Comparison of listeriolysin O and phospholipases PlcA and PlcB activities, and initial intracellular growth capability among food and clinical strains of Listeria monocytogenes.

AIMS: We investigated whether Listeria monocytogenes strains differ in their ability to escape from the primary phagosome after internalization into human intestinal epithelial cells. METHODS AND RESULTS: Food and clinical strains were used to study specific alleles; the activities of listeriolysin O (LLO) and phospholipases PlcA and PlcB, which promote rupture of the phagocytic vacuole; and initial intracellular bacterial growth in Caco-2 cells. Results showed no difference in LLO activities between food and clinical strains or among serotypes. In contrast, the LLO truncation mutant lacked detectable haemolytic activity and intracellular growth. PlcA and PlcB produced by the strains of serotypes 4b/4e and 1/2b exhibited significantly lower activities than those of serotypes 1/2a and 1/2c. In contrast, the strains of serotype 1/2b grew significantly faster than those of serotypes 4b/4e and 1/2a. Moreover, the PrfA truncation mutants lacked LLO and phospholipases activities and did not show intracellular growth. CONCLUSIONS: We determined that LLO and PrfA mutants exert a significant effect on intracellular growth, although it was unclear from this study whether PlcA and PlcB alleles affect escape from vacuoles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study estimates that low-virulence L. monocytogenes strains associated with escape ability from the primary vacuoles are not widely distributed among food strains.

Comparison of four enrichment broths for the detection of non-O157 Shiga-toxin-producing Escherichia coli O91, O103, O111, O119, O121, O145 and O165 from pure culture and food samples.

AIMS: We compared the efficiency of universal pre-enrichment broth (UPB), modified Escherichia coli broth containing novobiocin (mEC + n), modified Tryptic Soy Broth (mTSB) and mTSB with novobiocin (mTSB + n) for the enrichment of non-O157 Shiga-toxin-producing E. coli (STEC). METHODS AND RESULTS: Freeze-injured and control non-O157 STEC (O91, O103, O111, O119, O121, O145 and O165) strains were used to artificially contaminate beef and radish sprout samples, which were then cultivated in each of the four enrichment media. After incubation, STEC strains were detected by loop-mediated isothermal amplification (LAMP) and plating assays. Enrichment in mEC + n was least effective for facilitating the detection of uninjured STEC strains in radish sprouts, while mTSB + n was least effective for enriching freeze-injured non-O157 STEC strains from beef samples for detection by LAMP assay. UPB and mTSB were superior to mEC + n and mTSB + n for the enrichment of non-O157 STEC from food samples. CONCLUSIONS: The enrichment of non-O157 STEC was negatively affected by the addition of novobiocin to enrichment broths. SIGNIFICANCE AND IMPACT OF THE STUDY: Novobiocin should not be added to media used for the enrichment of non-O157 STEC in food when cell injury is anticipated.

Association of p53 gene mutation and telomerase activity in resectable non-small cell lung cancer.

PURPOSE: Mutation of the p53 gene and deregulation of telomerase may be essential for canceration in some malignant diseases. However, relationships between these occurrences have not yet been clarified. We examined the roles of p53 gene mutation and telomerase activity relative to the clinical and pathologic features of non-small cell lung carcinoma (NSCLC). METHODS: Frozen sections of 40 surgically resected NSCLC specimens were used. DNA extracted from fresh tumor specimens was analyzed with polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP) method, to screen alterations in the p53 gene. Exons showing aberrant band shifts on SSCP were reamplified, and the PCR products were directly sequenced. In addition, the telomerase activity of the same specimens was analyzed quantitatively with the fluorescence-based telomeric repeat amplification protocol assay, and the total product generated (TPG) method. Clinical and pathologic parameters were evaluated using a statistical analysis system. RESULTS: Mutations of the p53 gene relevant to an altered protein were confirmed in 19 of 40 specimens (47.5%). The TPG of 40 specimens was 75.24 +/- 15.55 (mean +/- SE). The TPG of the 19 specimens positive for p53 gene mutation was significantly higher than that of the 21 specimens negative for p53 gene mutation. Furthermore, the degree of cell differentiation was significantly correlated with both p53 gene mutation and high telomerase activity. CONCLUSIONS: p53 gene mutation and high telomerase activity cooperate to induce tumorigenesis and low-grade differentiation in NSCLC. Simultaneous occurrence of p53 gene mutation and high telomerase activity may be relevant to the grade of malignancy in NSCLC.

Importance of the control of lung recurrence soon after surgery of pulmonary metastases.

