Influence of cerebral fluid drainage on the pharmacokinetics of vancomycin in neurosurgical patients.

The objective of this study was to retrospectively investigate the influence of cerebral fluid drainage on the serum concentrations and pharmacokinetic parameters of vancomycin (VCM). We analyzed 55 patients with normal renal function who had been hospitalized in the neurosurgical ward and received intravenous infusions of VCM. We compared the daily doses of VCM, serum VCM concentrations, serum concentration/dose ratio (C/D ratio), and pharmacokinetic parameters calculated using the Sawchuk-Zaske method between patients who underwent cerebral fluid drainage (drainage group) and controls (non-drainage group). The patients in the drainage group showed a significantly lower trough concentration of VCM (5.8 ± 3.3 µg/mL) than that shown by the non-drainage group (9.9 ± 5.4 µg/mL, p = 0.017). Further, the patients in the drainage group showed a significantly lower trough C/D ratio (0.32 ± 0.17) than that shown by the non-drainage group (0.50 ± 0.31, p = 0.047). In conclusion, cerebral fluid drainage may influence VCM pharmacokinetics. Our findings strongly suggest that a high dose of VCM is required to maintain optimal serum concentrations of VCM in patients managed with cerebral fluid drainage.

Biocompatibility of cross-linked hyaluronate (Gel-200) for the treatment of knee osteoarthritis.

OBJECTIVE: To compare the biocompatibility and immunogenicity of two intra-articular hyaluronan formulations, Gel-200 (Gel-One(®)) and hylan G-F 20 (Synvisc(®) series). EXPERIMENTAL DESIGN: A comparison of the biocompatibility of Gel-200 and hylan G-F 20 was made using a rat subcutaneous air pouch model and the knee joint of normal rabbits. Immunogenicity was evaluated using a homologous passive cutaneous anaphylaxis (PCA) assay in guinea pigs. RESULTS: In the air pouch model in rats, characteristic fibrous belts formed in the subcutaneous tissue. Injection of hylan G-F 20 into the air pouch induced granulomatous nodules primarily composed of macrophages, multinucleated giant cells, and eosinophils accompanied with the test material in the center of the nodules in the fibrous belt. Furthermore, the thickness of the fibrous belt in the hylan G-F 20 group increased significantly compared to the saline group. Injection of Gel-200 into the air pouch induced neither granulomatous inflammation nor significant thickening of fibrous belt, while foamy macrophages containing the test material were observed. Intra-articular injection of hylan G-F 20 into the rabbit knee joints induced granulomatous inflammation, eosinophil infiltration, and significant increase in the number of cells in the synovial fluid, while these findings were absent in the Gel-200 group. In the immunogenicity assay, hylan G-F 20 induced a positive PCA reaction, but the Gel-200 did not. CONCLUSION: Gel-200 showed more favorable biocompatibility and less immunogenicity compared to hylan G-F 20. Gel-200 is expected to be a single injection hyaluronan product with less safety concerns for the treatment of knee osteoarthritis (OA) pain.

Tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in an ultra-short-term skin carcinogenesis bioassay using rasH2 mice.

Assessment of the skin tumor-promoting potential of 12-O-tetradecanoylphorbol-13-acetate (TPA) after initiation with 7,12-dimethylbenz[a]anthracene (DMBA) was conducted using rasH2 transgenic (Tg) mice and their nontransgenic (non-Tg) littermates. Mice were treated with DMBA (50 µg/100 µL acetone) on clipped back skin at the commencement of the study, and 1 week thereafter, TPA was applied at 8 µg/200 µL or 4 µg/200 µL acetone, once or twice weekly, for 7 weeks. Skin nodules were observed in the rasH2 Tg mice from week 4, and the incidence reached 100% at weeks 5 and 6. The number of skin nodules (multiplicity) in the 8-µg twice-weekly, 8-µg once-weekly, 4-µg twice-weekly, and 4-µg once-weekly groups was 62.4, 46.2, 62.6, and 36.9, respectively. The non-Tg mice also developed skin nodules, but the sensitivity to induction in the rasH2 Tg mice was higher. No nodules were observed in the acetone groups, but single nodules were apparent in the no-treatment rasH2 Tg and non-Tg groups. In conclusion, skin promotion effects could be detected within only 8 weeks in the rasH2 mice, and the concentration of 4 µg TPA once weekly was sufficient as a positive control. This short-term skin carcinogenesis bioassay using rasH2 mice could represent a useful tool for the assessment of drug and chemical safety with cutaneous treatment.

