Inhibition of autophagy with expression of NADPH oxidase subunit p22phox in preneoplastic lesions in a high-fat diet and streptozotocin-related hepatocarcinogenesis rat model.

To study the effects of autophagy inducer carbamazepine (CBZ) in a high-fat diet (HFD)/streptozotocin (STZ)-related early hepatocarcinogenesis model, we determined autophagic flux by immunohistochemical analysis of autophagy marker expression in preneoplastic liver foci and compared that with the expression of the NADPH oxidase subunit. Male F344 rats were fed a basal diet or HFD and subjected to two-stage hepatocarcinogenesis; diabetes mellitus was induced via STZ administration. Several STZ-treated, HFD-fed rats were administered CBZ (a total of five doses every one or two days) at week 7 and 8. STZ-treated, HFD-fed rats decreased ß cells in the islet of Langerhans and increased adipophilin-positive lipid droplets in the liver; moreover, they had a larger area of glutathione S-transferase placental form-immunopositive preneoplastic liver foci, which was associated with inhibition of autophagy and induction of the NADPH oxidase subunit, as demonstrated by increased immunohistochemical expression of an autophagosome receptor marker microtubule-associated protein light chain 3 (LC3)-binding protein p62, and of an NADPH oxidase subunit p22phox in the preneoplastic foci. An increased trend of an autophagy phagophore marker LC3 in preneoplastic foci was also detected. CBZ administration could induce autophagy and impair p22phox expression, as shown by altered expression of autophagy regulators (Atg5, Atg6, Lamp1, Lamp2, and Lc3), NADPH oxidase subunits (P22phox and P67phox), and antioxidant enzymes Gpx1 and Gpx2. These results suggest that inhibition of autophagy and induction of p22phox might contribute to HFD/STZ-related early hepatocarcinogenesis in rats; however, the effects of CBZ administration on the STZ/HFD-increased preneoplastic foci were marginal in this study.

Ectopically Localized Epithelial Cell Clumps in Ulcers Are Derived from Reserved Crypt Stem Cells in a Mouse Model of Ulcerative Colitis.

BACKGROUND: We previously reported that clumps of a few epithelial cells were scattered in ulcer regions in a dextran sulfate sodium (DSS)-induced mouse model of ulcerative colitis (UC). AIMS: To determine the ectopically localized epithelial clumps might be derived from stem cells or their daughter progenitor cells. METHODS: Female BALB/c mice were administered DSS in drinking water for 6 days, followed by withdrawal of DSS for 6 days. Histological and immunohistochemical examinations were conducted in the distal region and proximal region of the colorectum to determine expression of stem cell markers in the epithelial clumps. RESULTS: Similar to the characteristics of UC, the ulcers were more severe in the distal region close to the anus than in the proximal region of the colorectum. Quantitative analyses revealed that the epithelial clumps appeared in relation to the severity of the ulcer, and they expressed the cell adhesion molecules E-cadherin and ß-catenin. Among stem cell markers, the epithelial clumps primarily expressed +5 cell marker Dll1 as reserved intestinal stem cells, followed by +4 cell marker Bmi1 and crypt stem cell marker Lgr5 in that order. Nuclear expression of Sox9, but not nuclear ß-catenin, was identified in the clumps. CONCLUSION: The present results suggest that most epithelial clumps comprised crypt-derived, reserved stem cells, which might have potential for mucosal healing.

Metronidazole enhances steatosis-related early-stage hepatocarcinogenesis in high fat diet-fed rats through DNA double-strand breaks and modulation of autophagy.

Nonalcoholic fatty liver disease is a hepatic disorder with deposition of fat droplets and has a high risk of progression to steatosis-related hepatitis and irreversible hepatic cancer. Metronidazole (MNZ) is an antiprotozoal and antimicrobial agent widely used to treat patients infected with anaerobic bacteria and intestinal parasites; however, MNZ has also been shown to induce liver tumors in rodents. To investigate the effects of MNZ on steatosis-related early-stage hepatocarcinogenesis, male rats treated with N-nitrosodiethylamine following 2/3 hepatectomy at week 3 were received a control basal diet, high fat diet (HFD), or HFD containing 0.5% MNZ. The HFD induced obesity and steatosis in the liver, accompanied by altered expression of Pparg and Fasn, genes related to lipid metabolism. MNZ increased nuclear translocation of lipid metabolism-related transcription factor peroxisome proliferator-activated receptor gamma in hepatocytes, together with altered liver expression of lipid metabolism genes (Srebf1, Srebf2, Pnpla2). Furthermore, MNZ significantly increased the number of preneoplastic liver foci, accompanied by DNA double-strand breaks and late-stage autophagy inhibition, as reflected by increased levels of γ-H2AX, LC3, and p62. Therefore, MNZ could induce steatosis-related hepatocarcinogenesis by inducing DNA double-strand breaks and modulating autophagy in HFD-fed rats.

Leptospiral meningoencephalitis in a raccoon dog.

