Akira Kenjo

Cloning and Characterization of Novel Ficolins from the Solitary Ascidian, Halocynthia roretzi

Ficolins are animal lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding lectin (MBL) activates the complement lectin pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated ∼40 kDa (p40) and ∼50 kDa (p50) N-acetylglucosamine-binding lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizesN-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4–52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph lectin activity and the correlation of different functions with binding specificity.

A pancreaticoduodenectomy risk model derived from 8575 cases from a national single-race population (japanese) using a web-based data entry system: The 30-day and in-hospital mortality rates for pancreaticoduodenectomy

Objective:To create a mortality risk model after pancreaticoduodenectomy (PD) using a Web-based national database system. Background:PD is a major gastroenterological surgery with relatively high mortality. Many studies have reported factors to analyze short-term outcomes. Subjects and Methods:After initiation of National Clinical Database, approximately 1.2 million surgical cases from more than 3500 Japanese hospitals were collected through a Web-based data entry system. After data cleanup, 8575 PD patients (mean age, 68.2 years) recorded in 2011 from 1167 hospitals were analyzed using variables and definitions almost identical to those of American College of Surgeons–National Surgical Quality Improvement Program. Results:The 30-day postoperative and in-hospital mortality rates were 1.2% and 2.8% (103 and 239 patients), respectively. Thirteen significant risk factors for in-hospital mortality were identified: age, respiratory distress, activities of daily living within 30 days before surgery, angina, weight loss of more than 10%, American Society of Anesthesiologists class of greater than 3, Brinkman index of more than 400, body mass index of more than 25 kg/m2, white blood cell count of more than 11,000 cells per microliter, platelet count of less than 120,000 per microliter, prothrombin time/international normalized ratio of more than 1.1, activated partial thromboplastin time of more than 40 seconds, and serum creatinine levels of more than 3.0 mg/dL. Five variables, including male sex, emergency surgery, chronic obstructive pulmonary disease, bleeding disorders, and serum urea nitrogen levels of less than 8.0 mg/dL, were independent variables in the 30-day mortality group. The overall PD complication rate was 40.0%. Grade B and C pancreatic fistulas in the International Study Group on Pancreatic Fistula occurred in 13.2% cases. The 30-day and in-hospital mortality rates for pancreatic cancer were significantly lower than those for nonpancreatic cancer. Conclusions:We conducted the reported risk stratification study for PD using a nationwide surgical database. PD outcomes in the national population were satisfactory, and the risk model could help improve surgical practice quality.

An Ancient Lectin-Dependent Complement System in an Ascidian: Novel Lectin Isolated from the Plasma of the Solitary Ascidian, Halocynthia roretzi

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of ∼36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.

Risk stratification of 7,732 hepatectomy cases in 2011 from the National Clinical Database for Japan

BACKGROUND There has been no report on risk stratification for hepatectomy using a nationwide surgical database in Japan. The objective of this study was to evaluate mortality and variables associated with surgical outcomes of hepatectomy at a national level. STUDY DESIGN We analyzed records of 7,732 patients who underwent hepatectomy for more than 1 segment (MOS) during 2011 in 987 different hospitals, as identified in the National Clinical Database (NCD) of Japan. The NCD captured 30-day morbidity and mortality as well as 90-day in-hospital mortality outcomes, which were submitted through a web-based data entry system. Based on 80% of the population, independent predictors for 30-day mortality and 90-day in-hospital mortality were calculated using a logistic regression model. The risk factors were validated with the remaining 20% of the cohort. RESULTS The median postoperative length of hospitalization was 16.0 days. The overall patient morbidity rate was 32.1%. Thirty-day mortality and 90-day in-hospital mortality rates were 2.0% and 4.0%, respectively. Totals of 14 and 23 risk factors were respectively identified for 30-day mortality and 90-day in-hospital mortality. Factors associated with risk for 90-day in-hospital mortality were preoperative condition and comorbidity, operative indication (emergency surgery, intrahepatic/perihilar cholangiocarcinoma, or gallbladder cancer), preoperative laboratory data, and extent and location of resected segments (segment 1, 7, or 8). As a performance metric, c-indices of 30-day mortality and 90-day in-hospital mortality were 0.714 and 0.761, respectively. CONCLUSIONS Here we report the first risk stratification analysis of hepatectomy using a Japanese nationwide surgical database. This system would predict surgical outcomes of hepatectomy and be useful to evaluate and benchmark performance.

Increased IL‑17 production correlates with immunosuppression involving myeloid‑derived suppressor cells and nutritional impairment in patients with various gastrointestinal cancers

Although a causal relationship between inflammation and innate immunity of cancer is more widely accepted today, many of the precise cell mechanisms mediating this relationship have not been elucidated. Th17 cells, which produce the proinflammatory cytokine interleukin 17 (IL-17), have been recognized as one of the key factors in the regulation of inflammatory bowel disease and rheumatoid arthritis. This study demonstrated that, in patients with various types of gastrointestinal cancer, IL-17 production was correlated with myeloid-derived suppressor cell (MDSC) levels and with markers for nutritional impairment, immune suppression and chronic inflammation. IL-17 was significantly higher in patients with various types of gastrointestinal cancer compared to normal volunteers. In addition, IL-17 levels were significantly correlated with neutrophil counts and the neutrophil/lymphocyte ratio (NLR) and significantly inversely correlated with cell-mediated immune response indicators [lymphocyte phytohemagglutinin (PHA)-blastogenesis and IL-12 induction] and patient nutritional status (prealbumin levels). Circulating MDSC levels were significantly correlated with IL-17 production. These results suggest that, in human gastrointestinal cancers, chronic inflammation involving IL-17 may be an important mechanism contributing to disease progression through enhancement of immune suppression or cachexia. Controlling the activation of Th17 cells may prove to be a valuable strategy for the treatment of gastrointestinal cancer patients.