Establishing a laboratory animal model from a transgenic animal: RasH2 mice as a model for carcinogenicity studies in regulatory science.

Transgenic animal models have been used in small numbers in gene function studies in vivo for a period of time, but more recently, the use of a single transgenic animal model has been approved as a second species, 6-month alternative (to the routine 2-year, 2-animal model) used in short-term carcinogenicity studies for generating regulatory application data of new drugs. This article addresses many of the issues associated with the creation and use of one of these transgenic models, the rasH2 mouse, for regulatory science. The discussion includes strategies for mass producing mice with the same stable phenotype, including constructing the transgene, choosing a founder mouse, and controlling both the transgene and background genes; strategies for developing the model for regulatory science, including measurements of carcinogen susceptibility, stability of a large-scale production system, and monitoring for uniform carcinogenicity responses; and finally, efficient use of the transgenic animal model on study. Approximately 20% of mouse carcinogenicity studies for new drug applications in the United States currently use transgenic models, typically the rasH2 mouse. The rasH2 mouse could contribute to animal welfare by reducing the numbers of animals used as well as reducing the cost of carcinogenicity studies. A better understanding of the advantages and disadvantages of the transgenic rasH2 mouse will result in greater and more efficient use of this animal model in the future.

A larval medaka (Oryzias latipes) acute toxicity assay combined with solid-phase extraction to efficiently determine the toxicity of organic contaminants in river water.

Using a larval medaka (Oryzias latipes) acute toxicity assay combined with solid-phase extraction, we proposed a method for efficiently determining the fish toxicity of organic contaminants in river water. Organic toxicants were 10, 20, 50 and 100-fold concentrated from 4 L of the sample with adsorption cartridges. The lethal effect was observed by exposing every ten individuals of 48-72 h old larval medaka to 20 mL of each solution for 48h. The median lethal concentration rate (LCR50) was used as an indicator for the toxicity. With the developed toxicity test method, more than seven times difference was found in the LCR50 of the river water samples. LCR50 distribution profiles were compared with 125 samples in two typical rivers. The result revealed a lower toxicity level in the mainstream than in the confluences, and a lower toxicity level in Sagami River than in Ayase River. LCR50 proved unique as a toxicity indicator, which was impossible to speculate from the conventional water quality indicator of the dissolved organic carbon concentration. As an effective screening test for priority settings, the method can help us with an efficient planning for the environmental investigation and management.

Tsutsugamushi disease in Kanagawa Prefecture, Japan: clinical report of two cases and survey of other incidences in 1998.

Tsutsugamushi disease is characterized by the early appearance of a black crust at the bitten area and the subsequent development of macular or macropapular rush on the whole body with high fever. While treatment with tetracycline derivatives and chloramphenicols is effective, delayed diagnosis or inappropriate treatment will lead to fatality. In this report, we describe two typical cases of tsutsugamushi disease and discuss other incidences in Kanagawa Prefecture, Japan, in 1998. One of the present two patients was diagnosed to be a case of the new type by Kawasaki strain of Rickettsia tsutsugamushi, while responsible strain was not identified for the other case. Since the disease is spreading widely even to suburban areas, we emphasize the need to consider the possible diagnosis of tsutsugamushi disease in patients with generalized eruption and high fever.

Induction of drug metabolism-related enzymes by methylcholanthrene and phenobarbital in transgenic mice carrying human prototype c-Ha-ras gene and their wild type littermates.

