Tailoring Effective Phage Cocktails for Long-Term Lysis of Escherichia coli Based on Physiological Properties of Constituent Phages.

Background: Bacteriophage (phage) therapy has regained attention as an alternative to antimicrobial agents for eliminating bacteria; however, the emergence of phage-resistant bacteria during the therapy is a major concern. One method to control this emergence is to create a cocktail composed of multiple phages. Materials and Methods: In this study, we isolated 28 phages infecting Escherichia coli and evaluated their bacteriolysis (lysis) activity, lytic spectrum, adsorption rate constant, burst size, and titer of a 1-day incubation, followed by clustering of the phages based on these physiological characteristics. Results: The variation in lysis onset time and duration was more significant for cocktails of phages from different clusters than for phage cocktails from the same cluster. Conclusions: This suggests that a combination of phages with different physiological characteristics is necessary to create a cocktail that rapidly and continuously lyses bacteria over a prolonged duration while suppressing the emergence of resistant bacterial strains.

Increased Vancomycin Clearance in Patients with Solid Malignancies.

Vancomycin (VAN) is an anti-microbial agent used to treat a number of bacterial infections, which has a high incidence of nephrotoxicity. We examined the pharmacokinetics of VAN retrospectively based on trough concentrations at large scale and identified pharmacokinetic differences between Japanese patients having solid malignancy and non-malignancy patients. Data were analyzed from 162 solid malignancy patients and 261 non-malignancy patients, including the patient's background, VAN dose, and pharmacokinetics of VAN. We failed to detect differences in values for VAN clearance or shorter elimination half-lives between these two groups. In contrast, multiple regression analysis under adjusting for confounding factors by propensity score, showed that VAN clearance significantly increased in relation to solid malignancies in each stage. We conclude that VAN clearance in solid malignancy patients is increased and that the blood concentration of VAN becomes lower than expected. These results suggest that early monitoring of VAN levels in solid malignancy patients might be essential for maintaining desired effects without side-effects.

Augmented Renal Clearance of Vancomycin in Hematologic Malignancy Patients.

The pharmacokinetics of vancomycin (VAN) was retrospectively examined based on trough concentrations at large scale to identify pharmacokinetic differences between Japanese hematologic malignancy and non-malignancy patients. Data from 261 hematologic malignancy patients and 261 non-malignancy patients, including the patient's background, VAN dose, and pharmacokinetics of VAN estimated by an empirical Bayesian method, were collected and analyzed. Our results showed significantly higher values for VAN clearance and shorter elimination half-lives in patients with hematologic malignancies than non-malignancy patients. In addition, multiple regression analysis under adjusting for confounding factors by propensity score, showed that VAN clearance significantly increased in relation to hematologic malignancies. In conclusion, since in hematologic cancer patients VAN clearance is increased, the blood concentration of VAN becomes lower than expected and this may contribute to the survival of resistant bacteria when VAN is administered at low doses. These results suggest that early monitoring of VAN levels in hematologic cancer patients might be recommended to maintain desired effects without side-effects.

Analysis of [(2)H7]methionine, [(2)H4]methionine, methionine, [(2)H4]homocysteine and homocysteine in plasma by gas chromatography-mass spectrometry to follow the fate of administered [(2)H7]methionine.

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine-homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [(2)H(7)]methionine, [(2)H(4)]methionine, methionine, [(2)H(4)]homocysteine and homocysteine in rat plasma by gas chromatography-mass spectrometry (GC-MS). [(13)C]Methionine and [(13)C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water-ethanol-pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.

Usefulness of transcription factors Ad4BP/SF-1 and DAX-1 as immunohistologic markers for diagnosis of advanced adrenocortical carcinoma.

BACKGROUND: We examined the expressions of Ad4BP/SF-1 and DAX-1 in primary and metastatic lesions of advanced adrenocortical carcinoma with normal hormonal findings by immunohistochemistry and discussed their usefulness as immunohistochemical markers for diagnosis. PATIENTS AND METHODS: Four patients (average age 54.3 +/- 3.9) with retroperitoneal and metastatic tumors were enrolled in this study. All patients had normal hormonal findings. Ad4BP/SF-1 and DAX-1 underwent immunohistochemical study using needle biopsy specimens from the retroperitoneal tumor or metastatic tumor of these patients to diagnose primary adrenocortical carcinoma. RESULTS: Immunoreactivity for Ad4BP/SF-1 and DAX-1 indicated that it was localized exclusively in the nuclei. Ad4BP/SF-1 immunoreactivity was observed in the carcinoma cells of 2 patients, but not in the others, whereas DAX-1 immunoreactivity was observed in the carcinoma cells of all 4 patients. CONCLUSION: The expressions of Ad4BP/SF-1 and DAX-1 are considered essential to maintain the biological characteristics of adrenocortical cells even without abnormal hormonal findings or even after malignant transformation and metastasis. These markers are useful to distinguish other retroperitoneal tumors, especially in patients with bulky tumors, and to diagnose the metastatic site of origin as adrenocortical carcinoma in patients clinically presenting with widespread metastasis.

