Kyuya Ishibiki

Single‐cell Suspension Assay with an MTT End Point Is Useful for Evaluating the Optimal Adjuvant Chemotherapy for Advanced Gastric Cancer

One hundred and forty‐eight patients with gastric cancer admitted to Keio University Hospital between July 1988 and October 1992 underwent resection of the primary lesion, as well as single‐cell suspension assay of fresh surgical materials with 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT assay) for chemosensitivity evaluation. Fifty patients with histologically stage III or IV gastric cancer were enrolled in this study, among whom 10 received no chemotherapy after surgery while 40 received chemotherapy at equivalent dose levels after surgery. The patients given chemotherapy were divided into two groups consisting of an “Adapted” group treated with at least one agent identified as effective by the assay, and a “Non‐adapted” group treated with agents to which the cells were not sensitive in the assay, in order to identify the optimal cut‐off inhibition rate (IR) in MTT assay for evaluation of the appropriate adjuvant cancer chemotherapy after surgery. A cut‐off IR of 30% was optimal for differentiating the survival rates between the “Adapted” and “Non‐adapted” groups. Patients treated with drugs which showed more than 30% IR on their surgical specimens showed a better survival rate than patients treated with drugs which were ineffective in the assay.

Human breast carcinoma (MCF-7) serially transplanted into nude mice

The tumor cells (0.5 ml, 1×107) of MCF-7 line were inoculated into the subcutaneous tissue or intraperitoneum of female BALB/c nude mice. Primarily tansplanted mice were treated with 17β-estradiol dipropionate (E2) in a dose of 5 mg/kg and 17α-hydroxy progesterone caproate (Pg) in a dose of 250 mg/kg once a week. After the transferable strain was established, tumors were transplanted into female and male mice treated with E2, Pg, and E2+Pg. The tumors treated with E2 or E2+Pg grew exponentially while tumors in the other group regressed. Pg was assumed to play some role in the growth of MCF-7, in the presence of estrogen. Although cytosol estrogen receptors (ERc), nuclear estrogen receptors (ERn), and progesterone receptors (PgR) were detected by dextran coatedcharcoal method and exchange assay in the growing tumors, ERn and PgR of regressing tumors was usually negative. This MCF-7 strain in nude mice may be a promising animal model for studying chemo-hormone therapy for human breast carcinomas.

Rapid diagnosis of methicillin-resistant Staphylococcus aureus bacteremia by nested polymerase chain reaction

OBJECTIVE The purpose of this study was to establish a rapid and sensitive diagnostic method for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia in postoperative patients. SUMMARY BACKGROUND DATA As a result of diffusion and abuse of third-generation cephalosporin antibiotics in the 1980s in Japan, an outbreak of MRSA infection has been posed. In the field of surgery, severe postoperative infections with MRSA such as MRSA bacteremia, which may lead to multiple organ failure, have emerged with a high mortality. METHODS Thirty-five patients with high fever (above 38.5 C) or watery diarrhea or both within 2 weeks after gastrointestinal major surgery and 6 healthy volunteers were examined. Nested polymerase chain reaction was used to detect mecA and toxic shock syndrome toxin-1 (TSST-1) genes in blood specimens. RESULTS The mecA and TSST-1 genes were not detected in the blood samples of any of the six healthy volunteers. In all 12 samples from which MRSA colonies were isolated by blood culture, mecA and TSST-1 genes were detected. Although it took at least 48 hours to identify MRSA by the blood culture method, the presence of mecA and TSST-1 genes was determined by nested polymerase chain reaction method within only 3 to 4 hours after blood sampling. CONCLUSIONS This method, as a sensitive and rapid monitoring system for MRS bacteremia, would be clinically beneficial for prevention of cross infection and for early determination of appropriate treatment for infected patients.

Growth of human tumor xenografts in nude mice and mice with severe combined immunodeficiency (SCID)

Twenty-three fresh tumor specimens obtained at surgery and 5 serially transferable human tumor xenografts were implanted subcutaneously into nude mice and mice with severe combined immunodeficiency (SCID) to compare the take rates of the fresh surgical specimens and the growth rates of the transferable strains. The overall take rates were 65% for the SCID mice and 60% for the nude mice, without any significant difference, although colon carcinoma seemed to have higher acceptance in the SCID mice with a take rate of 6/8. All the serially transferable strains were successfully accepted in the SCID mice, their growth rates being essentially identical to those in the nude mice. These results indicate that the SCID mouse can be used as a human tumor xenograft-mouse system as well as the nude mouse.

Individualized fluid resuscitation based on haemodynamic monitoring in the management of extensive burns.

