A novel microplate-based spectrophotometric method for the quantitative assessment of freshwater bacterial coaggregation kinetics.

Coaggregation, the specific recognition and adhesion of genetically distinct bacteria, is proposed to contribute to the development of freshwater biofilms. This work aimed to develop a microplate-based system to measure and model the kinetics of freshwater bacterial coaggregation. Blastomonas natatoria 2.1 and Micrococcus luteus 2.13 were evaluated for coaggregation ability using 24-well microplates containing novel dome shaped wells (DSWs) and standard flat-bottom wells. Results were compared to a tube-based visual aggregation assay. The DSWs facilitated the reproducible detection of coaggregation via spectrophotometry and the estimation of coaggregation kinetics using a linked mathematical model. Quantitative analysis using DSWs was more sensitive than the visual tube aggregation assay and subject to substantially less variation than flat-bottom wells. Collectively these results demonstrate the utility of the DSW-based method and improve upon the current toolkit for studying freshwater bacterial coaggregation.

Organ donation from brain-dead and circulatory-dead donors: single-institution experiences.

Although the number of organ donations is extremely small in Japan, organ donation from brain dead (DBD) donors is increasing since the revised Law for Organ Transplantation was enacted on July 17, 2010. In our institution, organ donations had so far been performed from 247 donors (DCD 242, DBD 5), which is the largest number in Japan. In this study, we analyzed the status of organ donation before and after the enforcement of the revised law. After the enforcement of the revised law, the option of organ donation was shown to the more families of potential donors by the doctors or donor coordinators. However, the final number of donors was almost the same. The frequency of DBD donors of all donors increased (33.3%) as compared to 9.1% before the enforcement of the revised law. Reasons for rejection of donation from donor families were mainly based on the lack of understanding of brain death. To increase organ donation, we should promote social recognition of brain death, having the Organ Donation Card, and discussion of organ donation in each family.

Comparison of different methods for an accurate assessment of cytotoxicity in the in vitro micronucleus test. I. Theoretical aspects.

A decrease in the cytokinesis-block proliferation index (CBPI) or replication index (RI) is routinely used to determine cytotoxicity of a test compound and therefore the choice of its appropriate test concentration for the in vitro micronucleus (MN) test conducted in the presence of cytochalasin B. As a number of laboratories prefer to conduct the in vitro MN test in the absence of cytochalasin B, it is important that selected test concentrations, based on cytotoxicity, should be similar to what they would have been if cytochalasin B had been used, and should be relevant of a true cytotoxicity. By using models to analyse the dynamics of the cell cultures with and without cytochalasin B we have compared different methods for evaluation of cytotoxicity, and demonstrate that relative decrease in population doubling or relative increase in cell counts are the most appropriate measures of cytotoxicity to compare with reduction in CBPI or RI.

Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing.

This report summarizes the proceedings of the September 9-10, 2005 meeting of the Expert Working Group on Hazard Identification and Risk Assessment in Relation to In Vitro Testing, part of an initiative on genetic toxicology. The objective of the Working Group was to develop recommendations for interpretation of results from tests commonly included in regulatory genetic toxicology test batteries, and to propose an appropriate strategy for follow-up testing when positive in vitro results were obtained in these assays. The Group noted the high frequency of positive in vitro findings in the genotoxicity test batteries with agents found not to be carcinogenic and thought not to pose a carcinogenic health hazard to humans. The Group agreed that a set of consensus principles for appropriate interpretation and follow-up testing when initial in vitro tests are positive was needed. Current differences in emphasis and policy among different regulatory agencies were recognized as a basis of this need. Using a consensus process among a balanced group of recognized international authorities from industry, government, and academia, it was agreed that a strategy based on these principles should include guidance on: (1) interpretation of initial results in the "core" test battery; (2) criteria for determining when follow-up testing is needed; (3) criteria for selecting appropriate follow-up tests; (4) definition of when the evidence is sufficient to define the mode of action and the relevance to human exposure; and (5) definition of approaches to evaluate the degree of health risk under conditions of exposure of the species of concern (generally the human). A framework for addressing these issues was discussed, and a general "decision tree" was developed that included criteria for assessing the need for further testing, selecting appropriate follow-up tests, and determining a sufficient weight of evidence to attribute a level of risk and stop testing. The discussion included case studies based on actual test results that illustrated common situations encountered, and consensus opinions were developed based on group analysis of these cases. The Working Group defined circumstances in which the pattern and magnitude of positive results was such that there was very low or no concern (e.g., non-reproducible or marginal responses), and no further testing would be needed. This included a discussion of the importance of the use of historical control data. The criteria for determining when follow-up testing is needed included factors, such as evidence of reproducibility, level of cytotoxicity at which an increased DNA damage or mutation frequency is observed, relationship of results to the historical control range of values, and total weight of evidence across assays. When the initial battery is negative, further testing might be required based on information from the published literature, structure activity considerations, or the potential for significant human metabolites not generated in the test systems. Additional testing might also be needed retrospectively when increase in tumors or evidence of pre-neoplastic change is seen. When follow-up testing is needed, it should be based on knowledge about the mode of action, based on reports in the literature or learned from the nature of the responses observed in the initial tests. The initial findings, and available information about the biochemical and pharmacological nature of the agent, are generally sufficient to conclude that the responses observed are consistent with certain molecular mechanisms and inconsistent with others. Follow-up tests should be sensitive to the types of genetic damage known to be capable of inducing the response observed initially. It was recognized that genotoxic events might arise from processes other than direct reactivity with DNA, that these mechanisms may have a non-linear, or threshold, dose-response relationship, and that in such cases it may be possible to determine an exposure level below which there is negligible concern about an effect due to human exposures. When a test result is clearly positive, consideration of relevance to human health includes whether other assays for the same endpoint support the results observed, whether the mode or mechanism of action is relevant to the human, and - most importantly - whether the effect observed is likely to occur in vivo at concentrations expected as a result of human exposure. Although general principles were agreed upon, time did not permit the development of recommendations for the selection of specific tests beyond those commonly employed in initial test batteries.

