Ryohei Watanabe

Efficacy of Bacteriophage Therapy against Gut-Derived Sepsis Caused by Pseudomonas aeruginosa in Mice

ABSTRACT We evaluated the efficacy of bacteriophage (phage) therapy by using a murine model of gut-derived sepsis caused by Pseudomonas aeruginosa that closely resembles the clinical pathophysiology of septicemia in humans. Oral administration of a newly isolated lytic phage strain (KPP10) significantly protected mice against mortality (survival rates, 66.7% for the phage-treated group versus 0% for the saline-treated control group; P < 0.01). Mice treated with phage also had lower numbers of viable P. aeruginosa cells in their blood, liver, and spleen. The levels of inflammatory cytokines (tumor necrosis factor alpha TNF-α, interleukin-1β [IL-1β], and IL-6) in blood and liver were significantly lower in phage-treated mice than in phage-untreated mice. The number of viable P. aeruginosa cells in fecal matter in the gastrointestinal tract was significantly lower in phage-treated mice than in the saline-treated control mice. We also studied the efficacy of phage treatment for intraperitoneal infection caused by P. aeruginosa and found that phage treatment significantly improved the survival of mice, but only under limited experimental conditions. In conclusion, our findings suggest that oral administration of phage may be effective against gut-derived sepsis caused by P. aeruginosa.

KL-6 is another useful marker in assessing a micropapillary pattern in carcinomas of the breast and urinary bladder, but not the colon

To evaluate the peculiar “inside-out” pattern in micropapillary (MP) carcinoma, we investigated the usefulness of KL-6 antibody in the assessment of the MP pattern of cancers, in comparison with antibodies to epithelial membrane antigen (EMA), MUC1 (CD227), and CD 10. Immunohistochemical investigation was performed on specimens exhibiting an MP pattern obtained from 12 persons with cancer: 4 with breast carcinoma, 3 with carcinoma of the urinary bladder, and 5 with colonic carcinoma. Immunohistochemical study with KL-6, EMA, and MUC1 antibodies revealed similar continuous linear positive patterns restricted to the surface of the MP pattern in both breast and urinary bladder cancers, revealing the peculiar “inside-out” morphology. However, EMA also gave cytoplasmic positivity in most of the cases tested, and MUC1 was also present in the cytoplasm of some cases. In sharp contrast, immune reactions of colon carcinomas with these antibodies were negative, except for focal positivity for KL-6 and MUC1 antibodies in some cases. CD10 was only focally positive in an MP pattern in 4 of the 5 cases of colon carcinoma and in 1 case with carcinoma of the urinary bladder. These findings suggest that KL-6 is a useful marker to assess the MP character of breast and urinary bladder carcinomas; that MUC1 was similarly positive, with the addition of cytoplasmic positivity in some cases; and that the MP pattern of colon cancer, positive for CD 10, was different in character from both breast and urinary bladder carcinomas, although all these cancers seemingly exhibit similar MP patterns on histopathology. This heterogeneity of the MP pattern in various cancers needs to be investigated when more cases have been accumulated.

Osteoclast-like Giant Cell Tumor of the Pancreas with Metastases to Gallbladder and Lymph Nodes : A Case Report

Osteoclast-like giant cell tumor of the pancreas (OGTP) is a rare neoplasm, of which the histogenesis is still controversial. Here we report a case of OGTP involving the head of the pancreas in a 71-year-old woman with metastases to the gallbladder and lymph nodes. The primary and metastatic tumors had identical histopathological, immunohistochemical, ultrastructural and molecular biological features. Microscopically, the tumors were characterized by atypical, often pleomorphic mononuclear cells associated with the proliferation of benign-appearing osteoclast-like giant cells (OGCs). Electron microscopic observation provided ultrastructural evidence of epithelial differentiation of the mononuclear cells, including microvilli and desmosomes, which was not obtained for OGCs. On immunohistochemical study, OGCs stained for CD68 (KP-1), LCA and HAM56, whereas mononuclear cells only reacted with PCNA. These findings clearly suggest that mononuclear cells are capable of differentiation and proliferation and may have been the only true tumor cells in this neoplasm, and that OGCs may have been a paraneoplastic product of this rare tumor. On examination of DNA from dewaxed sections of the tumor, we found no p53 mutation in the tumor tissue, but found two K-ras mutations in codon 12; this pattern of mutation commonly occurs in pancreatic carcinoma, indicating a somewhat genetic relationship of OGTP to pancreatic carcinoma. Although OGTP often has a favorable prognosis, the outcome in the present case was poor due to early tumor spread, with less than two years postoperative survival.

