Evaluation of follicular development and oocyte quality in pre-pubertal cats.

Our study was conducted to assess the follicular development and availability of sound ovarian oocytes for in vitro production (IVP) of embryos in pre-pubertal cats. The relationship between body and ovarian weight was examined in 93 cats. The results revealed that ovarian weight rapidly increased until 100 days of estimated age. By histological evaluation of ovaries obtained from 11 pre-pubertal cats with estimated age of <20, 20-40 and 100-120 days, it was clarified that the increase in ovarian weight during kitten growth accompanied the increase in the number and size of antral follicles. The follicular diameter and percentage of normal oocytes in secondary/antral follicles also increased as estimated age (body weight) increased. The oocytes obtained from pre-pubertal cats with 100-120 days of estimated age were used for IVP of embryos. The results showed that the success rates of in vitro maturation, in vitro fertilization and development to blastocysts after in vitro culture in pre-pubertal cats were lower than in sexually mature cats. However, the percentage of blastocysts based on the cleaved embryos and cell number of blastocysts in pre-pubertal cats were comparable to those in mature cats. In conclusion, these results suggest that the ovaries of pre-pubertal cats with ≥100 days of age contain oocytes with in vitro developmental competence to blastocysts.

Effect of duration of in vitro maturation on nuclear maturation and fertilizability of feline oocytes.

This study was conducted to improve in vitro production of embryos from domestic cats using TCM-199 as an IVM medium. The time sequence of nuclear maturation and the optimal timing of in vitro insemination were examined. Most oocytes were at the germinal vesicle stage immediately after collection; however, 8.3% had already resumed meiosis before IVM culture. After 30 h of IVM culture, the percentage of oocytes at metaphase II (MII) reached a peak (75.5%) and did not change (P>0.05) from 30 to 48 h after IVM culture. The percentage of oocytes with two pronuclei was higher (P<0.05) for oocytes matured for 30 and 36 h (38.2 and 33.0%, respectively) than for those after IVM culture for only 24 h (18.5%). Total sperm penetration rate was highest (P<0.05) for oocytes that had been matured for 30 h (46.1%). After 30 h of IVM and 18 h of IVF culture, 66.3 and 24.8% of inseminated oocytes had cleaved and developed to the blastocyst stage, respectively. We concluded that IVM of feline oocytes for 30 h in TCM-199 resulted in optimal nuclear maturation and sperm penetration.

Spindle cell carcinoma of the pancreas.

We experienced a rare case of spindle cell carcinoma of the pancreas. The patient was a 74-year-old man who complained of abdominal pain and loss of weight. Ultrasonographic scans revealed a hypoechoic solid mass in the head of the pancreas, 4 cm in diameter, with a high echoic spot suggestive of central necrosis or hemorrhage. The mass was hypodense on enhanced computed tomographic scans and hypovascular on angiograms. At laparotomy, the tumor had invaded to the mesocolon, but dissemination and distant metastasis were not found. We therefore performed pylorus-preserving pancreatoduodenectomy and ascending colectomy. The patient was discharged on postoperative day 26 after an uneventful recovery. Two months later, he was readmitted because of ascites, with positivity of spindle cells shown on cytology; he died on day 92 after surgery. In the resected specimen, the tumor had a mostly sarcomatous component, consisting of spindle-shaped cells, and a small glandular component. Immunohistochemically, both components were positive for cytokeratin and epithelial membrane antigen, but negative for vimentin and desmin. The tumor was diagnosed as a spindle cell carcinoma of the pancreas. Perineural invasion, lymphatic permeation, and blood vessel invasion were found, but lymph node metastasis was not found. Although a curative operation was performed, the outcome in this patient was very poor.

Free 4-hydroxyproline content in serum of bedridden aged people is elevated due to fracture.

Hydroxyproline (Hyp) content in biological fluids is used as a parameter of collagen catabolism, especially bone resorption. In this paper, we examined the relationship between aging and serum free 4-Hyp and proline (Pro) content using a newly introduced analytical technique which is chemiluminescence determination with electrogenerated tris (2,2'-bipyridine) ruthenium(III). Serum was collected from 50 bedridden aged people and 131 normal subjects living in Yamato-son, Amamioshima, Kagoshima. In the free 4-Hyp content of normal serum, the change in females is more gentle than that males with aging. The free 4-Hyp content of bedridden aged serum is significantly (p<0.05) elevated compared to normal aged serum. Bedridden aged females and males also show levels significantly (p<0.05) elevated in comparison to normal. These changes are due to bone resorption based on bedridden patients' atrophy. In bedridden aged people, free 4-Hyp of those with a fracture was significantly (p<0.05) elevated than in people without a fracture. No relationship between low bone density and high free 4-Hyp in serum was observed. It seems that bone resorption or bedridden atrophy may be enhanced in bedridden aged people by a fracture. In free Pro content in serum, no significant change was observed. These results show that the free 4-Hyp content in serum is useful in the clinical or biological inspection of bone resorption, especially in aged people.

Mucin core protein expression in extrahepatic bile duct carcinoma is associated with metastases to the liver and poor prognosis.

