Enhancement of enterohemorrhagic Escherichia coli O157:H7 stress tolerance via pre-heating.

Enterohemorrhagic Escherichia coli O157:H7 infection causes several hundred cases of food poisoning every year in Japan. In severe cases, this type of food poisoning can be fatal. In the present study, we examined the induction of HSP70 in E. coli O157:H7 cells at various temperatures and the thermotolerance of E. coli O157:H7 cells alone and in contaminated food following pre-heating. We evaluated the possibility that thermotolerance by E. coli O157:H7 increases the likelihood of food poisoning. E. coli O157:H7 cells were heated at 43-51 °C, and the survival rate was examined. The temperature of highest induction of HSP70 was used as the pre-heating temperature. We measured the thermotolerance of E. coli O157:H7 cells following pre-heating as the survival after heating at 53 °C (lethal temperature). Additionally, we evaluated the thermotolerance of E. coli O157:H7 cells in ground beef following pre-heating. Heating at 47 °C for 30 min caused the highest induction of HSP70 and this temperature was selected as the pre-heating temperature. The survival rate was significantly higher for 0-90 min compared to that in cultures incubated at 53 °C without pre-heating indicating thermotolerance. Additionally, in ground beef, thermotolerance in E. coli O157:H7 cells was induced by pre-heating. We showed that E. coli O157:H7 cells acquired thermotolerance after pre-heating, which significantly increased survival after a lethal temperature, and increased the likelihood of food poisoning.

Lactate dehydrogenase, Gleason score and HER-2 overexpression are significant prognostic factors for M1b prostate cancer.

It has not been elucidated whether certain types of M1b prostate cancer (M1b PC) are associated with a poor outcome. The present study retrospectively identified predictive factors related to the outcome of M1b PC. The subjects were 104 patients who attended our hospital and received a diagnosis of M1b PC. The observation period ranged from 4 to 122 months (median, 43 months). The parameters investigated were: T classification, N classification, Gleason score (GS), pretreatment prostate-specific antigen (PSA) level, extent of disease (EOD) grade, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), calcium, and hemoglobin (Hb) levels, platelet count, and the status of HER-2 overexpression as determined with a Hercep Test(TM) Kit using initial needle biopsy specimens for diagnosis. Log-rank test and Cox univariate analysis identified the following factors with statistically significant differences: pretreatment PSA ≥ 192, N1, GS ≥ 8, EOD grade 3+4, high LDH, high ALP, low Hb, and HER-2 overexpression. Multivariate Cox proportional hazard analysis identified the factors GS ≥ 8, high LDH, and HER-2 overexpression with significant differences. The hazard ratio was 5.962, 2.465, and 2.907, respectively, and the probability value was P=0.0218, P=0.0207 and P=0.0090, respectively. When the subjects with GS ≥ 8, high LDH, and HER-2 over-expression were classified as the high-risk group, the 5-year cause-specific survival rate was 51.2, 29.6, and 20.0%, respectively. The present study showed that M1b PC patients with GS ≥ 8, high LDH, and HER-2 overexpression have a very poor outcome and thus, should be treated as a high-risk group requiring close follow-up.

Potential of molecular targeted therapy of HER-2 and Cox-2 for invasive transitional cell carcinoma of the urinary bladder.

