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We measured exposure to fine particles (PM2.5) and polycyclic aromatic hydrocarbons (PAHs), including carcinogenic PAHs, in multiple locations for a diverse population of participants who resided in Shizuoka, Japan. In summer and winter 2002 we surveyed personal concentrations, those of four primary indoor microenvironments-living room, bedroom, kitchen (summer only), and workplace--and those outside the subjects' houses. Concentrations of PM2.5 and PAHs tended to be higher during winter. Median PM2.5 concentration was highest in living room samples during winter but in personal samples during summer. The median PAH concentrations normalized to the cancer potency equivalence factor of benzo[a]pyrene (BaP-TEQ) was highest in the bedroom during winter but outdoors in summer. Personal exposure level profiles differed markedly between smokers and nonsmokers. Personal exposures to BaP ([BaP]p) and BaP-TEQ ([BaP-TEQ]P) in nonsmokers were strongly correlated. Personal exposures of nonsmokers, as calculated from the corresponding time-weighted indoor and outdoor concentrations, were consistent with measured levels of BaP but not PM2.5. Personal exposure of nonsmokers to BaP, as calculated from the time-weighted living room, bedroom, and either workplace or outdoor concentrations, accounted for 92-107% of the measured levels of BaP-TEQ.

Notch signaling is required for the early steps in T cell development. However, distribution of Notch ligands in the thymus anlages is not clear. We investigated the expressions of Delta like (Dll) 1 and Dll4 in the mouse thymus anlages. In the normal thymus anlages on embryonic day 13, Dll4 is strongly expressed throughout the epithelial region, but Dll1 is expressed only in scattered cells. In contrast, epithelial cells of the nude thymus anlages express neither Dll1 nor Dll4. These results indicate that expressions of Dll1 and Dll4 in thymic epithelial cells are regulated by Foxn1 transcriptional factor.

It remains controversial whether the thymus-colonizing progenitors are committed to the T cell lineage. A major problem that has impeded the characterization of thymic immigrants has been that the earliest intrathymic progenitors thus far identified do not necessarily represent the genuine thymic immigrants, because their developmental potential should have been influenced by contact with the thymic microenvironment. In the present study, we examined the developmental potential of the ontogenically earliest thymic progenitors of day 11 murine fetus. These cells reside in the surrounding mesenchymal region and have not encountered thymic epithelial components. Flow cytometric and immunohistochemical analyses demonstrated that these cells are exclusively Lin(-)c-kit(+)IL-7R(+). Limiting dilution analyses disclosed that the progenitors with T cell potential were abundant, while those with B cell potential were virtually absent in the region of day 11 thymic anlage. Clonal analyses reveled that they are restricted to T, NK, and dendritic cell lineages. Each progenitor was capable of forming a large number of precursors that may clonally accommodate highly diverse TCRbeta chains. These results provide direct evidence that the progenitors restricted to the T/NK/dendritic cell lineage selectively immigrate into the thymus.

Polycomb group (PcG) proteins play a role in the maintenance of cellular identity throughout many rounds of cell division through the regulation of gene expression. In this report we demonstrate that the loss of the PcG gene mel-18 impairs the expansion of the most immature T progenitor cells at a stage before the rearrangement of the TCR beta-chain gene in vivo and in vitro. This impairment of these T progenitors appears to be associated with increased susceptibility to cell death. We also show that the expression of Hes-1, one of the target genes of the Notch signaling pathway, is drastically down-regulated in early T progenitors isolated from mel-18(-/-) mice. In addition, mel-18(-/-) T precursors could not maintain the Hes-1 expression induced by Delta-like-1 in monolayer culture. Collectively, these data indicate that mel-18 contributes to the maintenance of the active state of the Hes-1 gene as a cellular memory system, thereby supporting the expansion of early T progenitors.

The atmospheric levels of 12 chlorinated polycyclic aromatic hydrocarbons (ClPAHs) associated with particulates at an urban site in Japan were investigated. Only 7 of the 12 species studied were detected in air samples collected monthly during 2002. 1-Chloropyrene (1-ClPy) was detected at the highest concentration (7.5 pg m(-3) (annual mean)), followed by 6-chlorobenzo[a]pyrene (6-ClBaP; 5.6 pg m(-3)) and 9,10-dichlorophenanthrene (5.1 pg m(-3)). The concentrations of the ClPAHs tended to be higher in winter than in summer, with the exception of the 6-ClBaP concentration, which was high in both summer and winter. Correlation analysis also indicated that the behavior of 6-CIBaP differed significantly from that of the other ClPAHs. Comparison of the atmospheric ClPAH concentration profile normalized to 1-CIPy concentration with that for a traffic air sample revealed significant differences between the profiles. The behavior of the atmospheric ClPAHs was also influenced by the origin of the associated particulates, which varied according to season. The positions of chlorination in the detected ClPAHs were consistent with those where the frontier electron density was high. This means that the atmospheric ClPAHs were formed by secondary reactions with chlorine atoms. The photostabilities of the ClPAHs were also investigated in laboratory experiments using a chemical model system. The ClPAHs decayed according to first-order reaction rate kinetics, with photostabilities increasing in the order 6-ClBaP < 1-ClPy < 7-ClBaA < ClPhe < ClFluor, consistent with the trend for the parent PAHs. The photolyses of chlorophenanthrenes and 7-chlorobenz[a]anthracene were confirmed to proceed by initial abstraction of chlorine, followed by oxidative degradation.