Phase I trial of preoperative intratumoral injection of immature dendritic cells and OK-432 for resectable pancreatic cancer patients

PurposeTo determine the feasibility, safety and histological change of preoperative endoscopic ultrasound-guided fine-needle injection (PEU-FNI) of immature DCs (iDCs) with OK-432 in pancreatic cancer patients.MethodsNine patients enrolled in the trial (DC group) and were compared with 15 patients operated on without iDC injection (non-DC group). Adverse events of PEU-FNI and postoperative complications were evaluated according to CTC-AE ver.3.0 and the Clavien–Dindo classification/ISGPF definition, respectively. Histological changes within the tumor and lymph nodes were evaluated by immunohistochemical examination of infiltrating inflammatory cells (CD4+, CD8+, Foxp3+ and CD83+).ResultsThere were no severe toxicities following PEU-FNI, except for one transient grade 3 fever, and there were no significant differences in the incidence of postoperative complications between the two groups. Colliquative necrosis and diffusely scattered TUNEL-positive cells were observed at the injection sites. CD83+ cells significantly accumulated in the regional lymph nodes of the DC group as well as Foxp3+ cells in the regional and distant lymph nodes. The two DC group patients, one of which was stage IV with distant lymph node metastasis, survived more than 5 years without requiring adjuvant theraphy.ConclusionPEU-FNI was feasible and safe, and further study needs to confirm and enhance antitumor responses.

HER2 peptide-specific CD8[+] T cells are proportionally detectable long after multiple DNA vaccinations

We prepared a plasmid encoding 147 amino acid residues from the N terminus of c-erbB-2/HER2/neu (HER2), which included both a cytotoxic T lymphocyte (CTL) epitope (HER2p63) and a helper epitope (HER2p1), using the mammalian expression vector pCAGGS-New (pCAGGS147HER2). In a parallel analysis with a Tetramer assay and CTL assay, good specificity and sensitivity of a quantitative enzyme-linked immunospot (ELISPOT) assay to detect functional HER2p63-specific CD8+ T cells were demonstrated after intramuscular immunization of pCAGGS147HER2. In an ELISPOT assay for HER2p63, spots of IFNγ-producing cells were first detected 10 days after the first immunization, and additional immunizations increased the number of spots. HER2p63-specific CD8+ T cells were detected over a period of more than 10 months after the last immunization. In hosts receiving more than three immunizations, surprisingly high numbers of specific CD8+ T cells were persistently detectable. HER2 protein-specific antibodies of IgG class with dominance of IgG2a remain detectable 6 months after single or multiple immunizations. The antibodies however, were not reactive with cell surface HER2 antigens. Total suppression of tumor growth was observed when syngeneic HER2+ tumor cells (2 × 106) were injected subcutaneously 14 days after a single immunization with pCAGGS147HER2. Furthermore, the number of pulmonary metastases decreased significantly when DNA vaccination was initiated on the day of, or 3 days after, intravenous injection (1 × 106 cells).

Ischemic preconditioning enhances regenerative capacity of hepatocytes in long-term ischemically damaged rat livers

Background and Aims:  Ischemic preconditioning (IPC) protects tissues against ischemia and reperfusion (I/R) injury. The aim of this study was to examine the impact of IPC on protection and regeneration of hepatocytes after prolonged I/R injury.

A model to evaluate toxic factors influencing islets during collagenase digestion: the role of serine protease inhibitor in the protection of islets.

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.

Successful treatment of pseudoaneurysms of celiac and superior mesenteric arteries by combined endovascular and surgical approach

Pseudoaneurysm after pancreas resection poses serious complications, including rupture and hemorrhage. Here we report a case of delayed massive hemorrhage from celiac and superior mesenteric arteries, which was successfully treated with a combined endovascular and surgical approach. The patient was a 52-year-old man who presented with pseudoaneurysms of the celiac and superior mesenteric arteries after distal pancreatectomy. Following the detection of sentinel bleeding from the abdominal drain, emergency angiography of the celiac and superior mesenteric arteries revealed stenosis of the celiac artery and pseudoaneurysms in the superior mesenteric artery. We occluded these lesions with a platinum coil, using an interventional radiological technique combined with bypass grafting between the abdominal aorta and the SMA, using the saphenous vein. However, re-bleeding into the abdominal cavity occurred from the proximal SMA pseudoaneurysm. We inserted an endoluminal stent-graft into the abdominal aorta and completed bypass grafting between the aorta and bilateral renal arteries. The hemorrhage ceased and the postoperative course was uneventful. The patient was discharged 34 days after the treatment (149 days after the initial operation). In conclusion, this combined endovascular and surgical approach is feasible and seems appropriate for pseudoaneurysms arising from proximal sites in visceral arteries.