Transgenic mice hemizygously carrying human c-Ha-ras proto-oncogene, Tg-rasH2 show very sensitive and facilitated carcinogenicity to various carcinogens. In this study, activities of certain enzymes related to drug metabolism and energy metabolism were measured in microsome and cytosol fractions of livers of Tg-rasH2 mice and their wild type littermates with both sexes treated with 3-methylcholanthrene (MC) and phenobarbital (PB). Aminopyrine N-demethylase activities increased significantly in livers of all mice treated with PB. MC and PB treatments induced significant increases in activities of UDP-glucuronosyltransferase and S-adenosyl homocysteinase compared to those in the non-treated groups in microsome fractions from all mice. In cytosol fractions of livers of all mice, glutathione S-transferase activity was significantly induced in the PB treated groups. There were no significant differences in activities of lactate dehydrogenase, glucose 6-phosphate dehydrogenase, pyruvate kinase and glucose 6-phosphatase related to energy metabolism in livers and kidneys among all mice. Tg-rasH2 mice showed stable activities of enzymes related to drug detoxication and energy metabolism similar to those of non-transgenic mice. These results suggest that the human c-Ha-ras transgene may not affect drug metabolism-related enzymes, and the facilitated carcinogenic response in the Tg-rasH2 mouse is not due to these enzymatic disorders.

Convenient dioxin measuring method using an efficient sampling train, an efficient HPLC system and a highly sensitive HRGC/LRMS with a PTV injector.

An efficient sampling method for dioxins from flue gas, an efficient automatic purification and fractionation method by a new HPLC system using a sulfuric acid-silica/silica column, a Nitro column and a PGC column, and sensitive determination method by an economical HRGC/LRMS using a programmable temperature vaporization (PTV) injector were developed. It was confirmed that the dioxins could be easily collected by the proposed simple sampling train consisting of only two bottles, and the extracted sample could be purified sufficiently and separated as mono-ortho PCBs, non-ortho PCBs and PCDDs/PCDFs completely with the proposed HPLC system. The peak areas of GC/MS were increased linearly with the injection volume up to 40 microl by the PTV technique, and the sensitivity could be increased to 20 times higher than usual. This convenient measuring method can drastically reduce operation time, consumption of hazardous solvent and cost.

Method for measuring mutagen formation potential (MFP) on chlorination as a new water quality index.

A novel water quality index, the mutagen formation potential (MFP) is proposed for use in evaluation of the quality of drinking water which may contain pollutants capable of forming mutagens when chlorinated under the conditions used in water purification processes. A method for measuring MFP was established as follows. The water sample to be tested is diluted until the TOC reaches 3-4 mg l-1, the pH is adjusted to 7.0 +/- 0.2, sodium hypochlorite is added to obtain conditions where Cl/TOC = 3-4 mg Cl (mg C)-1, and the water sample is left standing for 24 +/- 2 h at room temperature. Thereafter, 21 of the chlorinated water sample at pH 2.0 +/- 0.1 is passed through a Sep-Pak Plus CSP-800 cartridge to adsorb any mutagens formed, and DMSO is applied to the cartridge to desorb the mutagens. Then, a 2 ml sample of the eluate is collected after the DMSO had begun to flow out of the cartridge and evaluated by the Ames Salmonella mutagenicity assay (preincubation method).

Overexpression of human H-ras transgene is responsible for tumors induced by chemical carcinogens in mice.

Level of human prototype H-ras transgene expression in tumors induced by chemical carcinogens (N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea) was analyzed in human H-ras transgenic mice (CB6F1-TgrasH2 Jic mice). All forestomach tumors examined revealed about 2-fold overexpression of the human H-ras transgene with or without point mutation at codon 12 or codon 61. However, endogenous mouse H- and K-ras genes exhibited neither point mutation nor overexpression. These results suggested that increased levels of ras gene products in the cell played an important role in facilitating chemical carcinogenesis in transgenic mice.

Convenient substitute indices to toxic equivalent quantity for controlling and monitoring dioxins in stack gas from waste incineration facilities.

