. Mouse HCC cells were co-cultured with ß-HB, and the numbers of tumor cells were counted at days 3 and 7. RESULTS: In the first set of experiments, the tumor count observed in the control group was significantly blunted in the MCT group. Maximum tumor diameter also decreased in the MCT group compared to the control group. The expression of inflammatory cytokines and chemokines was significantly decreased by MCT. Furthermore, expression of 4-hydroxynonenal was lower in the MCT group compared to the control group. In the second set of experiments, hypertrophy of the adipocytes was suppressed, and the concentration of adiponectin and leptin in the adipose tissue decreased by MCT. In the third set of experiments, plasma ß-HB concentration increased in the MCT group as expected. ß-HB significantly inhibited the proliferation of HCC cells. CONCLUSION: MCT administration markedly suppresses the incidence of chemically-induced HCC by inhibition of inflammation and increase of ketone bodies.

M-CSF Receptor Antagonists Inhibit the Initiation and Progression of Hepatocellular Carcinoma in Mice.

BACKGROUND/AIM: The aim of this study was to investigate the effects of the macrophage colony-stimulating factor (M-CSF) receptor antagonist on hepatic carcinogenesis in mice. MATERIALS AND METHODS: Mice were injected with diethylnitrosamine (DEN) and treated with M-CSF receptor antagonist GW2580 (GW) or a saline vehicle just after (early treated group) or 2 weeks after (late treated group) DEN injection. Animals were sacrificed after 28 weeks and incidence of tumor was assessed. Isolated Kupffer cells were co-cultured with M-CSF in the presence or absence of GW, and the concentration of VEGF was measured. RESULTS: The incidence of tumors was significantly blunted both in the early- and the late-treated groups. In addition, angiogenesis within the tumor was also suppressed in both groups. The concentration of VEGF increased in Kupffer cells treated with M-CSF compared to those cultured without M-CSF. This increase was blunted by GW. CONCLUSION: M-CSF and its receptor could be novel molecular targets for hepatocellular carcinoma.

Expression of podoplanin in stromal fibroblasts plays a pivotal role in the prognosis of patients with pancreatic cancer.

PURPOSE: To investigate the role of podoplanin (PDPN) expression in invasive ductal carcinoma of the pancreas (IDCP) in humans. METHODS: Tumor samples were obtained from 95 patients with IDCP. Immunohistochemical staining was done to evaluate the expression of PDPN in cancer tissues. RESULTS: PDPN was detected predominantly in stromal fibroblasts, stained with α-smooth muscle actin. The cutoff value of PDPN-positive areas was calculated according to a histogram. There was no significant difference in clinicopathologic factors between patients with high vs. those with low PDPN expression. The high PDPN group showed significantly poorer disease-free and disease-specific survival rates than the low PDPN group. Among patients from the high PDPN group, those with lymph node metastases and those with a tumor larger than 20 cm in diameter had significantly poorer prognoses than similar patients from the low PDPN group. Multivariate Cox proportional hazards analysis indicated that a high expression of PDPN was an independent risk factor for disease-specific survival. CONCLUSIONS: PDPN expression in cancer-related fibrotic tissues is associated with a poor prognosis, especially in patients with large tumors or lymph node metastases.

Feasibility and Validation of Single-Port Laparoscopic Surgery for Simple-Adhesive or Nonadhesive Ileus.

A single incisional laparoscopic surgery (SILS) approach is increasingly being used, taking advantage of the minimally invasive technique. The aim of this study was to evaluate the feasibility and the validation of SILS procedure for small bowel obstruction (SBO). Sixteen consecutive patients with SBO who underwent SILS release of ileus between April 2010 and March 2015 were compared with the conventional multiport laparoscopic treatment group of 16 patients matched for age, gender, and surgical procedure. Laparoscopic treatment was completed in a total of 14 patients in SILS group and 13 in multiport laparoscopic group. Two cases and 3 cases were converted to multiport laparoscopic surgery or open surgery. Eight patients with nonscar and nonadhesive ileus, such as internal hernia, obturator hernia, gallstone ileus, and intestinal invagination, were treated successfully in the laparoscopic procedure. There was no mortality in either of the groups. The mean procedural time was 105  minutes in the SILS group and 116  minutes in the multiport laparoscopic group. The mean amount of blood loss was not statistically different in either of groups (15  ml vs. 23  ml). Patients resumed oral intake after a mean of 2 days in the SILS and 3 days in the multiport groups with the statistically difference. The length of hospital stay was shorter in the SILS group (5 days vs. 7 days) with no statistically difference. Perioperative morbidity was seen in 2 patients in the SILS group and 3 patients in the multiport group. SILS approach has superior and/or similar perioperative outcomes to multiport approach for SBO. SILS release of ileus as an ultra-minimal invasion technique is feasible, effective, and offers benefits with cosmesis in simple adhesive or scar-less nonadhesive ileus patients.

Single-port laparoscopic adhesiolysis for torsion ileus.

Single-incisional laparoscopic surgery (SILS) has emerged as an attempt to further enhance the cosmetic benefits and reduce the morbidity of minimally invasive surgery. We present an approach of SILS adhesiolysis to adhesive strangulated ileus. A 70-year-old female patient, who had undergone laparoscopic low anterior resection 6 years before, underwent SILS adhesiolysis to a midline surgical incision wound adhesion site. The surgery was performed with only a 2.5-cm left-side transrectus incision using the SILS port. Laparoscopy revealed intestinal torsion and congestion with adhesion of the ileum to the previous surgical incision wound. SILS adhesiolysis was successfully carried out. The patient was discharged 4 days after surgery. SILS adhesiolysis is a feasible and efficient procedure in certain cases.

Successful management of a blunt pancreatic trauma by endoscopic stent placement.

The present study reports a case involving a 17-year-old man who was brought to the emergency department of our hospital with severe upper abdominal pain following a blow received in a rugby game. Emergency computed tomography (CT) revealed severe pancreatic neck injury, and the patient was subsequently given conservative treatment in the High Care Unit. Forty-eight hours later, follow-up enhanced CT revealed that the pancreas was clearly lacerated and the amount of peripancreatic fluid was increasing; furthermore, serum amylase and elastase levels were elevated. Endoscopic retrograde pancreatography revealed that contrast medium in the main pancreatic duct (MPD) had leaked to the parenchyma, indicating an MPD injury. To prevent traumatic pancreatitis from worsening, a stent was inserted endoscopically to a site distal to the injured portion of the MPD. Thereafter, the patient's condition dramatically improved, and his serum amylase levels returned to normal. CT revealed that the apparent pancreatic edema and peripheral fluid were also decreased. During a short-term follow-up period of 6 months, removal of the stent was uneventfully carried out and the patient did not develop any exocrine or endocrine insufficiency. We suggest that, in some cases, endoscopic management of traumatic pancreatic duct disruption is feasible and effective.