Purpose: Although intussusception has been reported as quite a rare cause of jejunoileal atresia (JIA), pediatric surgeons have noted the frequent presence of intussusception as well as volvulus at surgery. The aim of this study was to investigate the contribution of intrauterine intussusception and volvulus to the development of JIA., Methods: In 48 newborns (24 boys and 24 girls) treated for JIA at our hospital between 1978 and 2004, the operative and pathologic findings were reviewed., Results: Intussusception was responsible for gap and cord type atresia in 12 cases (25%). The cord showed an atrophic intestinal lumen in 2 cases. Volvulus was observed in 13 cases. Volvulus and intussusception were simultaneously noted in 1 case. This suggested that intussusception was the cause of the atresia, whereas volvulus was a secondary event. Neither intussusception nor volvulus was observed in high jejunal, apple peel, or multiple atresia., Conclusions: Intrauterine volvulus and intussusception were commonly observed in single mid- and low JIA. Thus, intrauterine intussusception may be a common cause of gap and cord type JIA. Volvulus may not only cause JIA but also result from anatomic changes after the development of JIA in some cases.

The occurrence of a mutagenic compound, 1-chloropyrene (Cl-Py), in extracts of ambient particulate matter at an urban site in Japan has been investigated. Samples were collected with a high-volume air sampler for 24 h periods over the course of 1 week in winter (February), spring (May), summer (August), and autumn (November) 2002. The Cl-Py levels showed seasonal variation, ranging from 2.4 pg/m(3) (summer) to 18.9 pg/m(3) (winter). This variation would indicate that the lower temperatures in winter results in an increased distribution of Cl-Py from vapor phase to the particle phase. In addition, there is also the possibility that ambient Cl-Py is emitted from seasonal sources or is susceptible to photodegradation by sunlight, or both. The photodegradation of Cl-Py in a laboratory experiment was conducted to simulate the compound's fate on airborne particle surfaces. The degradation of Cl-Py proceeded by a first-order reaction with a rate constant of 0.72 h(-1). In the presence of a radical sensitizer, 9,10-anthraquinone (AQ), the photodegradation rate of Cl-Py was elevated in comparison with the rate in the absence of AQ. In addition, the dechlorination of Cl-Py (i.e., the formation of Py) occurred in the presence of AQ.

Background/purpose: The mechanisms of intrauterine vascular disruptions that result in the development of jejunoileal atresia (JIA) are not fully understood. Monochorionic twinning with fetal death of a cotwin is known to be correlated with the development of JIA in the survivor through placental communication. The aim of this study was to evaluate whether other placental vascular compromises might contribute to the development of JIA., Methods: Forty-five newborns (23 boys and 22 girls) who were treated for JIA at Tsukuba University Hospital from 1978 to 2003 were reviewed. Placental findings were informative in 23 cases., Results: No or slight abnormality of the placenta was found in 19 cases. Significant placental abnormalities were found in 4 patients who also had a low birth weight. One patient with apple peel atresia (APA) had excessive torsion of the umbilical cord (UC), which was inserted at the margin of the placenta, and there was an adjacent area of infarction. One patent with multiple atresia (MA) was a surviving monochorionic twin with intrauterine fetal death of the other. Another case of MA showed marginal insertion of the UC. Severe placental abnormalities including wide infarction, cyst formation, and marginal insertion of the UC were found in 1 case of MA. These 3 cases of MA were complicated with other anomalies including brain anomaly., Conclusions: Placental vascular compromises were involved infrequently in JIA but might possibly be responsible for the development of JIA as well as associated anomalies and a low birth weight as chronic insults since an early stage of gestation in some cases.