It is necessary to develop technical systems for controlling and monitoring dioxins in stack gas from waste incineration facilities. However, it required much labor, high technical skill and extreme cost to measure dioxins for obtaining the toxic equivalent quantity (TEQ) values. In this study, the positive correlations of the WHO-TEQ values of PCDDs/PCDFs/Co-PCBs or PCDDs/PCDFs with the conventional I-TEQ values of PCDDs/PCDFs were confirmed beforehand for a large number of incineration facilities in a wide concentration range of a hundred thousand times. The relationships between the TEQ values and the concentrations of total PCDDs/PCDFs, each toxic isomer of PCDDs/PCDFs, total Cl4-6BZs, or each homologue of Cl4-6BZs were investigated for various incineration facilities. And it was found that positive correlations of the TEQ values with the concentrations of total PCDDs/PCDFs, 2,3,4,7,8-P5CDF, or Cl5BZ for a large number of different incineration facilities and in a wide concentration range. Consequently, the concentrations of total PCDDs/PCDFs, 2,3,4,7,8-P5CDF and Cl5BZ, whose measuring methods are much easier than that of obtaining the TEQ values, could be used as convenient substitute indices to the TEQ values for controlling and monitoring dioxins in stack gas from various waste incineration facilities.

A measuring method of chlorobenzenes as a convenient substitute index of dioxins in stack gas from waste incineration facilities.

Much labor, high technical skill and extreme cost are required to measure dioxins for obtaining the toxic equivalent quantity (TEQ) values. On one side, it was found that there were high amounts of chlorobenzenes (CBZs) in the flue gas from waste incineration facilities, and they are also thought to be one of the principal precursors for dioxins. The concentrations of CBZs could be used as a convenient substitute index to the TEQ values for controlling and monitoring dioxins in stack gas from waste incineration facilities as shown in another report. In this study, collection efficiencies in the sampling performed by a convenient train which was composed of two bottles containing water and diethylene glycol in a cooling box, recoveries in the evaporation of the extract by an evaporator, proper procedure of the concentration by nitrogen purge were investigated for measuring CBZs. It was found that tetra-, penta-, and hexa-chlorobenzenes (Cl4-6BZs) whose boiling points are higher than ca. 240 degrees C could be sufficiently collected and recovered in the sampling and the evaporation. For sufficient recoveries of Cl4-6BZs in the concentration by the nitrogen purge, the final volume should be larger than 200 microliters with a proper receiver of the K-D concentrator at 35 degrees C. However, Cl1-3BZs were lost in each of the procedures.

Histologic study of the regeneration process of human hair follicles grafted onto SCID mice after bulb amputation.

This study examines histologically the degeneration and subsequent regeneration processes of human hair follicles whose bulb is severely damaged. Human scalp hair follicles were isolated and grafted onto immunodeficient mice after their bulb was amputated. On day 14, thickening and corrugation of the vitreous membrane, apoptosis of follicular keratinocytes, and regression of the lower portion of the follicles were observed. By day 20, mesenchymal cells had accumulated around the lower end of the follicles. From day 14 through 50, the follicular regression and apoptosis continued, and between days 30 and 40 the follicles became maximally shortened, and the vitreous membrane disappeared. By day 50 the lower end of the follicles had become cup-shaped, and the cup surrounded an aggregate of mesenchymal cells that corresponded to the dermal papilla. By day 60, all the grafted follicles had developed into anagen VI follicles, and the apoptosis had ceased. These results indicate that human scalp hair follicles whose bulb is completely destroyed enter into dystrophic telogen after restoration of the dermal papilla, then into anagen, and that the duration of the dystrophic telogen is shorter than that of the normal hair cycle.

Histologic and cell kinetic studies of hair loss and subsequent recovery process of human scalp hair follicles grafted onto severe combined immunodeficient mice.