Expression of HER-2 and COX-2 was determined to assess the potential of molecular-targeted therapy against human epidermal growth factor receptor-2 (HER-2) and cyclooxygenase-2 (COX-2) for the treatment of invasive bladder cancer. The subjects were 46 patients who attended Aichi Medical University Hospital between January 2001 and August 2008, underwent total cystectomy with a diagnosis of M0 bladder cancer, and received a pathological diagnosis of invasive transitional cell carcinoma of the urinary bladder (pT2-pT4). Expression of HER-2 and COX-2 was determined by immunohistochemical staining, and the results were interpreted by two pathologists by classifying HER-2 expression into four grades, and considering COX-2 positive when 10% or more of the tumor cells were stained. In HER-2 immunostaining, 10 subjects (21.7%) were positive, all of whom had a Grade 3 tumor. Staging classification identified 2 subjects (2/22, 9.1%) with pT2 stage, 3 (3/16, 18.8%) pT3 stage, and 5 (5/8, 62.5%) pT4 stage. There was a correlation between HER-2 positivity and tumor stage (P=0.007). Lymph node metastasis was detected in 13 subjects, 3 of them (3/8, 37.5%) with pN2 metastasis were HER-2 positive. The 5-year cause-specific survival rate was 51.4% for HER-2-positive subjects and 83.4% for HER-2-negative subjects. The outcome was poorer in HER-2-positive subjects, but the difference in survival rate was not statistically significant (P=0.218). In COX-2 immunostaining, 27 subjects (58.7%) were found to be positive. Three (3/4, 75.0%) showed a Grade 2 tumor and 24 (24/42, 57.1%) a Grade 3 tumor. Staging classification identified 13 subjects (13/22, 59.1%) with pT2 stage, 9 (9/16, 56.3%) pT3 stage, and 5 (5/8, 62.5%) pT4 stage. There was no correlation between COX-2 positivity and tumor grade or stage (P=0.488 and 0.089, respectively). Classification by the extent of lymph node metastasis revealed that 6 subjects (6/8, 75.0%) with pN2 were COX-2 positive. There was a correlation between COX-2 positivity and the extent (pN1 or pN2) of lymph node metastasis (P=0.008). The 5-year cause-specific survival rate was 84.0% for COX-2-positive subjects and 71.7% for COX-2-negative subjects. However, the difference in survival rate was not significant (P=0.407). Seven subjects (7/46, 15.2%) were positive for both HER-2 and COX-2, and there was no statistically significant correlation between the status of HER-2 expression and that of COX-2 expression (P=0.2195). The present study failed to show any association between HER-2 or COX-2 positivity and outcome in subjects with invasive bladder cancer. However, HER-2-positive subjects tended to have a poorer outcome. This finding suggests that molecular-targeted therapy against HER-2 could be an effective therapy. Further studies involving a larger number of subjects are required.

Investigation of risk factors for prostate cancer patients with bone metastasis based on clinical data.

It has not yet been determined whether certain types of prostate cancer with bone metastasis (M1b PC) are associated with a poor outcome. The present study retrospectively assessed the potential significance of various clinical data in predicting the outcome of M1b PC. The subjects were 104 patients who attended our hospital and received a diagnosis of M1b PC between January 1998 and December 2006. The age of the subjects ranged from 51 to 91 years (median 74). The observation period ranged from 4 to 122 months (median 43). The parameters investigated were T classification, N classification, Gleason score (GS), pre-treatment prostate-specific antigen (PSA) level, extent of disease (EOD) grade, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), calcium and hemoglobin (Hb) levels and platelet count. The 5-year cause-specific survival rate was 56.6% and the 10-year cause-specific survival rate was 34.9%. Log-rank test and Cox univariate analysis identified the following factors with statistically significant differences: pre-treatment PSA level ≥192, N1, GS ≥8, EOD grade 3+4, high LDH, high ALP and low Hb. Multivariate Cox proportional hazard analysis identified the factors GS ≥8 and high LDH with significant differences. The hazard ratio was 4.967 and 2.728, respectively, and the probability value (P) was 0.029 and 0.004, respectively. When the subjects with GS ≥8 and high LDH were classified as the high-risk group, the 5-year cause-specific survival rate was 24.6%. The outcome was significantly poorer in this group (P<0.0001) than in the other group, which had a 5-year cause-specific survival rate of 67.7%. The present study showed that patients with M1b PC with GS ≥8 and high LDH have a very poor outcome and thus should be treated as a high-risk group requiring close follow-up.

Preliminary results of M-VAC chemotherapy combined with mild hyperthermia, a new therapeutic strategy for advanced or metastatic transitional cell carcinoma of the urothelium.

OBJECTIVE: We evaluated the efficacy and safety of M-VAC chemotherapy combined with mild hyperthermia, a new therapeutic strategy for advanced metastatic transitional cell carcinoma of the urothelium. SUBJECTS AND METHODS: The subjects were 12 patients diagnosed with advanced metastatic transitional cell carcinoma of the urothelium. For mild hyperthermia, the patients' oral temperature was elevated to about 38 degrees C by heating for 20 min and retaining the heat for 20 min with a far-infrared heater. The antitumor effect was evaluated according to the RECIST, while adverse drug reactions were assessed based on the NCI-CTC. RESULTS: The antitumor effect was rated as partial remission (PR) in 10 of the 12 patients and stable disease in 2 patients, with an efficacy rate of 83% (10/12). All 10 patients who had achieved PR received three courses of treatment. Of the 12 patients, 5 died during the observation period, with survival for 9-23 months (mean: 15.6 months). Adverse drug reactions included myelosuppression in all patients (Grade 3 in 4 patients, Grade 4 in 8), and gastrointestinal toxicity, such as nausea or vomiting, which was mild (Grade 0 in 2 patients, Grade 1 in 8, Grade 2 in 1, Grade 3 in 1). CONCLUSIONS: The results of the present study suggest that M-VAC chemotherapy combined with mild hyperthermia, which potentiates the anticancer effect and reduces adverse drug reactions such as gastrointestinal symptoms, is a useful and safe method for the treatment of advanced transitional cell carcinoma of the urothelium.