Efficacy of bacillus Calmette-Guérin in the treatment of superficial bladder cancer: the impact of previous intravesical treatment.

OBJECTIVE: To clinically evaluate the role of intravesical bacillus Calmette-Guérin (BCG) in the management of superficial bladder cancer, focusing on dose, age, high grade/stage, and pretreatment episodes with other therapies in long-term follow-up patients. METHODS: A total of 213 patients with superficial (Ta-T1) transitional cell carcinoma of the bladder received 6-8 weekly instillations of 40, 60 or 80 mg of Tokyo strain BCG. Further 6-8 monthly applications were given in some cases. RESULTS: Recurrence-free and progression-free survival rates were compared with reference to dose, age, previous treatment, grade, stage, and high risk categories. Overall recurrence-free rates for 3, 5, and 10 years were 70.8%, 67.1%, and 57.6%, with progression-free survival rates of 94.5%, 93.6%, 90.6%, respectively. In univariate analyses, younger cases demonstrated higher recurrence-free survival rates, but without statistical significance (P = 0.1229). Recurrence-free rates were significantly higher for cases without previous intravesical treatment (P = 0.0010). There was no significant BCG dose-dependence. High grade and high stage patients were confirmed as having higher recurrence and progression rates. CONCLUSIONS: Intravesical instillation of BCG is the most effective in the long term when chosen as the initial prophylactic therapy for the prevention of recurrence in superficial bladder cancer.

Azoospermia with Klippel-Feil anomaly.

We present a case of azoospermia with Klippel-Feil anomaly. Klippel-Feil anomaly is characterized by the fusion of two or more cervical vertebrae and a short neck, limitation of head movement, and low posterior hairline. The association of this anomaly with MURCS (Müllerian-duct aplasia, renal agenesis, and cervical somite dysplasia) is traditionally regarded as being limited to females, but it has been hypothesized that men displaying the combination of azoospermia, segmentation abnormalities of the cervicothoracic spine, and renal anomalies have a male analog of MURCS. Here we describe the first case of MURCS in a male in whom testicular sperm extraction was carried out.

Simulated conditions of microgravity increases progesterone production in I-10 cells of Leydig tumor cell line.

BACKGROUND: The prospect of long-term residence in outer space is becoming a reality. Previously we found that testosterone secretion and spermatogenesis is reduced in model mice on which the shift of body fluids in space is simulated. The present study examines the effects of the microgravity (muG) environment on steroidogenesis of Leydig cells. METHODS: I-10 cells were cultured for 24 h under a simulated muG environment. Progesterone production was evaluated as steroidogenesis. Western blots of the enzyme, 3betahydroxy dehydrogenase (HSD) as well as of the transcription factors, Ad4bp/SF-1 and DAX-1, the amount of adenosine3',5'-cyclic monophosphate (cAMP) and cell morphology were investigated. The muG environment was simulated using clinostat rotation (CR). Rotational (Cont-1) and stationary (Cont-2) controls always received a regular gravity effect with and without rotatory motion, respectively. RESULTS: Progesterone levels in cells under CR were significantly increased compared with those of both controls. Increased progesterone production was not related to the expression of either of the transcription factors or the enzyme. Although the cytoplasm expanded, the amount of cAMP produced under CR did not significantly differ from that produced under both control environments. CONCLUSIONS: Our findings showed that I-10 cells produced more progesterone under a muG, than under control environments. Since the amounts of 3betaHSD, Ad4bp/SF-1, DAX-1 and cAMP were not altered, the increase in steroid production under CR might be due to the expansion of cytoplasm, where steroidogenesis occurs. Steroidogenesis should be investigated in more detail to predict the effects of outer space on humans.

Effect of microsurgical repair of the varicocele on testicular function in adolescence and adulthood.