To establish a model for studying human scalp hair, individually isolated hair follicles were grafted onto back skin of severe combined immunodeficient mice. Histologic changes and cell kinetics in the hair loss and subsequent recovery process were investigated. In the dystrophic stage (from day 7 to 30), all the hair shafts became dystrophic and were shed. Thickening and corrugation of vitreous membrane, apoptosis, and regression of the lower part were observed in the grafted hair follicles. 5-bromo-2'-deoxy-uridine-labeled cells were not detected in the lower end of the follicles, and keratin 19-positive cells appeared there. At the end of this stage their lower part was maximally retracted, secondary germ remained beneath the bulge, and the vitreous membrane disappeared. In the regeneration stage (from day 30 to 50), the same histologic findings as those at the end of the dystrophic stage were observed. The keratin 19-positive cells in the secondary germ, however, were replaced with keratin 19-negative and 5-bromo-2'-deoxy-uridine-labeled cells. Then, differentiation into an inner root sheath and a hair shaft began, and apoptosis was terminated. In the stable growth stage (from day 40 to at least 150), the grafted follicles were immunohistochemically and light microscopically identical with the normal anagen hair follicles except for the presence of melanin incontinence. These findings suggest that the grafted hair follicles entered into dystrophic catagen, subsequently dystrophic telogen, then returned to normal anagen follicles, and that stem cells or their close progeny in the secondary germ play an important part in the recovery process.

Decontamination of human xenotransplantable tumor with mouse hepatitis virus by implantation in nude rat: a case report.

A human tumor xenograft contaminated with mouse hepatitis virus (MHV) was implanted in a nude rat in order to decontaminate the tumor line. The decontamination failed in the first trial, but succeeded in the second trial. The difference between the two trials was the duration of implantation of the tumor in the nude rat, i.e., 12 days in the first and 24 days in the second trial. Duration of implantation might be a factor in the decontamination of transplantable tumors infected with MHV by passaging in the nude rat.

Adsorption equilibriums of principal herbicides on paddy soils in Japan.

Herbicides used in paddy fields during the flooding season can easily cause pollution by run-off into rivers or by other routes. It is very important to know the adsorption characteristics that influence their fate in the soil. The adsorption equilibriums have often been expressed by Henry equations, and the values of equilibrium constant, Kd, are estimated from the adsorption constants, K(OC), based on organic carbon contents of soils. There is little information concerning the equilibrium values expressed by the Freundlich equations, and insufficient information on the actual concentration levels in the paddy field. Therefore, adsorption equilibriums of the five principal herbicides: esprocarb, mefenacet, pretilachlor, simetryn and thiobencarb, on five kinds of paddy soil in Japan were investigated. It was found that their equilibrium values were better expressed by the Freundlich equation for concentration levels for the paddy fields, and that the values for the adsorption coefficient, n, varied from 1.0 to 1.6. Values for the coefficient, k, were in the range of 29-420 mg(1 - 1/n) l(1/n)/kg-dry, and the values were poorly related to solubilities in water or to the octanol-water partition coefficients of the herbicides. For each herbicide, except for simetryn, the values of k among the soils differed by 2-3 times, and no correlation could be found with the organic carbon contents, specific surface areas, pH, cation exchange capacity or major minerals of the soils. The adsorption equilibriums calculated from the values of adsorption constant Kd by the values of K(OC) in the literature were found to be very different from the experimental equilibriums. From the experimental values of coefficient k and n of the Freundlich equation, the maximum runoff concentrations of the herbicides were preliminarily estimated by a simple equilibrium model.

The quantitative and qualitative defect of CD4+ CD45RO+ memory-type T cells are involved in the abnormality of TH1 immunity in atopic dermatitis patients.

Peripheral blood mononuclear cells (PBMCs) obtained from atopic dermatitis (AD) patients produced low levels of IFN-gamma in response to Dermatophagoides farinae antigen (Der f Ag) plus IL-2 or OKT3 MoAb in contrast with PBMCs obtained from healthy donors. The reduced IFN-gamma production in AD patients' T cells appeared to be derived from the defect of CD4+ T cells but not CD8+ T cells. Indeed, from the cytoplasmic staining analysis of cytokines, it was demonstrated that the frequency of IFN-gamma producing CD4+ T cells (TH1 cells) in AD patients was markedly lower than that of healthy donors. From the phenotypic analysis using flow cytometry, it was also found that the number of CD4+ CD45RO+ memory type T cells was significantly reduced in AD patients compared with that of healthy donors. In addition to quantitative defect of memory type CD4+ T cells, functional defect of CD4+ CD45RO+ memory type T cells was also demonstrated in AD patients. Enriched CD4+ CD45RO+ T cells obtained from AD patients, who exhibited greatly reduced delayed-type hypersensitivity (DTH) response in tuberculin test, showed no significant TH1 immunity in terms of IFN-gamma production by stimulation with OKT3 MoAb or purified protein derivative (PPD). Thus, the immunological abnormality of TH1 immunity in AD patients appeared to be induced in concomitant with both the quantitative and qualitative defect of memory type CD4+ T cells.