An evaluation has been made on the effectiveness and feasibility of the individualized fluid replacement programme based on intensive haemodynamic monitoring using a Swan-Ganz catheter. Twenty-one extensively burned patients with an average burn of 60.8 per cent BSA were resuscitated with lactated Ringers and colloid solutions. The rate of fluid administration was adjusted to maintain the optimal ranges of the various haemodynamic parameters including cardiac index and left ventricular stroke work index. Two patients failed to respond to fluid resuscitation possibly due to inadequate emergency procedures given before arrival at our institute. The remaining patients survived the shock phase, with the amount of fluid given to the 18 adult patients being (3.38 /+- 1.02 ml/kg) x (percentage burn) for the first 24 hours. A negative correlation existed between the amounts of lactated Ringers solution and colloid solution used for the fluid resuscitation. Dopamine was effective in 4 out of 5 patients who showed depressed myocardial function. The individualized fluid programme was shown to be effective and reliable for the management of critically burned patients.

Cell kinetic study of human carcinomas using bromodeoxyuridine

Abstract. Cell kinetics in human malignant tumours were studied in vivo using bromodeoxyuridine (BrdU) and immunohistochemistry. BrdU was administered to twenty‐four patients with gastric cancer at a dose of 1 g pre‐operatively. Specimens were obtained during the operation, fixed in 70% ethanol and embedded in paraffin. BrdU‐incorporating cells were detected by immunohistochemical staining using anti‐BrdU monoclonal antibody. the labelling index (LI), determined by counting tumour cells microscopically, ranged from 4.0 to 41.4%. the LI was higher at the site of invasion than in the central area of the tumour, but no correlation was found between histological differentiation and LI. the LI of stage I gastric cancer was statistically lower than that of stage II, III and IV gastric cancers (P < 0.005). This technique, which is less cumbersome and time‐consuming than using radioactive isotopes of thymidine, appears to be useful for studying cell kinetics of human malignant tumours.

Collagen biosynthesis in gastric cancer: immunohistochemical analysis of prolyl 4-hydroxylase.

The mechanism of the desmoplastic response in gastric carcinoma tissues is largely unknown. The objective of this study is to determine the localization of prolyl 4‐hydroxylase (PH), an enzyme that plays a crucial role in collagen biosynthesis.

A metastatic model of human colon cancer constructed using cecal implantation of cancer tissue in nude mice

COL-2-JCK, a human colon cancer xenograft line able to be transplanted into nude mice, was implanted in the subserosal layer of the cecum, either as cancer tissue or as a single cell suspension. When cancer tissue was used for the cecal implantation, 100% extensive local tumor growth and a high incidence of metastases to the regional lymph nodes, peritoneum, liver, and lung was observed. In contrast, when the cell suspension of this line was injected into the cecal wall, no metastases were observed, with significantly reduced local tumor growth. The use of cancer tissue maintaining the original cancer tissue structure is therefore considered imperative for allowing full expression of the biological characteristics of cancer cells. This nude mouse model using the cecal implantation of cancer tissue should thus prompt further study on the biology of human colon cancer.

The antitumor effects of adriamycin entrapped in liposomes on lymph node metastases

Adriamycin (ADM) entrapped in liposomes (Lip-ADM) was prepared and its therapeutic effects studied using the mouse leukemia cell line, P388, which metastasized to axillary lymph nodes after inoculation into the foot pads of CDF1 mice. Lip-ADM injections (7.5 mg/kg) were given into the foot pad at two-day intervals. Two series of experiments were performed; one in which Lip-ADM was administered on days 1, 3 and 5 following tumor inoculation, and the other in which it was administered on days 5 and 7. Both Lip-ADM injection regimens significantly inhibited metastases to the lymph nodes as compared with mice given injections of saline solution. Furthermore, the therapeutic effects of three Lip-ADM injections were significantly greater than the effects of free ADM. Histological examinations of lymph nodes revealed that three injections of Lip-ADM completely eliminated tumor cells, whereas viable tumor cells were still observed in the lymph nodes after treatment with free ADM. The results of this study suggest that Lip-ADM is useful for the treatment of lymph nodes metastases and that the local injection of Lip-ADM, through such means as endoscopy, would be recommended as a clinical mode of application.

Experimental and clinical studies on the intraperitoneal administration of cis-diamminedichloroplatinum (II) for peritoneal carcinomatosis caused by gastric cancers.

The effectiveness of the intraperitoneal administration of cis-diamminedichloroplatinum (II) (DDP) on peritoneal carcinomatosis caused by gastric cancers was evaluated. Seventeen patients were treated with one of three protocols, consisting of the intraperitoneal injection (ip) of DDP at doses of 70 and 110 mg/m2, with or without sodium thiosulfate (STS) rescue. The area under the curve (AUC) of DDP for sufficient anticancer activities against cultured human cell lines in vitro was estimated at 240 μg h/ml, which was equivalent to the AUC gained by 110 mg/m2 ip DDP in the clinical studies. The cytotoxic activity of DDP was reduced by approximately 50% with 100-fold STS in the AUC in the experimental studies. However, this was achieved only in urine, and not in either the peritoneal cavity or in plasma in the clinical studies. Three cases of a partial response against peritoneal carcinomatosis were seen from a total of four evaluable cases treated with 110 mg/m2 DDP, and no renal toxicities were observed in those treated with the STS rescue. The results of this study led us to conclude that high-dose ip DDP treatment combined with the STS rescue would be useful chemotherapy against peritoneal carcinomatosis caused by gastric cancers.