Prophylactic antibiotics given within 24 hours of surgery, compared with antibiotics given for 72 hours perioperatively, increased the rate of methicillin-resistant Staphylococcus aureus isolated from surgical site infections

The purpose of this research was to find which method better prevented MRSA isolation from postoperative infection sites: the administration of postoperative infection control agents within 72 h of surgery, including the day of surgery, or the administration of these agents within 24 h of surgery. More than 3000 patients who underwent elective surgery of the digestive system were studied. Cefazolin or cefotiam was used as the prophylactic antibiotic. The number of patients, sex, age, clinical stage, incidence of surgical site infection (SSI), isolated bacteria, distal pancreatectomy with or without gastrectomy, the rate of laparoscopic surgery, and the rate of abdominoperineal resection (APR) were examined in a prospective controlled study over three time periods. There were no significant differences in the demographics of patients in the three periods. The duration of antibiotic administration was 96.1 ± 11.2 h in period A, 18.2 ± 2.7 h in period B, and 66.9 ± 11.1 hours in period C (P < 0.05). There was no significant difference in the incidence of SSI in the three periods. Methicillin-resistant Staphylococcus aureus (MRSA) was isolated from the infectious site in 0.47% of patients in period A, and from 2.1% and 0.34% of patients in periods B and C, respectively, and the incidence of MRSA was significantly higher in period B as compared with periods A and C (P < 0.01). The isolation rates of MRSA and methicillin-sensitive S. aureus (MSSA) were both significantly higher in period B patients (P < 0.005). We concluded that the administration of prophylactic antibiotics within 24 h of surgery increased the rate of isolation of MRSA.

Vav3 is linked to poor prognosis of pancreatic cancers and promotes the motility and invasiveness of pancreatic cancer cells

BACKGROUND/OBJECTIVES The aim of this study was to investigate the role of the guanine nucleotide exchange factor Vav3 in the motility and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells. METHODS Immunohistochemistry was used to determine whether high Vav3 expression in human PDAC tissues is correlated with poor prognosis. Immunocytochemistry was used to determine the association and intracellular distribution of Vav3, Rac1 and Akt in PDAC cells. Phosphoprotein array analysis was performed to determine the Vav3-associated intracellular signaling pathways. Immunocytochemistry and Matrigel invasion assays were used to examine the effects of Vav3 on the formation of cell protrusions and PDAC cell invasion. RESULTS Expression of Vav3 in PDAC tissue was significantly correlated with overall survival. Vav3 was localized in cell protrusions of migrating PDAC cells. Knockdown of Vav3 inhibited the motility and invasiveness of PDAC cells through a decrease in cell protrusions. The levels of active Rac1 or active Akt were not associated with the concentration of Vav3 in cell protrusions. The Vav3-dependent promotion of motility and invasiveness was not modulated by Rac1 or Akt. Additionally, knockdown of Vav3 increased phosphorylated WNK1 in PDAC cells, and knockdown of WNK1 inhibited the motility and invasiveness. This study suggests that Vav3 can be a useful marker for predicting the outcome of patients with PDAC and that Vav3 can promote PDAC cell motility and invasion through association with dephosphorylation of WNK1. CONCLUSIONS Vav3 was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and thereby increased the motility and invasiveness of PDAC cells.