The spatial distribution and concentration profiles of 39 vapor and particulate polycyclic aromatic hydrocarbons (PAHs) have been investigated in two Japanese industrial cities (Fuji and Shimizu; a summer and winter season in each). The concentrations of particulate PAHs in winter tended to be higher than those in the summer, but for vapor PAHs, this was not the case. Significant correlations (p < 0.01) were found between most of the PAH concentrations monitored in winter, suggesting the presence of common emission sources. To identify PAH spatial distributions and emission sources in the area, we created contour maps for PAHs monitored; this indicates that the distinctive local distributions correspond to the emission sources. PAH profiles based on benzo[e]pyrene (BeP) concentration, especially for certain relatively heavy molecular weight PAHs, showed differential behaviors among divided areas related to potential regional emission sources such as paper-making plants, power plants, and traffic. We conclude that the origins of atmospheric PAHs in the surveyed areas were dominated by not only traffic but also by stationary emission sources such as paper-making plants and power plants and that local distributions were dependent on the local wind direction.

A highly sensitive analytical method for the simultaneous determination of 39 gaseous and particulate polycyclic aromatic hydrocarbons (PAHs) was used to determine the PAH composition of indoor and outdoor air in Shimizu, Japan. In both indoor and outdoor air, gaseous PAH concentrations were higher in summer than in winter, whereas particulate PAH concentrations were higher in winter than in summer. Correlation analysis indicated that indoor PAH compositions, especially the gaseous PAH composition, differed significantly from outdoor PAH compositions. Gaseous PAH concentrations indoors were significantly affected by insect repellents and heating sources. Particulate PAH concentrations indoors were significantly affected by cigarette smoking, the age and type (wood) of the house, and outdoor PAH concentrations. Inhalation risk associated with carcinogenic PAHs was estimated by using toxic equivalency factors based on the potency of benzo[a]pyrene (BaP). The carcinogenicity of the indoor PAH mixture was dominated by naphthalene followed by BaP and dibenz[a,h]anthracene.

Development of a mature T-cell repertoire in the thymus depends on lympho-stromal interaction between thymocytes and stromal cells. To facilitate intercellular contact, the epithelium in the thymus has differentiated into a unique three-dimensionally (3D)-oriented network. Here we analyze factors influencing induction and maintenance of the 3D configuration of the epithelial network in fetal thymic lobes in vitro. We show that the 3D configuration of the thymic stroma depends on (a) the oxygen pressure in vitro and (b) permanent physical contact between stromal cells and developing thymocytes. This latter feature is demonstrated by incubation of fetal thymic lobes with deoxyguanosine (d-Guo), inducing a 2D-organized thymic stroma, with thymic cysts appearing. Reconstitution of d-Guo-treated lobes with a limited number of flow-sorted T-cell progenitors restores the 3D configuration of the thymic epithelium, but only at high oxygen pressure. This study underlines the plasticity of thymic epithelium and shows that the unique organization of the thymic epithelium is dependent on both oxygen and crosstalk signals derived from developing thymocytes.

The functions of a chemokine CXC chemokine ligand (CXCL) 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its physiologic receptor CXCR4 in T cell development are controversial. In this study, we have genetically further characterized their roles in fetal and adult T cell development using mutant and chimeric mice. In CXCL12(-/-) or CXCR4(-/-) embryos on a C57BL/6 background, accumulation of T cell progenitors in the outer mesenchymal layer of the thymus anlage during initial colonization of the fetal thymus was comparable with that seen in wild-type embryos. However, the expansion of CD3(-)CD4(-)CD8(-) triple-negative T cell precursors at the CD44(-)CD25(+) and CD44(-)CD25(-) stages, and CD4(+)CD8(+) double-positive thymocytes was affected during embryogenesis in these mutants. In radiation chimeras competitively repopulated with CXCR4(-/-) fetal liver cells, the reduction in donor-derived thymocytes compared with wild-type chimeras was much more severe than the reduction in donor-derived myeloid lineage cells in bone marrow. Triple negative CD44(+)CD25(+) T cell precursors exhibited survival response to CXCL12 in the presence of stem cell factor as well as migratory response to CXCL12. Thus, it may be that CXCL12 and CXCR4 are involved in the expansion of T cell precursors in both fetal and adult thymus in vivo. Finally, enforced expression of bcl-2 did not rescue impaired T cell development in CXCR4(-/-) embryos or impaired reconstitution of CXCR4(-/-) thymocytes in competitively repopulated mice, suggesting that defects in T cell development caused by CXCR4 mutation are not caused by reduced expression of bcl-2.

The purpose of this study was to compare the tensile bond strengths (TBSs) and failure mode of 4-META/MMA-TBB resin to 60 degrees C-heated and unheated bovine dentin, especially to investigate the influence of ferric chloride contained in citric acid pre-conditioning. In addition, the effect of HEMA priming for heated dentin was also evaluated. The TBSs to heated dentin were significantly lower than those to unheated dentin. Adhesive failures were observed in most specimens of the heated and HEMA-non primed group. HEMA application to heated dentin significantly increased the TBSs in each acid conditioning, which were also significantly higher than those of the unheated and ferric chloride-contained citric acid-conditioned group. It was clarified that heating dentin decreased the bond strength without HEMA priming even if the dentin surfaces were acid conditioned with 10-3, while HEMA priming after acid conditioning recovered the bond strength.