Neuroleptospirosis is a rare disease caused by pathogenic Leptospira interrogans in humans; however, it has not been fully studied in animals. A young wild raccoon dog was found convulsing in the recumbent position and died the next day. Histologic examination revealed nonsuppurative meningoencephalitis in the cerebrum, cerebellum, midbrain, and medulla oblongata. The lesions consisted of mixed infiltrates of Iba1-positive macrophages and CD3-positive T cells, with a small number of CD79α-positive B cells and myeloperoxidase-positive neutrophils. In the frontal cortex, perivascular cuffs and adjacent microglial nodules were distributed diffusely, especially in the molecular layer. Glial nodules were comprised of Iba1- and myeloperoxidase-positive activated microglia. Immunohistochemistry revealed leptospires in mononuclear cell perivascular cuffs, but not in glial nodules. Neuroleptospirosis was accompanied by Leptospira-related nonsuppurative interstitial nephritis, pulmonary edema and hemorrhage, and coronary periarteritis, as well as Toxocara tanuki in the small intestine and nonspecific foreign-body granulomas in the lungs and stomach.

Squamous cell carcinoma in a digit of the hind limb with systemic metastasis in a 17-year-old female koala.

We encountered a case of cutaneous squamous cell carcinoma (SCC) in a 17-year-old female koala at a zoo. A fragile, papillary, elevated mass was found on the third digit of the right hind limb. SCC was identified histopathologically: squamous cell-like polygonal tumor cells showed a nest-like growth pattern with epidermal down growth, central keratinization and necrotic foci, and invaded dermal connective tissues. Metastatic lesions were observed in various organs, including the lung and axillary lymph node: in the lung, multiple metastatic foci similar to the primary lesion, and in the axillary lymph node, individual polygonal tumor cells infiltrated the sinusoids. Immunohistochemistry revealed that the tumor cells were positive for proliferating cell nuclear antigen, which exhibited 32-33% of labeling indices in the tumor cells. To our knowledge, this is the first report of a case of SCC in a digit of a koala.

Establishment of 2.5D organoid culture model using 3D bladder cancer organoid culture.

Three-dimensional (3D) organoid culture holds great promises in cancer precision medicine. However, Matrigel and stem cell-stimulating supplements are necessary for culturing 3D organoid cells. It costs a lot of money and consumes more time and effort compared with 2D cultured cells. Therefore, the establishment of cheaper and Matrigel-free organoid culture that can maintain the characteristics of a part of 3D organoids is demanded. In the previous study, we established a dog bladder cancer (BC) 3D organoid culture system by using their urine samples. Here, we successfully isolated cells named "2.5D organoid" from multiple strains of dog BC 3D organoids using 2.5 organoid media. The cell proliferation speed of 2.5D organoids was faster than parental 3D organoid cells. The expression pattern of stem cell markers was close to 3D organoids. Injection of 2.5D organoid cells into immunodeficient mice formed tumors and showed the histopathological characteristics of urothelial carcinoma similar to the injection of dog BC 3D organoids. The 2.5D organoids had a similar sensitivity profile for anti-cancer drug treatment to their parental 3D organoids. These data suggest that our established 2.5D organoid culture method might become a reasonable and useful tool instead of 3D organoids in dog BC research and therapy.

Extraskeletal chondrosarcoma in the abdominal cavity of a cow.

A 25-month-old female crossbred cow presented with astasia, emaciation, and stunted growth. Macroscopic examination revealed a large mass in the abdominal cavity, approximately 100 × 30 × 30 cm. Microscopic examination revealed that the mass consisted of multilobular mature and immature cartilaginous matrices with chondrocytic cells, surrounded by spindle to pleomorphic mesenchymal tumor cells. The cartilaginous matrices consisted of hyaline and elastic cartilages, as confirmed with Azan stain, and Victoria Blue and Van Gieson stain. Immunohistochemistry revealed that the chondrocytic and mesenchymal cells both expressed S-100. The tumor was diagnosed as an extraskeletal chondrosarcoma in the abdominal cavity of this cow.

Clinical and pathological characteristics of acute myelogenous leukemia in a female koala with diabetes mellitus.

A female koala presented with hyperglycemia related to diabetes mellitus diagnosed at 9 years and treated with insulin. She presented with nasal hemorrhage, anemia, leukocytosis, and tachypnea at 10 years. A blood smear examination revealed scattered, atypical large myeloid cells and a clinical diagnosis of myelogenous leukemia was made. White blood cell count reached a maximum of 295 × 102/µl, with evidence of severe regenerative anemia and thrombocytopenia. Grossly, systemic lymph node enlargement, fragile liver with hemorrhage, and bloody ascites were observed. Histopathologically, atypical myeloid cells, including myelocytic and metamyelocytic cells, were scattered in the vasculature and surrounding tissues throughout the organs. The patient was infected with a koala retrovirus, which might have caused the myelogenous leukemia.

Improvement of high-sensitivity inflammation-based Glasgow prognostic score by gastrectomy is a favorable prognostic factor in patients with gastric cancer.