Immunohistochemical characterisation of a case of insular thyroid carcinoma

Summary A case of insular thyroid carcinoma arising in the right lobe of a 54‐year‐old male is reported. The tumour exhibited an invasive growth pattern with regional lymph node metastasis. Microscopically, the tumour was characterised by well‐defined solid nests like insulae, including mature follicles containing colloid and immature follicles without colloid. Immunohistochemically, tumour cells in follicular areas predominantly exhibited immunopositivity to antithyroglobulin antibody. On the other hand, diffuse immunoreactions with anti‐neuron‐specific enolase (NSE), S‐100 protein and Leu‐7 were detected mainly in the tumour cells of solid areas. In addition, clear cytoplasmic immunoreactivity with anti‐myelin basic protein (MBP) antibody was exhibited in a number of tumour cells. Ultrastructurally, many tumour cells possessed dense vacuoles, apparently containing colloid material, but intracytoplasmic neurosecretory granules were absent. The histopathological and ultrastructural characteristics of the tumour as well as its anti‐thyroglobulin antibody immunoreactivity support the classical hypothesis that this neoplasm is a variant of poorly differentiated thyroid carcinoma. The positive immunohistochemical reactions for NSE, S‐100, MBP and Leu‐7 raise the possibility of aberrant differentiation, for example neural.

CCDC88A, a prognostic factor for human pancreatic cancers, promotes the motility and invasiveness of pancreatic cancer cells.

BackgroundCoiled-Coil Domain Containing 88A (CCDC88A) was identified as a substrate of the serine/threonine kinase Akt that is capable of binding to the actin cytoskeleton. The aim of this study was to investigate the potential role of CCDC88A in the migration and invasiveness of pancreatic ductal adenocarcinoma (PDAC) cells.MethodsImmunohistochemistry was performed to determine whether high CCDC88A expression in human PDAC tissues is correlated with poor prognosis. Immunoprecipitation, immunoblotting and immunocytochemistry were performed to determine the intracellular distribution of CCDC88A, and its association with the serine/threonine kinase Akt and actin-filaments in PDAC cells. Phosphoprotein array analysis was performed to determine CCDC88A-associated intracellular signaling pathways. Finally, immunofluorescence analyses and Matrigel invasion assays were performed to examine the effects of CCDC88A on the formation of cell protrusions and PDAC cell invasion.ResultsExpression of CCDC88A in PDAC tissue was significantly correlated with overall survival. CCDC88A was co-localized with peripheral actin structures in cell protrusions of migrating PDAC cells. Knockdown of CCDC88A inhibited the migration and invasiveness of PDAC cells through a decrease in cell protrusions. Although CCDC88A has been previously reported to be a binding partner and substrate of Akt, the level of active Akt was not associated with the translocation of CCDC88A towards cell protrusions. CCDC88A-dependent promotion of cell migration and invasiveness was not modulated by Akt signaling. Knockdown of CCDC88A decreased phosphorylated Src and ERK1/2 and increased phosphorylated AMPK1 in PDAC cells. Knockdown of AMPK1 inhibited the migration and invasiveness of PDAC cells. The combined data suggest that CCDC88A may be a useful marker for predicting the outcome of patients with PDAC and that CCDC88A can promote PDAC cell migration and invasion through a signaling pathway that involves phosphorylation of Src and ERK1/2 and/or dephosphorylation of AMPK1.ConclusionsCCDC88A was accumulated in cell protrusions, contributed to the formation of membrane protrusions, and increased the migration and invasiveness of PDAC cells.