1. The accuracy of measurements of flow rate and concentrations of O 2 and CO 2 in expiratory gas by indirect calorimetry (IC) using paediatric and adult circuits were assessed by mass spectrometry and a pneumotachogram, which have been proven as the most reliable instruments for these purposes. 2. In the paediatric circuit, all measurements were demonstrated to be reliable: error rates for flow rate, O 2 concentration and CO 2 concentration were +3.13, +2.66 and -6.63%, respectively. All were within the 10%, which is acceptable as a biological error. 3. However, in the adult circuit of IC, all measurements were reliable except for measurements of CO 2 concentration: error rates for flow rate, O 2 concentration and CO 2 concentration were +2.82, +1.64 and +11.42%, respectively. 4. A fluctuation phenomenon of expiratory gas concentration was observed only in IC. Mass spectrometry did not show this phenomenon. This phenomenon seems to be derived from the IC machine itself.

1. Biliary atresia (BA), as a common disease in Japan, and cystic fibrosis (CF), as an extremely uncommon disease in Japan, were selected to assess the clinical significance of measurement of energy expenditure (EE). 2. Energy expenditure was significantly higher in children with BA than in normal children. 3. Measurement of EE in BA lead to clues to resolving its mechanism by novel assessment of interleukin-6 and leptin. 4. Energy expenditure in children with CF is also higher, but this has been addressed by nutritional intervention with additional calories. 5. Individualization of EE measurement is necessary in the analysis of pathological mechanisms and nutritional management of patients with both common and uncommon diseases.

The purpose of this study was to evaluate the priming effect of 2-hydroxyethylmetaclirate (HEMA) following acid treatment on resin bonding to prototype Er:YAG laser-irradiated dentine. Extracted bovine dentine following laser irradiation was acid treated by aqueous solution of 10% citric acid (10-0) or 10% citric acid/3% ferric chloride (10-3), and additionally treated with 35% HEMA. Pre-treated dentines were bonded to the polymethyl-methacrylate (PMMA) rod with 4-META/MMA-TBB resin (Super Bond C & B) and miniaturized dumbbell-shaped bonded specimens were prepared. These specimens profiled for tensile bond testing and fractured surfaces were observed by scanning electron microscopy (SEM). Cross-sections of resin-dentine interface were also examined. The HEMA treatment following acid conditioned by 10-3 or 10-0 for both laser-irradiated and non-irradiated dentines was significantly higher than that without HEMA treatment. SEM view of a fractured specimen showed some cohesive failure in cured resin, but almost all of the fractured surface shows boundary failure between the penetrated resin and underlying dentine. A cross-sectional view of the interface showed a very thick hybrid layer between the hybridized dentine and underlying dentine. It was concluded that HEMA treatment following acid conditioning provided a slightly higher bond strength for both the Er:YAG laser-irradiated and non-irradiated dentines. However, the bond strength of Er:YAG laser irradiated dentine was significantly lower than that of the non-irradiated dentine.

In this study, the authors assessed air quality and health effects of the 1997 haze disaster in Indonesia. The authors measured carbon monoxide, carbon dioxide, sulfur dioxide, nitrogen dioxide, ozone, particulate matter with diameters less than or equal to 10 microm, inorganic ions, and polycyclic aromatic hydrocarbons. The authors also interviewed 543 people and conducted lung-function tests and determined spirometric values for these individuals. Concentrations of carbon monoxide and particulate matter with diameters less than or equal to 10 microm reached "very unhealthy" and "hazardous" levels, as defined by the Pollution Standards Index. Concentrations of the polycyclic aromatic hydrocarbons were 6-14 times higher than levels in the unaffected area. More than 90% of the respondents had respiratory symptoms, and elderly individuals suffered a serious deterioration of overall health. In multivariate analysis, the authors determined that gender, history of asthma, and frequency of wearing a mask were associated with severity of respiratory problems. The results of our study demonstrate the need for special care of the elderly and for care of those with a history of asthma. In addition, the use of a proper mask may afford protection.

An analytical method was established for the determination of benzene and 13 of its alkyl derivatives. The method was applied to a survey of indoor pollution that investigated the usefulness of the method, concentration levels, seasonal variations, profiles, correlations between compounds, and factors that affected indoor pollution by these compounds. The survey was performed in 21 houses in the summer of 1999 and 20 houses in the winter of 1999-2000 in Fuji, Japan. All the target compounds were detected in the indoor and outdoor air of all houses. Outdoor concentrations of benzene ranged from 0.779 to 3.17 microg/m3 in summer and from 1.35 to 6.04 microg/m3 in winter, whereas indoor concentrations of benzene ranged from 0.694 to 3.11 microg/m3 in summer and from 1.65 to 6.89 microg/m3 in winter. Indoor concentrations of the target compounds, except for benzene, were elevated, compared with outdoor concentrations. Because indoor and outdoor concentrations of benzene and its derivatives in summer were lower than in winter, the emission of these compounds may be increased by use of a heater and other variables present in winter. Profiles of the compounds, correlations between the compounds, and factors that affected indoor pollution (determined by multiple regression analysis) were investigated. These results suggested that indoor benzene predominantly penetrated from outdoors and that other benzene derivatives were emitted from indoor sources, such as paint solvents and kerosene heaters.