Development of a novel solid phase extraction method for the analysis of bacterial quinones in activated sludge with a higher reliability.

A novel analytical method for analysis of microbial quinones in activated sludge sample was developed with improved reliability compared to the conventional method. The operating conditions for the extraction of quinones from activated sludge sample with a methanol-chloroform mixture and hexane were optimized. A solid phase extraction method using the Sep-Pak Plus Silica, a small column packed with silica gel, was employed for the purification and separation of quinones instead of column chromatography and thin layer column chromatography used in the conventional method. The analytical operation was simplified and the analytical time was shortened by a half or two-thirds of that required for the conventional method. The recoveries of quinones were markedly increased from 30-60% with the conventional method to as high as 90-101% with the improved method, demonstrating that the reliability of the analytical results of the improved method is significantly higher than that of the conventional method. The quinone content value obtained using the improved method was twice as high as that obtained using the conventional method for the same activated sludge sample, but the quinone profiles expressed as the mole fraction of each quinone type were the same for each method.

Statistical procedures for estimating the detection limit and determination limit of the Ames Salmonella mutagenicity assay.

Novel and flexible procedures for estimating the detection limit as well as the determination limit of the Ames mutagenicity assay were proposed to evaluate the genetoxicity of a water sample. The accumulated data under the test conditions of TA 100-S9 by our group were taken as examples and analyzed to estimate the detection limit and the determination limit. The detection limit was estimated at 1.7 as the MR value when duplicate plates were used in the negative control test. However, it decreased to 1.4 as the MR level when quadruple plates were used in the negative control test. Therefore it was found that the sensitivity of the Ames mutagenicity assay was improved very easily by increasing the number of plates for the negative control test from two to four. The application of the conventional twofold rule to the data obtained with the strain TA100 was considered too conservative. The determination limit was regarded at 2.2 as the MR value under the following conditions: (a) quadruple plates were used in the negative control test; (b) three dose-steps including negative control step were designed at regular intervals; and (c) duplicate plates were used for each dose-step. It was proved by comparing data of two students that the detection limit and the determination limit estimated in this study were considered acceptable to any well trained students.

Mutagenic activities of exhaust gas and ash from sludge incineration plants.

We have found that the exhaust gas from many incineration plants of municipal solid wastes (MSW) show significant mutagenic activities. The mutagenic activities of exhaust gas from incineration plants of the other wastes have not been studied in detail. Here, we analyzed the mutagenic activities and compounds in exhaust gas and ash from seven sludge incineration plants. Some samples of the exhaust gas from the sludge incineration plants showed high mutagenic activities; although, none of the ash residues showed mutagenic activities. There was no relationship between the mutagenic activities and furnace types, the plant size or the apparent residence time of the gas in the furnace. The mutagenic activities of the exhaust gas were produced during incomplete combustion at lower temperatures. Direct mutagenic activities without S9 mix were higher than indirect mutagenic activities with S9 mix which was made from rat liver homogenate and was used to test mutagenic activity after metabolism. These results are different to those of MSW incineration plants. We analyzed the mutagenic compounds in the exhaust gas by GC/MS after fractionation by HPLC, but they could not be identified. We found that the mutagenic compounds in the exhaust gas were different from the compounds that were produced from the MSW incineration plants. We believed that these mutagenic compounds might be non-volatile and more polar than the heterocyclic amines, Trp-P-2, Trp-P-1 and Glu-P-1, which are typical mutagenic compounds in sewage and sludge.