OBJECTIVE: Many reports on varicoceles suggest improved spermatic findings and increased pregnancy rates after correction of these lesions. Early repair during adolescence has been advocated, since clinically apparent varicoceles may affect testicular volume and sperm production in the future. We examined the efficacy of microsurgical varicocelectomy, and aimed to establish predictive parameters useful for ascertaining whether varicocele repair provides any benefits in adolescents and adults. METHODS: We carried out microsurgical varicocelectomy on nine boys and 19 men. In adolescents, catch-up growth of the testis, expressed by pre- and postoperative ratios of left and/or right testicular volume, grade and serum level of follicle-stimulating hormone (FSH) were evaluated. In adults, the ratio of sperm concentration improvement, grade, testicular volume, preoperative sperm concentration and serum FSH level were evaluated. All subjects were followed for 12 months. RESULTS: Catch-up growth was seen in 62.5% of boys. FSH level was significantly lower in boys with catch-up growth than in boys without catch-up growth. Improved sperm concentration was seen in 73.6% of adults. FSH level was significantly lower in adults with improved sperm concentration than in patients without improved sperm concentration. No correlations were seen between other parameters and catch-up growth in adolescents, or sperm concentration improvement in adults. CONCLUSIONS: Microsurgical varicocelectomy as a treatment for varicoceles with low FSH might be effective and the relevant predictive parameter for testicular development and function after surgery might be serum FSH level in both adults and adolescents.

Gene transfer to mouse testes by electroporation and its influence on spermatogenesis.

We transferred the adventitious gene pCAGGS-lacZ to mouse testes with the use of a square-wave electroporator and investigated the efficiency of gene transfer (GT) and the influence of the procedure on testicular damage and spermatogenesis. Mice were divided into 5 groups: (1-2) injection of gene/phosphate-buffered saline (PBS) into the interstitial space followed by electroporation (EP), (3) EP alone, (4-5) injection of gene/PBS without EP. The presence of the lacZ gene was determined by X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) staining and the polymerase chain reaction (PCR). The influence of transfer on spermatogenesis was assessed by evaluating the seminiferous tubules according to the Johnsen score (JS). TdT-mediated dUTP-biotin nick end-labeling (TUNEL) staining was performed for the detection of apoptosis in the testes to evaluate the testicular damage caused by GT, and fertilization ability was assessed by mating male mice from each group with normal female mice at 1, 2, 4, 6, and 8 weeks after the procedure. LacZ expression was detected by X-gal staining and PCR for 4 weeks after GT in group 1. But in group 4, LacZ expression was not detected for all times. In groups 1 through 3, the JSs decreased gradually until 4 weeks and recovered at 6 and 8 weeks after GT. The JSs were significantly decreased at 4 weeks for groups 1 through 3 compared with groups 4 and 5. In groups 1 through 3, apoptotic cells were significantly more numerous at 1, 2, and 4 weeks after the procedure, and there were significant differences in their numbers between groups 1 through 3 and groups 4 and 5 until 4 weeks after the procedure. The number of offspring did not differ significantly between all groups. These results suggest that although spermatogenic damage caused by EP could present problems, GT by EP might be effective for transfecting germ cells or somatic cells and could be applicable for in vivo gene therapy for male infertility in the future.

Human sperm motility in a microgravity environment.

Background and Aims: We carried out clinostat and parabolic flight experiments to examine the effects of a microgravity (µG) environment on human sperm motility. Methods: Semen samples were obtained manually from 18 healthy men (aged 27.4 ± 5.4 years) who had given their informed consent. In clinostat experiments, samples that were left stationary were used as a stationary control. Samples rotated vertically and horizontally were used as a rotation control and a clinostat rotation, respectively. In parabolic flight experiments using a jet plane, sperm motility was compared for each parameter at µG, 1G and 2G. The state of 1G during the flight was used as a control. Sperm motility was determined using an automatic motility analyzer HT-M2030 in a microgravity environment. Results: All parameters of sperm motility tended to be lower in clinostat rotation compared with rotation control at both low-speed and high-speed, but the differences were not statistically significant. In parabolic flight, sperm motility and parameters of linear movement were decreased (P < 0.05). There was no significant difference between µG and 2G, but sperm motility was significantly decreased at µG than at 1G. Conclusions: Our findings suggest that sperm motility is reduced under µG. (Reprod Med Biol 2005; 4: 161-168).