An analytical method was established for the simultaneous determination of 39 polycyclic aromatic hydrocarbons (PAHs) in air. The method was applied to a survey of gaseous and particulate PAHs in household indoor air. The survey was performed in 21 houses in the summer of 1999 and in 20 houses in the winter of 1999-2000 in Fuji, Japan. Thirty-eight PAHs were determined in indoor and outdoor air in the summer, and 39 PAHs were determined in indoor and outdoor air in the winter. The concentrations of gaseous PAHs in indoor air tended to be higher than those in outdoor air in the summer and winter. The concentrations of particulate PAHs in indoor air were the same as or lower than those in outdoor air in the summer and winter. PAH profiles, correlations between PAH concentrations, and multiple regression analysis were used to determine the factors affecting the indoor PAH concentrations. These results showed that gaseous PAHs in indoor air were primarily from indoor emission sources, especially during the summer, and that indoor particulate PAH concentrations were significantly influenced by outdoor air pollution.

Background: The trimethadione (TMO) tolerance test was performed to evaluate its usefulness in the assessment of hepatic functional reserve in patients with biliary atresia., Method: Nineteen patients with biliary atresia after hepatic portoenterostomy (age range: 2 months to 25 years; sex: 6 males and 13 females) were studied. The study was performed in the morning after a 12-h fast. TMO was given orally, at a dose of 4 mg/kg, with 5 mL of 5% glucose 2 h before breakfast. Blood samples (0.5 mL) were collected to determine serum TMO and dimethadione (DMO), a metabolite of TMO, levels 4 h after the administration of TMO. TMO and DMO were measured by a gas-liquid chromatographic method., Results: A higher total bilirubin level (over 1 mg/dL) in patients with jaundice was reflected in the smaller serum DMO/TMO ratio 4 h after the oral administration of TMO. In addition, these patients with total bilirubin levels of 1 mg/dL or less had a significantly lower DMO/TMO ratio than the control group (healthy subjects). The serum DMO/TMO ratio showed a close correlation with the Child-Pugh score, which is used for overall evaluation of severity of cirrhosis and Mayo risk scores for primary biliary cirrhosis in adults (0.856, P < 0.01 and 0.788, P < 0.01, respectively). The TMO tolerance test shows the benefit of performing a relatively early test of dynamic liver function to evaluate hepatic functional reserve in pre- and post-operative biliary atresia patients.

Thymic epithelial cells, which create a three-dimensionally organized meshwork structure peculiar to the thymus, develop from simple epithelia of the third pharyngeal pouch and cleft during organogenesis. We comparatively investigated the thymus anlages of normal and nude mice by immunohistochemical analysis with regard to epithelial organization and distribution of hematopoietic progenitor cells at early stages of organogenesis. Our results show that development of the mouse thymus anlage at early stages can be subdivided into at least two stages by the differences in epithelial organization, i.e. stratified epithelial stage on embryonic day (Ed) 11 and clustered epithelial stage on Ed12. At the former stage, hematopoietic progenitor cells are accumulated in the mesenchymal layer of the thymus anlage, and at the latter stage progenitor cells enter the epithelial cluster and proliferate. In nude mice, hematopoietic progenitor cells are found in the mesenchymal layer on Ed11.5, but they are not observed among epithelial cells on Ed12, even though epithelial cells form a cluster structure. The present results suggest that aberrant development of the nude mouse thymus anlage occurs at the clustered epithelial stage and that epithelial cells of the nude anlage lack the ability to induce the entrance of hematopoietic progenitor cells into the epithelial cluster.

A case of carcinosarcoma of the urinary bladder in a 2-year-old girl is reported. The tumor, measuring 34 x 20 x 18 mm, was located in the peri-trigone area of the urinary bladder with polypoid features. Histologic examination revealed transitional cell carcinoma at the tumor surface with downward invasion. Concurrently, a sarcomatous area was found beneath the carcinoma, with these two different malignant components sharing on apparent transition without distinct boundaries. Sarcomatous components included immature round cells focally showing rhabdoid features. No rhabdomyomatous component was observed. Immunohistochemistry disclosed vimentin and cytokeratin-double positive cells at the transposition between carcinoma and sarcomatous components. In addition, ultrastructural analysis revealed that the epithelial cells had a distinct junctional complex, and the sarcomatous cells occasionally had a meshwork of cytoplasmic intermediate filaments, indicating bidirectional cytodifferentiation to epithelial and mesenchymal elements. The extremely young age at which this case of carcinosarcoma occurred suggests that the tumor may be of mesodermal stem cell origin.