An attempt to study the economic loss arising from Schistosoma japonicum infection and the benefits derived from treatment.

From the northern and southern portions of Leyte Province, which are endemic for schistosomiasis, a total of 801 infected individuals were interviewed, examined, and classified into mild, moderate, severe and very severe forms of disease with an assumed loss of working capacity for each category. The frequency rate or number of spells of illness for the past year under observation were correlated with the degree of incapacity to get the total days lost per person per year. Following a series of computations, of which the disability rate was considered as the most important, a total of 45.4 days lost per infected person per year was arrived at. Treatment of the disease with praziquantel was carried out and the patients were followed up one year after treatment, at which time the same methodology was applied. The results show that the 45.4 days lost prior to treatment went down to 4 days lost. There was an economic gain of 41.4 days as a result of treatment. This can be expressed in terms of financial value if we consider half of the infected cases as breadwinners receiving a minimum wage. It should be noted that a number of assumptions in this study were made. It is, however, hoped that this work will serve as a guide and a starting point for others to carry out related studies on economic loss and subsequent economic benefits to justify budgetary requests/allocations for the implementation of various preventive and control measures.

Simultaneous determination of nineteen hallucinogenic tryptamines/beta-calbolines and phenethylamines using gas chromatography-mass spectrometry and liquid chromatography-electrospray ionisation-mass spectrometry.

To investigate the trend of non-controlled drugs of abuse, simultaneous analytical methods were developed using GC-MS and LC-ESI-MS for 8 tryptamines/beta-carbolines, 6 phenethylamines of typically non-controlled substances in Japan, and, additionally, five legally controlled tryptamines and phenethylamines originally found in fungi or plants. Moreover, the proposed methods were applied to analyses of these drugs in 99 kinds of products (a total number of 123 products purchased at adult shops or via the Internet over the past 2 years in Japan), which potentially advertised psychotropic/psychoactive effects. The samples were extracted with methanol under ultrasonication. After centrifugation, the extracts were filtered prior to injections. GC-MS analysis was performed using a DB-5MS capillary column. Regarding the LC-ESI-MS analysis; the separation of the target drugs was optimized on an ODS column in acetonitrile/MeOH (7:3)-10 mM ammonium formate buffer (pH 3.5)/acetonitrile (95:5) by a linear gradient program and a quantitative analysis was carried out by the monitoring of each [M+H]+ in the positive ion mode of ESI-MS. As a result of the analyses using GC-MS and LC-ESI-MS, 5-MeO-DIPT (the synthetic substance known by the street name "Foxy") was found in 8 out of the 99 kinds of products. Additionally, AMT (from brown powder), DMT (from dried plant), harmine and harmaline (from dried plant) were also found in some of the 99 products. These analytical methods could be useful for the investigation of the distribution of the non-controlled psychotropic tryptamines/beta-carbolines and phenethylamines in the market.