Combining anti-tumor drugs with mild hyperthermia increases the cytotoxic effects of drugs on human leukemia cells in vitro.

Although the benefits of using a combination of hyperthermia and chemotherapy or radiotherapy in the treatment of cancer have been theoretically established, the use of such combination therapy is not widespread at the clinical level, as the application of hyperthermia is complex and maintaining a tumor temperature of 43°C or higher is exceedingly difficult. Consequently, in the present study, the effects of chemotherapy combined with mild hyperthermia at 41°C (which is easier to apply than standard hyperthermia) were examined in the NALM-6 leukemia cell line. The results were as follows: i) NALM-6 leukemia cells, like most cells, survived mild hyperthermia at 41°C, but were killed at temperatures over 43°C. ii) Low concentrations of adriamycin (0.1 µg/ml) or mild hyperthermia applied separately did not have a visible effect on the survival rate of NALM-6 cells, whereas combined treatment with these therapies decreased the survival rate of NALM-6 cells in a time-dependent manner. The anti-tumor effect after 5 h of the combination of 0.1 µg/ml adriamycin and mild hyperthermia was the same as that observed with a 10-fold higher concentration (1 µg/ml) of adriamycin alone. iii) Another anti-tumor drug, vincristine, exhibited the same behavior as adriamycin. The anti-tumor effect after 1 h of the combination of 5x10-11 M vincristine and mild hyperthermia was the same as that observed with a 10-fold higher concentration (5x10-10 M) of vincristine alone. The results indicate that it may be possible to reduce the required concentrations of anti-tumor drugs by using them in combination with mild hyperthermia. In this way, the side effects of chemotherapy may be reduced in clinical settings. Mild hyperthermia is a useful and practical heating method, and could result in the increasing clinical application of hyperthermia in the treatment of cancer.

First Streptococcus agalactiae isolates highly resistant to quinolones, with point mutations in gyrA and parC.

Three isolates of Streptococcus agalactiae highly resistant to multiple fluoroquinolones were isolated in Japan. Compared with susceptible strains of S. agalactiae, these quinolone-resistant strains had double point mutations within the quinolone resistance-determining regions of gyrA and parC; Ser-81 was changed to Leu (TCA --> TTA) in the amino acid sequence deduced from gyrA, and Ser-79 was changed to Phe (TCC --> TTC) in the amino acid sequence deduced from parC. Comparative sequence analysis revealed the possibility of gene transfer between S. agalactiae and another beta-hemolytic streptococcus, Streptococcus difficile.

[Expression of stress protein (HSP 70) in adrenalectomized mice].

We have already reported that the expression of HSP 70 in the adrenal gland was the most apparent among all organs in whole body-heated mice. In this paper, we examined the relation between the induction of HSP 70 and the adrenal gland using adrenalectomized mice. The expression of HSP 70 in some organs of adrenalectomized mice was significantly lowered in comparison with control mice. Adrenalectomy attenuated whole body heating-induced HSP 70 induction in some organs. Low expression of HSP 70 in adrenalectomized mice was a result to high-mortality caused by endotoxin shock. These results indicate that the adrenal gland plays an important role in the response to stress, leading to the induction of HSP 70.

[Radioprotective effects of natural beta-carotene on villi and crypts in abdominally radiated mice].

The protective effect of beta-carotene against radiation injury to the small intestine of abdominally radiated mice (15 Gy) was examined with administration given pre-radiation, during (pre- and post-) radiation, and post-radiation. In the beta-carotene group, the ratio of villus length to crypt was significantly greater in comparison with the radiation only group at 2 days after radiation. At 7 days after radiation, the ratio of necrotic cells in the crypt vs. the total was significantly lower, and the ratio of necrotic cells in the villus vs. the total was significantly greater with beta-carotene administration, which indicated that beta-carotene accelerated recovery from radiation injury. Each group administered beta-carotene showed a significant radioprotective effect, with pre-radiation administration yielding a smaller effect than administration during radiation and post-radiation. It is concluded that pre-, during, and post-radiation administration of beta-carotene protected against radiation injury of the small intestine and accelerated recovery from it.