PURPOSE: In this study, we investigated factors that determined prognosis in patients who underwent surgery for metastatic lung tumors, focusing on early relapse of metastatic lung lesions after surgery, and considered countermeasures for improving long-term results based on this study. PATIENTS: This study was performed in patients with metastatic lung tumors who underwent surgery during the 22 years after November 1975 in this department. RESULTS: The 1-year, 3-year, and 5-year survival rates in all patients were 70%, 42%, and 37%, respectively. On comparison among the groups, there were no significant differences by gender, age, organ with the primary lesion, disease-free interval, number of metastases, or surgical procedure. However, prognosis was significantly poorer in patients with recurrent metastatic lung lesions. Prognosis was especially poor in patients with recurrence within 6 months after pneumonectomy, and this was an important factor that worsened the surgical results. CONCLUSIONS: As the mechanism of early recurrence of lung metastasis after surgery for metastatic lung tumor, multiple micrometastases (dormancy) that cannot be detected during surgery for metastatic lung tumor may be present in the lung. Establishment of a method of controlling an increase in dormant metastasis may lead to improvement of surgical results of metastatic lung tumors.

An availability of video-assisted thoracic surgery for the resection of pulmonary metastases.

We report the use of video-assisted thoracic surgery (VATS) as a treatment for pulmonary metastases. Eight patients with metastatic lung cancer were treated with VATS techniques. These patients included 5 males and 3 females whose ages ranged from 34 to 61 years (average: 47.4). Their primary diseases were seminoma (n = 3), renal cell carcinoma (n = 2), colon carcinoma (n = 1), mammary carcinoma (n = 1) and choriocarcinoma (n = 1). Computed tomography (CT) scans with current generation scanners were performed preoperatively and revealed one metastatic lesion in each of five cases and two lesions in each of the other three cases. In one case, one lesion was located in each of the lungs. Tumor size ranged from 5 to 30 mm in diameter, and all lesions were in the peripheral field of the lung. VATS was carried out with three surgical ports for three cases, two surgical ports for one case, and only one port for two cases. For all cases, an endo-stapler was utilized. In two cases, the preoperative point-marking technique for tumors was employed under the guidance of CT imaging. All patients were discharged from the hospital with no complications, and were followed up with no evidence of lung recurrence. Our criteria in selecting patients for the VATS removal of metastatic lung tumors are as follows: 1) tumors are less than 30 mm in diameter in the peripheral lung, 2) the number of tumors detected by CT scan, etc., is one or two. We conclude that VATS is a good candidate for the resection of lung metastases in the selected cases. Long term outcome remains to be solved in the future.

The prognostic factor of the surgically treated metastatic lung cancer.

Surgical treatment for metastatic lung tumor has also been aggressively performed to treat multiple or bilateral lesions recently. However, in some patients, metastatic pulmonary foci have recurred after surgery for metastatic lung tumor. These foci could not be controlled even after performing thoracotomy several times in some patients. In this study, we examined prognostic factors in patients undergoing surgery for metastatic lung tumor with respect to early relapse of metastatic pulmonary foci after surgery, and discussed strategies for improving long-term results. This study included 120 patients who underwent surgery for metastatic lung tumor in our department between November 1975 and November 1997. Overall, the 5-year survival rate was 37.1%. When results were compared among groups, there were no significant differences related to age, gender, primary organ, DFI, number of metastatic foci or surgical technique. However, the prognosis was significantly poorer in patients with recurrent metastatic pulmonary foci after surgery. Especially in patients with early relapse within 6 months after resection of the lung, the prognosis was markedly poor. Early relapse was an important factor involved in unfavorable surgical outcomes. The mechanism involved in the early relapse of metastatic pulmonary foci after surgery for metastatic lung tumor may be associated with the presence of several micrometastases that could not be recognized during surgery for metastatic lung tumor, that is, dormancy, in the lung. Surgical outcomes in patients with metastatic lung tumor will be improved if a method of controlling this increase in dormant metastases is established.

Establishment and characterization of a new canine B-cell leukemia cell line.

A new cell line derived from a spontaneous canine leukemia was established and designated GL-1. The cells have been cultured in a floating fashion and passaged for over two years. They were round with rich cytoplasm containing many rough endoplasmic reticula and mitochondria. Peroxidase staining was negative. The nuclei of many cells were round, but segmented nuclei were seen frequently. The doubling time of the cells was 27.3 hr and they had 78 chromosomes. Surface marker analysis using monoclonal antibodies (MABs) and flowcytometry revealed that GL-1 possessed CD45 and surface IgG. However, the cells did not react with MABs detecting T-cell markers. These results indicate that GL-1 has a lymphocytic lineage and is derived from a B-cell leukemia.