Assessment of preferential Th1 or Th2 induction by low-molecular-weight compounds using a reverse transcription-polymerase chain reaction method: comparison of two mouse strains, C57BL/6 and BALB/c.

The aim of this study was to examine whether the RT-PCR method for various Th1/Th2 cytokines is appropriate for determination of response to allergens using C57BL/6 and Balb/c mice, which are known to preferentially demonstrate Th1 and Th2 responses, respectively. To this end, both strains of mice were sensitized by skin painting with the contact allergen dinitrochlorobenzene (DNCB) or the respiratory allergen trimellitic anhydride (TMA). We used the sensitizing protocol adopted by Kimber and coworkers (Toxicology 103, 63-73, 1995). At various time points after the last application, the levels of mRNA expression for Th1-type cytokines IFN-gamma, IL-18, and IL-12p40, as well as receptor IL-18R, and the Th2-type cytokine IL-4 and the receptor ST2L, in lymph nodes were measured. The results suggest that differential expression of IL-12p40 and IL-4 mRNA after 24 h allows clear discrimination between DNCB and TMA in C57BL/6 mice, more obviously than in Balb/c mice. Furthermore, to examine this method, C57BL/6 mice were exposed to OXA, DNFB, and TNCB (Th1-predominant allergens) or PA, TDI, and MDI (Th2-predominant allergens). Elevation of IL-12p40 expression was significant with the Th1 inducers, while the level of IL-4 was higher with Th2-predominant allergens. The results of the present study demonstrate, for the first time, that differential expression of IL-12p40 and IL-4 in C57BL/6 mice may be useful as a parameter for assessing influence of contact and respiratory allergens.

[Assessment of stage IIB lung cancer from the pathological factors].

In our experience, the prognosis of patients with pathological T3N0M0 lung cancer is generally poor, the 5-year survival rate being almost the same as that of patients with stage IIIA disease. Thus, we assessed patients with stage IIB disease by examining the pathological factors, lymphatic invasion, vessel invasion, histological type, differentiation, tumor size, and node dissection. Lymphatic invasion was found to be positive in 20 of 21 cases, patients with T3N0M0 lung cancer, and all of those with positive vessel invasion had a significantly poor prognosis. This indicates that positive lymphatic and vessel invasion could be a prognostic factor predicting a poor outcome. Patients with T3N0M0 lung cancer that are found to have this poor prognostic factor may not be diagnosed as having stage IIB disease.

Rapid immunologic diagnosis of classic late infantile neuronal ceroid lipofuscinosis.

OBJECTIVE: To establish a new method for rapid diagnosis of late infantile neuronal ceroid lipofuscinosis (LINCL, CLN2) using specific polyclonal antibodies against the CLN2 gene product. METHODS: Cells and tissues were obtained from five patients with LINCL, two with variant type NCL, three with other lysosomal storage diseases, and eight control subjects. Two antibodies were raised against N- and C-terminal peptide fragments of the normal product of the CLN2 gene. The authors examined the possibility of diagnosis of LINCL with immunostaining and immunoblotting using specific antibodies made of the recently identified defective gene in LINCL. RESULTS: Immunoreactivity with these antibodies showed the absence or marked reduction of CLN2 immunoreactivity in the lymphocytes, lymphoblasts, and fibroblasts of all five patients with LINCL examined. CONCLUSIONS: These results indicate the usefulness of this diagnostic method based on the changes in CLN2 immunoreactivity. This relatively simple, specific, and cost-effective method is a promising diagnostic tool for this disease, although additional studies are necessary.

Clinical assessment of collagen cross-linked N-telopeptides as a marker of bone metabolism in patients with primary hyperparathyroidism.

This study assesses the clinical usefulness of collagen cross-linked N-telopeptides (NTx), a new marker for bone resorption, as a marker for primary hyperparathyroidism. The study was conducted on 114 patients who had undergone parathyroidectomy for primary hyperparathyroidism. Urine NTx levels were longitudinally determined by ELISA. Longitudinal serum bone Gla protein (BGP) determinations and dual energy X-ray absorptiometry (DEXA) examinations were also performed before and after parathyroidectomy. Before parathyroidectomy, the mean urine NTx level in primary hyperparathyroidism was 149.0 +/- 116.8 pmol BCE/mumol Cr (normal: < 55 pmol BCE/mumol Cr), and the positive rate was 88.5%. The mean BGP level was 33.5 +/- 59.1 ng/mL (normal: 12.7 ng/mL), and the positive rate was 77.7%. The NTx positive rate was higher than the BGP positive rate. NTx values were significantly correlated with bone mineral density (P < 0.002) and BGP values (P < 0.005). Two weeks after parathyroidectomy the NTx levels had already decreased significantly to 65.1 pmol BCE/mumol Cr (P < 0.001), and by one month postoperatively they had fallen to 55.9 pmol BCE/mumol Cr, reaching the normal range much sooner than BGP. The NTx normalization rate two weeks postoperatively was 37%, and by 12 months postoperatively it was normal in 83% of patients. BGP levels decreased more gradually than the NTx levels, and only became normal in 9% of cases two weeks postoperatively, as opposed to 74% by 12 months. It is concluded that NTx reflects bone turnover in primary hyperthyroidism, and that it is an excellent marker for bony lesions before and after parathyroidectomy.