AIM: The aim of the present study was to clarify the efficacy of inflammation-based Glasgow prognostic score after surgery in patients with gastric cancer and to determine clinicopathological factors affecting score improvement. PATIENTS AND METHODS: Participants in this retrospective study were 236 patients with gastric cancer who underwent gastrectomy at the Fukuoka University Hospital. The high-sensitivity inflammation-based Glasgow prognostic score (HS-GPS) (cut-off values: 0.3 mg/dl for C-reactive protein; 3.5 g/dl for albumin) were calculated before and 1 month after surgery, and correlated to clinicopathological parameters and prognosis after surgery. RESULTS: HS-GPS was classified as normal (score 0) in 162 patients and abnormal (score 1 or 2) in 74 patients. Out of the 162 patients with normal HS-GPS before surgery, 62 showed abnormal HS-GPS after surgery, while 26 of the 74 patients with abnormal HS-GPS before surgery improved to normal HS-GPS postoperatively. Abnormal HS-GPS before (p<0.0001) and after (p=0.0002) surgery were unfavorable prognostic factors in univariate analysis. HS-GPS after surgery was an independent prognostic factor (p=0.0324) in multivariate analysis, but HS-GPS before surgery was not. In the sub-group with abnormal HS-GPS before surgery (but not normal HS-GPS before surgery), improved HS-GPS after surgery had a favorable prognostic impact in both uni- (p=0.0039) and multivariate analyses (p=0.0032). CONCLUSION: HS-GPS after surgery may be a valuable prognostic factor in patients with gastric cancer. Supplemental therapy represented by adjuvant chemotherapy might be required for gastric cancer patients showing no improvement in HS-GPS after gastrectomy.

Gastric cancer arising from the remnant stomach after distal gastrectomy: a review.

Gastric stump carcinoma was initially reported by Balfore in 1922, and many reports of this disease have since been published. We herein review previous reports of gastric stump carcinoma with respect to epidemiology, carcinogenesis, Helicobacter pylori (H. pylori) infection, Epstein-Barr virus infection, clinicopathologic characteristics and endoscopic treatment. In particular, it is noteworthy that no prognostic differences are observed between gastric stump carcinoma and primary upper third gastric cancer. In addition, endoscopic submucosal dissection has recently been used to treat gastric stump carcinoma in the early stage. In contrast, many issues concerning gastric stump carcinoma remain to be clarified, including molecular biological characteristics and the carcinogenesis of H. pylori infection. We herein review the previous pertinent literature and summarize the characteristics of gastric stump carcinoma reported to date.

The high-sensitivity modified Glasgow prognostic score is superior to the modified Glasgow prognostic score as a prognostic predictor in patients with resectable gastric cancer.

AIM: The aim of the present study was to evaluate the superiority of the high-sensitivity modified Glasgow prognostic score (HS-mGPS) before surgery in patients with gastric cancer. PATIENTS AND METHODS: The participants of this retrospective study comprised 552 patients with gastric cancer who underwent gastrectomy at the Fukuoka University Hospital. The HS-mGPS was calculated before surgery based on cutoff values of 0.3 mg/dl for C-reactive protein and 3.5 g/dl for albumin, and correlations between the HS-mGPS and the clinicopathological parameters and prognosis were evaluated. In addition, the superiority of the HS-mGPS to the mGPS as a prognostic indicator was examined in detail. RESULTS: The mGPS was 0 in 494 patients, 1 in 24 patients and 2 in 34 patients. In contrast, the HS-mGPS was 0 in 411 patients, 1 in 75 patients and 2 in 66 patients. Both the mGPS (p < 0.0001) and HS-mGPS (p < 0.0001) were good prognostic predictors in gastric cancer patients who underwent gastrectomy. Of the 494 patients with an mGPS of 0 before surgery, 51 and 32 exhibited an HS-mGPS of 1 and 2, respectively. The patients who exhibited migration in the HS-mGPS demonstrated a significantly more unfavorable prognosis than the patients with an HS-mGPS of 0 (p < 0.0001). The prognostic impact of the HS-mGPS was especially clear in stage I and IV patients (p = 0.0027, p = 0.017). The HS-mGPS was found to be a superior prognostic predictor compared to the mGPS in a multivariate analysis (p = 0.0002). CONCLUSIONS: The HS-mGPS before surgery is a superior prognostic predictor in patients with gastric cancer.

Successful Treatment of Septic Shock due to Spontaneous Esophageal Perforation 96 Hours after Onset by Drainage and Enteral Nutrition.

Spontaneous esophageal perforation is relatively uncommon, but carries a high mortality rate if diagnosis or treatment is delayed. We report the case of a 68-year-old man with spontaneous esophageal perforation who was successfully treated over 96 h after onset by thoracic drainage and jejunostomy for enteral nutrition. He vomited after drinking alcohol, soon followed by epigastralgia. Heart failure was suspected on admission to another hospital. Spontaneous esophageal perforation was diagnosed 48 h after admission. Chest tube drainage was performed, but his general condition deteriorated and he was transferred to our hospital. Emergent surgery was performed and esophageal perforation combined with pyothorax and mediastinitis was identified on the left side of the lower esophagus. The left thoracic cavity was rinsed and thoracic drainage was performed. Feeding jejunostomy was performed for postoperative enteral nutrition. Effective drainage and sufficient nutrition management appear extremely valuable in treating spontaneous esophageal perforation.