Composite paraganglioma and ganglioneuroma in the retroperitoneum: a case report

Reports of a composite paraganglioma (PG) and ganglioneuroma (GN) in the retroperitoneum are rare. In the present case, dynamic computed tomographic (CT) findings showed a 30 × 22 × 20 mm tumor that was located in the retroperitoneum and which was dissociated from pancreatic tissue and the left adrenal gland. The markedly reddish tumor showed a clear margin and central multicystic changes on the cut surface. The tumor was composed of two major components, the PG and the GN. The paraganglionic cells in the PG component, which were arranged in a nested pattern, occupied the main and central part of the tumor. Both ganglionic cells and Schwann cells in the GN were located in a unorganized pattern in the periphery. The paraganglionic cells exhibited a Zellballen pattern, which consisted of an association of edematous vascular-rich stroma and focal hemorrhage, resulting in multicystic changes. These centrally located tumor cells were pleomorphic in part and did not have mitotic figures. In the periphery, Schwann cells, which were arranged in an obscure and fascicular pattern that was intermingled with large ganglionic cells, were located adjacent to the PG component with a mostly sharp margin. With higher magnification, the border was not as sharp, as revealed especially with chromogranin-A immunostain, in which both the PG and GN components were focally intermingled with each other. The histogenesis of the composite PG and GN was thought to be extraadrenal neural crest cells in the retroperitoneum because the tumor was not located in the adrenal gland or the Zuckerkandl organ, according to the CT findings. The immunohistochemical findings of this rare case of a composite PG and GN in the retroperitoneum are reported with a focus on the peculiar arrangement of these two components.

Usefulness of KL-6 in the subtyping of intraductal papillary mucinous neoplasia of the pancreas, including carcinoma, dysplasia, and hyperplasia

KL-6 is known as a useful serum biomarker of the disease activity in interstitial pneumonias. We investigated its usefulness as a biomarker for subtyping intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. IPMNs are generally divided into 4 subtypes, namely pancreatobiliary (PB), intestinal (INT), gastric (GS), and oncocytic (ONC). Aside from the KL-6 antibody, the MUC1, MUC2, MUC5AC, MUC6, and MIB-1 antibodies were also examined. Eighteen IPMN cases were examined, including 12 cases of intraductal papillary mucinous carcinomas (IPMCs) simultaneously associated with dysplasia (IPMDs) and hyperplasia (IPMHs) and 6 IPMD cases with IPMH. KL-6 antibody was positive in the 8 IPMC cases, corresponding to a MUC2-negative PB subtype, but negative in 4 IPMC cases, corresponding to the INT subtype, which is positive for MUC2. IPMD of moderate-to-severe degree positively stained for the KL-6 antibody in the IPMC cases of the PB subtype but not in those of the INT subtype. The IPMH cases were mostly negative for KL-6, similar to the mild IPMD cases. In the 6 cases of mild IPMD and/or IPMH, KL-6 and MUC2 expressions were mostly negative. In conclusion, the KL-6 antibody is immunohistochemically a good biomarker of the PB subtype of IPMC, but not the INT subtype. Identifying IPMN subtypes based on KL-6 stainability would be useful. Clinicopathological studies with more IPMC cases might be needed for further progress in this field of study.

Open surgery versus laparoscopic surgery after stent insertion for obstructive colorectal cancer

PurposeTo compare the outcomes of laparoscopic surgery vs. open surgery after insertion of a colonic stent for obstructive colorectal cancer.MethodsBetween April 2005 and August 2013, 58 patients underwent surgery after the insertion of a colonic stent for obstructive colorectal cancer. We analyzed the outcomes of the patients who underwent laparoscopic surgery vs. those who underwent open surgery.ResultsWe compared blood loss, operative time, hospital stay, and complications in 26 patients who underwent laparoscopic surgery and 32 patients who underwent open surgery. Blood loss was significantly less in the laparoscopic surgery group, but operative time was significantly shorter in the open surgery group. The length of hospital stay was shorter in the laparoscopic surgery group than in the open surgery group, but the difference was not significant. There was no significant difference in postoperative surgical complications between the groups.ConclusionThe patients who underwent laparoscopic resection had less blood loss, although no significant difference was found in postoperative morbidity or mortality. Thus, laparoscopic resection after stent insertion is a feasible and safe option for patients with obstructive colorectal cancer.