Health information system.

Outline of selected ones among a number of nationwide surveys related to health and welfare services (90 surveys as of 1995) conducted mainly by the Ministry of Health and Welfare periodically in Japan is described. That is, history, purpose, coverage, date, contents, data collection procedure, tabulation and publication were explained in regard to : (1) Population Census, (2) Vital Statistics, (3) Patient Survey, (4) Infectious Disease Surveillance System, (5) Comprehensive Survey of Living Conditions of People on Health and Welfare, (6) Census on Medical Care Institutions, (7) Hospital Report, (8) Survey on Physicians, Dentists and Pharmacists, (9) Statistical Report on Public Health Administration Services, (10) Survey on Social Welfare Institutions, and (11) National Nutrition Survey. On-line data service system of the Ministry of Health and Welfare is also briefly mentioned.

Assessment of right-to-left shunt flow in atrial septal defect by transesophageal color and pulsed Doppler echocardiography.

To investigate the clinical significance and problems of right-to-left (R-L) shunt flow dynamics in atrial septal defects, we performed transesophageal color and pulsed Doppler echocardiography in 30 patients with atrial septal defects of the ostium secundum type. The 30 patients consisted of 20 with a pulmonary artery systolic pressure of less than 40 mm Hg, four with a pressure of 40 to 60 mm Hg, three with a pressure of 90 mm Hg or more, two patients with pulmonic stenosis, and one patient with Ebstein's anomaly. R-L shunting was determined by the presence of a shunt flow signal across the defect during each cardiac cycle. The time of R-L shunt flow was compared with the various parameters obtained by echocardiography and cardiac catheterization. R-L shunt flow signals were detected at the following times: (1) at the onset of ventricular contraction or the closing phase of the tricuspid valve in five patients with isolated atrial septal defect. These patients showed an increase of mean right atrial pressure but had no severe pulmonary hypertension; (2) during ventricular systole in five of 26 patients with tricuspid regurgitation and one patient with Ebstein's anomaly. The tricuspid regurgitant signal was directed toward the ostium of the defect in three patients and was massive in the other patients; (3) during middiastole in three patients without pulmonary hypertension. These patients showed massive left-to-right shunt flow from end systole to early diastole; and (4) during atrial systole in three patients with severe pulmonary hypertension and two patients with pulmonic stenosis. The former, in particular, showed the aliasing signal as a high-speed shunt flow. In two of the three patients with severe pulmonary hypertension, R-L shunting continued from atrial systole to early ventricular systole and was also observed in early diastole. R-L shunt flow was detected in patients with atrial septal defects not only with pulmonary hypertension but also without pulmonary hypertension and was influenced by the right atrial pressure in the phase of tricuspid valve closing, the volume or direction of tricuspid regurgitation, rebound flow caused by massive left-to-right shunt flow, the grade of right ventricular distensibility or the complication of pulmonary hypertension, and complications with other cardiac anomalies. Thus R-L shunt flow in patients with atrial septal defects was detected easily by transesophageal color and pulsed Doppler echocardiography because of the high efficiency of this method for its detection.

[Differences in inpatient medical costs for the aged within one prefecture].