A simple and reliable method was developed for analysis of 18 volatile organohalogen compounds (VOHCs) both indoors and outdoors, consisting of VOHC collection by a passive sampler, extraction with toluene by mechanical shaking, and automatic separation analysis by capillary gas-chromatography with electron capture detector (GC/ECD). The passive sampler is a porous polytetrafluoroethylene (PTFE) tube (30.30+/-0.37 mm net collection length, 5.0 mm inside diameter, 0.990 g weight) uniformly packed with activated charcoal (194.4+/-3.8 mg). The procedure was applied to a field survey on indoor and outdoor VOHC pollution in Shizuoka, Japan. Ten VOHCs, including trichloroethylene, tetrachloroethylene, chloroform, carbon tetrachloride, and p-dichlorobenzene, were detected from indoor and outdoor air samples. The ratios of maximum to minimum VOHC concentrations, both outdoors and indoors, were large. The indoor and outdoor concentrations of 1,1-dichloroethylene, dichloromethane, 1,1,1-trichloroethylene, carbon tetrachloride and trichloroethylene were found to be similar. Indoor concentrations of trihalomethanes, p-dichlorobenzene and tetrachloroethylene were higher than those of outdoors.

The authors propose a new surgical approach for neonates and infants with hydrometrocolpos caused by double vagina and imperforate hemivagina. Usually for these patients, laparotomy is a common approach used to decompress the obstructed hemivagina. The authors compared the characteristics of three options used to relieve obstruction of the hemivagina using endoscopic, transvaginal, and laparotomy approaches. An endoscopic septotomy (colposcopic approach) using the Storz neonatal resectoscope is less invasive and less expensive than other methods. We emphasize that endoscopic septotomy is a feasible surgical method to relieve the obstructive symptoms related to imperforate hemivagina.

Lympho-stromal interactions in the thymus crucially de- termine the fate of developing T cells. Epithelial cells, inter- digitating reticular cells, macrophages and fibroblasts all play a role in the shaping of the T cell repertoire. Recently published evidence shows that lympho-stromal interaction acts bi-directional. Developing T cell themselves, at different stages of differentiation, control the microarchitecture of thymic microenvironments, a phenomenon designated as 'crosstalk'. This paper reviews experiments showing that developing T cells crosstalk to different thymic epithelial cells in a stepwise fashion. In this way, correctly organized thymic microenvironments guarantee normal thymopoiesis., (Copyright 1999 Academic Press.)

Previously, we have shown that embryonic day 12 thymus anlage cultured alone cannot develop into the mature organ but degenerates. In the present study, we investigated the cause of this insufficient organogenesis of embryonic day 12 thymus anlage in organ culture. We cocultured embryonic day 12 thymus anlages with various cell lines as pellets formed by centrifugation. In coculture with fibroblastic cell lines, but not with thymic epithelial cell lines, embryonic day 12 thymus anlages developed to support full T cell differentiation, and expressed mature stromal cell markers, Ia and Kb. By pellet culture of thymus anlages and fibroblastic cell lines transfected with a beta-galactosidase expression vector, we analyzed the distribution of added fibroblastic cells in pellets. The added fibroblastic cells constituted neither thymic capsule nor septa but disappeared after about 2 weeks in culture. Moreover, immunohistochemical studies indicated that added fibroblastic cells were adjacent to mesenchymal cells of thymus anlage. Our results strongly suggest that added fibroblastic cells support the development of the thymus anlage through interaction with its mesenchymal cells.

Using SCID-hu mice, it was tested whether humanized mAb Rmu5.5 could prevent infection by HIV-1 i.v. inoculation. The Ab that recognizes the IHIGPGRAFYT motif in the principal neutralizing determinant (PND) of HIV(MN), as well as the original mouse mAb mu5.5, neutralized HIV(MN) with high activity. Seven primary field isolates from Japanese hemophiliacs seropositive for HIV-1 clade B were compared for their reactivities to Rmu5.5. Rmu5.5 was effective, particularly against the viruses that matched amino acid sequences of the PND region of HIV-1, and it completely neutralized primary isolates. Moreover, the passive transfer of the Ab elicited protection against challenge by the primary isolates in SCID-hu or hu-PBL-SCID mice after i.v. inoculation with the virus by both quantitative PCR and PBMC-based virus isolation in vitro. Further, inoculation with the Ab also prevented the atrophic change in the medulla of the thymic transplant that was induced by i.v. inoculation of the virus. Thus, the humanized neutralizing Ab Rmu5.5 appears to protect SCID-hu mice from infection by primary field isolates.