BACKGROUND: Curative resection does not guarantee long term survival for the patient with extrahepatic bile duct carcinoma because of the possibility of metastases to the liver (LM). Expression of mucin core protein-1 (MUC1), sialyl-Le(x), and sialosyl-Tn in bile duct carcinoma was determined and compared with LM and prognosis. METHODS: Immunohistochemical expression of MUC1, sialyl-Le(x), and sialosyl-Tn in 73 extrahepatic bile duct tumors was analyzed using the DF3, FH6, and TKH2 monoclonal antibodies, respectively. Scoring was based on the percentage of immunoreactive cells: negative, low expression (25%). RESULTS: High expression of MUC1, sialyl-Le(x), and sialosyl-Tn was observed in 68.5%, 34.2%, and 54.8%, respectively. of 73 cases. Patients with tumors showing high expression of MUC1 had a higher rate of LM (48.9%) and a significantly shorter survival period (median survival time, 17.8 months) compared with patients with tumors showing low (incidence of LM, 9.1%; median survival time, >100 months) or negative (incidence of LM, 11.1%; median survival time, 52.9 months) expression of MUC1 (P < 0.01). However, the survival period of patients with tumors showing high, low, or negative expression of sialyl-Le(x) or of sialosyl-Tn did not differ significantly. High MUC1 expression correlated with LM by logistic regression analysis and emerged as an independent prognostic factor in stepwise multivariate analysis. CONCLUSIONS: The results of the current study demonstrate that high expression of MUC1 correlates with LM and poor outcome in patients with extrahepatic bile duct carcinoma.

Molecular detection of circulating cancer cells during surgery in patients with biliary-pancreatic cancer.

BACKGROUND: It remains unclear whether surgical treatment for biliary-pancreatic cancers provokes the hematogenous dissemination of cancer cells. The aim of this study was to detect circulating cancer cells in the blood stream before and during tumor resection for biliary-pancreatic cancer. METHODS: We analyzed blood samples obtained perioperatively from the portal vein, peripheral artery, and superior vena cava, using a carcinoembryonic antigen (CEA)-specific nested reverse transcriptase-polymerase chain reaction. RESULTS: CEA-mRNA expression was detected in the blood of 21 (52.5%) of 40 patients with biliary-pancreatic cancer. The patients with detectable CEA-mRNA expression included 8 (42.1%) of 19 with bile duct cancers and 13 (61.9%) of 21 with pancreatic cancers. CEA-mRNA expression was not detected in blood obtained from 15 healthy volunteers and 15 patients with benign disease. The positive rate of CEA-mRNA of advanced clinical stage (TNM pStage III and IV) showed higher than that of early stage (pStage I and II; P <0.05). Tumor resection increased significantly the positive rates of CEA-mRNA in the blood stream of three kinds of vessel. CONCLUSIONS: Surgical procedures provoke the hematogenous dissemination of cancer cells perioperatively. Therefore, new strategies during operations to prevent liver metastases are needed to improve the survival of patients with biliary-pancreatic cancer.

Liver metastases after curative resection in patients with distal bile duct cancer.

BACKGROUND: Curative resection cannot ensure long-term survival for patients with distal bile duct cancer. The aim of this study was to determine the most frequent mode of recurrence after curative resection and to analyse its risk factors. METHODS: Clinical details for 64 consecutive patients with distal bile duct cancer who underwent surgical resection between 1980 and 1997 were reviewed. Modes of recurrence and clinicopathological findings were analysed based on the residual tumour (R) classification. RESULTS: The overall actuarial 5-year survival rate was 32 per cent. Of 42 patients undergoing R0 resection 10 were alive at 5 years, of 17 having R1 resection one was alive at 5 years, and none of the five patients having R2 resection survived for 5 years (R0 versus R1, P = 0.02). In the R0 group, the incidence of liver recurrence (14 of 42 patients) was similar to that in the R1 resection group (six of 17), although there were fewer lymph node and peritoneal recurrences than in patients who had R1 resection (P < 0.05). The high-risk factor for liver metastasis was microscopic vascular involvement. Important factors for survival were lymph node metastasis, microscopic vascular involvement and age. In addition, microscopic vascular involvement was the only independent factor for survival in the R0 group. CONCLUSION: R0 resection provided significant survival benefit but had no effect on liver recurrence. Therefore, new agents or strategies to prevent liver metastasis are necessary for improvement of survival.

Comparison of relapse and long-term survival between pylorus-preserving and Whipple pancreaticoduodenectomy in periampullary cancer.

BACKGROUND: Whether long-term survival and type of recurrence in periampullary cancer are influenced by the type of surgical procedure or not is still obscure. The purpose of this review was to analyze the results at a single institution. METHODS: Between 1982 and 1996, 113 consecutive patients with either distal bile duct or ampullary cancers underwent either pylorus-preserving pancreaticoduodenectomy (PPPD) or the Whipple procedure. The median follow-up was 30 months (range 12 to 150). Patient records were reviewed to determine type of recurrence and survival. RESULTS: In distal bile duct or ampullary cancers, comparison of actuarial survival curves between PPPD and the Whipple operation revealed no significant differences. Even in node-positive patients, the 5-year survival rates did not differ significantly between those undergoing PPPD or the Whipple operation. Tumor recurrence was not correlated with the operative method, although among types of tumor recurrence, the rate of blood-borne metastasis was higher in distal bile duct or ampullary cancers. CONCLUSIONS: PPPD is an acceptable alternative to the Whipple procedure in the treatment of periampullary cancer. Long-term survival and type of recurrence were not influenced by these types of surgical procedures.

Determination of catecholamines by radioenzymatic assay using ion-pair liquid chromatography.

A simple radioenzymatic method for the simultaneous determination of norepinephrine, epinephrine and dopamine in human serum was developed. catecholamines were converted to normetanephrine (NMN), metanephrine (MN), 3-methoxytyramine (3-MT) and their 4-O-methyl isomers by catechol O-methyl transferase with S-adenosylmethionine-(3H-methyl) ([3H]SAM). After addition of unlabelled NMN, MN and 3-MT as carriers, the methyl derivatives were isolated from the reaction mixture by passing through a small boric acid gel column and separated from each other by ion-pair liquid chromatography. Interference by tailing of radioactivity of [3H] SAM and its degradation products eluted earlier was minimized by the use of a precolumn and a branched flow path.