The rapid increase in the proportion of the elderly population has made the problem of medical costs increasingly important. This problem, however, is not uniform over all regions in Japan. In order to determine what factors are associated with per capita medical care expenditure of inpatients aged 70 years and over, in fiscal 1991, correlation analyses were performed using several indices of medical services, socioeconomic factors and medical supplies among 208 municipalities in one prefecture. Data from a survey in 1992 conducted by the Ministry of Health and Welfare was used in the analysis. Requests were sent to all 218 municipalities in one prefecture, and 208 responses were received a response rate of 95.4%. The main findings were as follows: 1) There were large differences in per capita medical care expenditure for those aged 70 years and over among the secondary medical areas in the prefecture. 2) Both the inpatient expenditures per person per year and bed-days in hospitals per person per year were positively correlated with number of hospital beds per 1,000, the aging population rate and death rate of those aged 70 years and over; but correlated negatively with the primary industry population rate for both sexes. 3) Inpatient expenditure per person per day was negatively correlated with the aging population rate, especially for men. 4) Three major factors that correlated significantly and positively with per capita medical care expenditure for those aged 70 years and over were bed days of inpatients per receipt, the total receipts per inpatient per year, and the number of the admission days of inpatients per year. These three major factors were negatively correlated but not significantly, with the per capita medical care expenditure per day. 5) Multiple regression analyses indicate that the aging population rate, the number of hospital beds per 1,000 of those aged 70 years and over, and death rate of those aged 70 years and over were independently correlated with the inpatient expenditure per person per year for both sexes. 6) Results of this analysis indicate that data from secondary medical regions within the prefecture rather than the municipalities yield a stronger possibility of clarifying those factors contributing to differences in per capita medical care expenditure.

Initial experiences and future directions for transgenic mouse mutation assays.

Transgenic mice have introduced new possibilities in the field of mutation research and safety testing. Using lacZ transgenic mice (Muta Mouse), we have combined the peripheral blood micronucleus assay with the transgenic mouse mutation assay, enabling the concomitant detection of gene mutations and micronucleus induction in vivo in the same animals (Suzuki et al., 1993). Several mutagens, i.e., mitomycin C (MMC), ethyl nitrosourea (ENU), ethyl methanesulfonate (EMS) and diethyl nitrosamine (DEN), were tested in this combined assay. All of them increased the lacZ mutant frequency in bone marrow or liver, and all except DEN induced micronuclei in peripheral blood. These initial studies demonstrated that genotoxicity in vivo could be detected with these two endpoints and, more importantly, that some specificity exists among these tissues analyzed. Although transgenic mouse mutation assays have many potential applications in in vivo mutation research, several problems stand in the way of wider use. Paramount among these are cost and labor intensiveness. The color screening systems for lacZ or lacI mutation detection require large numbers of plates and tedious scoring processes. In order to make significant advances in this field, it will be necessary to use positive selection for induced mutants, such as has been described recently for the lacZ and lacI transgenic mouse models.

Statistical analysis of data in mutagenicity assays: rodent micronucleus assay.

To evaluate chemical safety, many kinds of short-term mutagenicity assays are performed together with long-term assays in animals. Rationales and methodology for these assays have been well discussed and documented. No statistical method, however, has been singled out as the method of choice for the evaluation of mutagenicity assay data, although a number of reports on statistical methods to evaluate such data have been published. Among the mutagenicity assays, the micronucleus assay using mouse bone marrow erythropoietic cells have been widely used to assess cytogenetic activities of test chemicals. A statistical evaluation procedure for this assay is proposed herein, combining the use of historical control data and dose-response relationships. The probability of type I errors and the power of this method are compared with those of some other conventional methods by Monte Carlo simulation.

[Residency regulations and the situation for foreign workers in Japan]. / Regime de sejour et situation des travailleurs etrangers au Japon.

PIP: The author discusses the status of foreign laborers in Japan. The focus is on the impact of government regulations concerning labor migration, particularly denial of entry to workers without professional skills. (SUMMARY IN ENG AND SPA)^ieng

On the use of historical controls in the analysis of laboratory data.

Our purpose is to propose a method of data analysis incorporating historical controls. It consists of three steps of hypothesis testing procedure. The first step is the check of concurrent controls. The second is the significance testing of treatment groups in comparison with historical controls which are composed of negative and positive controls. The third is the significance testing of a linear trend of the dose-response relationship. The superiority of the proposed procedure is also verified.

Assessment of vascular permeability increase in the mouse by dye leakage during paw edema.

Vascular permeability increase induced by histamine, bradykinin, platelet-activating factor (PAF), or phorbol myristate acetate (PMA) in the mouse paw was assessed by the dye leakage method. The amount of dye extracted from the paw showed a clear dose-response relationship to the dose of each agonist injected into the paw. Among the autacoids used, PAF showed the most potent activity in the mouse paw. The results are consistent with those seen in the rat dorsal skin as previously reported. Involvement of histamine, 5-hydroxytryptamine and PAF is suggested in the vascular permeability increase induced by PMA in the mouse paw.