A method for the quantitation of polycyclic aromatic hydrocarbons (PAHs) in indoor and outdoor air by high-performance liquid chromatography (HPLC) with a spectrofluorometric detection and programmed excitation and emission wavelength pairs is proposed. The mobile phase is a linear gradient of methanol-water. The relative standard deviations (n = 5) are in the range 0.38-1.7% at concentration levels of 0.69-11.40 ng ml(-1). The determination limits (S N = 10 ) are 0.5-15.9 pg. The proposed method was successfully applied to quantitate 12 PAHs in gas phase and particulates in indoor and outdoor air. The recoveries of PAHs from gas phase and particulates were 95.7-117.5 and 94.8-112.4%, respectively. This highly sensitive automatic HPLC analysis for PAHs both in gas phase and particulates can be applied to indoor and outdoor survey.

Human fetal thymus/liver engrafted SCID mice were constructed and studied for its susceptibility to HIVBRU infection by i.v. inoculation which seemed to represent an appropriate route of HIV infection in vivo. By the i.v. inoculation of HIV, the medulla in the engrafted thymus narrowed significantly when compared with that of the human thymic implant from virus-uninoculated mice. Further, immunohistochemical staining indicated the presence of HIV antigen predominantly in thymic epithelial cells in medulla of the engrafted thymus. Polymerase chain reaction (PCR) assays resulted in amplifications of HIV genome in the implanted grafts as well as in lymph nodes and PBMC. The virus infections to the implants were confirmed biologically by coculturing with PHA-stimulated human PBMC and the graft cells from the HIV-inoculated SCID-hu mice. Thus, the i.v. inoculation of HIV into Thy/Liv SCID-hu mice induce narrowing of medulla of the engrafted thymus and may become an efficient and useful tool for screening candidate anti-HIV agents.

Oxytocin-like and vasopressin-like immunoreactive cells, and the cells expressing mRNAs for these peptides in the spleen of the C57BL/6 mouse were studied by immunohistochemistry, immuno-electron microscopy and in situ hybridization. Immunoreactive cells were distributed mainly in the splenic cord and marginal zone, whereas there were few in the lymphocyte-packed periarteriolar-lymphoid sheath, lymphoid follicle and germinal center. More numerous vasopressin-positive cells were seen in the splenic cord. The colocalization of oxytocin-like and vasopressin-like immunoreactivity in the same cells was identified by the investigation of mirror sections. By the pre-embedding immuno-electron-microscopic method using antisera against oxytocin and vasopressin, immunopositive reaction products were localized in the matrix around the specific granules, small clear vesicles and mitochondrial membrane of the eosinophils. No immunoreactivity to these peptides was found within the specific granules of the eosinophils. In situ hybridization with synthetic oligonucleotide probes labeled with 32P revealed the presence of mRNAs for oxytocin and vasopressin in the cells of the spleen, the distribution of the mRNAs for these peptides being the same as that of immunopositive cells. These observations suggest that eosinophils synthesize both oxytocin and vasopressin and store them in the matrix. Possible differences in the mechanism of synthesis and storage of these peptides between peripheral eosinophils and hypothalamic neurons are discussed.

Previous studies have demonstrated that murine thymus separates from the pharynx during 11.5-12 days of gestation, and that the proliferation of thymic cells starts at this age. We characterized embryonic day 12 thymus in terms of the surface phenotype of the thymus cells, the function of the lobe in supporting T cell development in organ culture, and the precursor activity of the thymus cells in a mixed culture with deoxyguanosine-treated lobes. The phenotype of the major population of embryonic day 12 thymus cells was HSA+, CD44+, c-kit+, Thy-1-, CD25-, CD4-, CD8-, TcR-, and Sca-1-. In organ culture of embryonic day 12 thymus lobes, most of the lobes did not develop well and failed to generate CD4+CD8+, CD4+CD8-, or CD4-CD8+ cells, even when embryonic day 14 thymus cells were added. However, thymus cells on embryonic day 12 contained T cell precursors that developed into mature T cells in co-culture with deoxyguanosine-treated fetal thymic lobes. The majority of the stromal cells in deoxyguanosine-treated embryonic day 14 thymus lobes expressed the surface molecules I-A and H-2D, whereas these cells in embryonic day 12 thymus lobes were negative for these surface molecules. Thus, our findings suggest that the embryonic day 12 thymus lobe contains T cell precursors, but that the undeveloped thymic stromal cells are insufficient to support full T cell development.

Recently antigenic heterogeneity in human Pneumocystis carinii (Pc) isolates was observed in several laboratories. Monoclonal antibodies (MAb) were produced to human Pc (PcH) from a lung autopsy sample from a non-AIDS patient (MAb Group I, n = 10), or from bronchoalveolar lavage (BAL) fluid from AIDS patients (MAb Group II, n = 8). To detect Pc antigen from specimens, indirect immunofluorescence and immunoblotting techniques were used. The reactivity was evaluated by using one autopsy sample from the non-AIDS patient and 14 BAL samples from AIDS patients. The MAb in group I (C5-9, E9) stained a part of PcH from all isolates. On the other hand, several MAb in group II (L20-5, M34-2, M78-3, M79-5, N23-4) stained all PcH from all isolates. Some MAb (C5-9, E9, M34-2, M78-3) stained cysts as well as trophozoites. Immunoblot studies detected a 92 kDa molecule as a common antigen by all of these MAb. Therefore, we have found a common antigenic epitope on PcH and MAb that recognize this epitope may become useful for diagnosis of infection and for biological characterization studies on the organism.

Pneumocystis pneumonia is the most serious opportunistic infection in immunocompromised patients, particularly those with AIDS. Approved therapy is limited to pentamidine and inhibitors of folic acid synthesis, but these drugs show a high rate of adverse reactions in AIDS patients emphasizing the urgent need for additional effective therapies. Progress has, however, been hindered by lack of knowledge about this parasite's cellular characteristics. Previously we reported that beta (1,3)glucan is a major component of the Pneumocystis carinii cyst wall. This study shows that administration of aculeacin A, an inhibitor of beta (1,3)glucan biosynthesis, affects cyst wall formation, inhibits cyst maturation, and prevents severe pneumonia in steroid-treated rats. Thus this study not only demonstrates that beta (1,3)glucan is indispensable for growth of the parasite in rats, but suggests a new therapeutic strategy for human pneumocystosis.

We examined the effects of cyclosporin A (CsA) administered in vivo on the capacity of peripheral blood mononuclear cells (PBMC) from kidney transplant recipients to express IL-2 receptor (IL-2R) gene at the level of mRNA after mitogen stimulation in vitro. There were no differences in the percentage of IL-2R+ cells among the groups of normal individuals, azathioprine-prednisolone treated, and CsA-prednisolone-treated recipients, using FITC-labelled monoclonal anti-IL-2R antibody (anti-alpha chain): 40.3 +/- 10.1% and 62.8 +/- 11.1% of normal PBMC (n = 18), 37.0 +/- 9.3% and 61.7 +/- 5.8% of PBMC from azathioprine-prednisolone-treated recipients (n = 20), and 37.7 +/- 9.6% and 60.7 +/- 12.7% of PBMC from CsA-prednisolone-treated recipients (n = 20) expressed IL-2R after 24 h and 48 h of phytohaemagglutinin stimulation, respectively. However, in a study of Northern blotting using cDNA for IL-2R (anti-alpha chain specific), both the 3500 and 1400 bp families of IL-2R mRNA were remarkably decreased in PBMC from CsA-prednisolone-treated recipients compared with azathioprine-prednisolone-treated recipients and normal individuals. These studies demonstrated that CsA could inhibit IL-2R gene expression at the level of mRNA at physiological concentration.

Schwann cell cultures prepared from murine sciatic nerves (MuSN), rat sciatic nerves (RSN) and murine dorsal root ganglia (MuDRG) were transformed by the introduction of oncogenes (v-arc, c-Ha-ras, and v-mos). The transformation was carried out by infection with helper-free retroviruses containing the oncogene and the neomycin-resistant gene which were produced in psi-2 cells. After G418 selection and cloning of cells showing spindle shape morphology, six different transformed cell clones, i.e., MuSN-arc, MuDRG-arc, MuDRG-ras, MuDRG-mos, RSN-arc, and RSN-mos, were established. All of the clones were labeled with antibodies to the S-100 protein and the laminin, which are specific markers for Schwann cells. Fibronectin and vimentin were not detected in these cell clones, in contrast to fibroblasts as 3T3 and Rat-2. These cell clones have been maintained with characteristics of Schwann cells for over 18 months. When RSN-mos, one of the Schwann cell clones, and rat retinas were cocultured direct cellular interaction was demonstrated. Furthermore, by the addition of conditioned medium of the RSN-mos, a prominent activity promoting neurite outgrowth in PC12 pheochromocytoma cells was observed.

The monoclonal antibody A7 (MoAb A7), which belongs to IgG1, was digested with pepsin to yield F(ab')2 fragments. The maximum binding to the human colon cancer cell line, SW1116, was 27% with 125-I labeled whole MoAb A7 and 24% with 125-I labeled F(ab')2 fragments using an in vitro binding assay. The results showed that the binding activity of F(ab')2 to SW1116 was practically the same as that of whole MoAb A7. The preferred localization of the fragments to tumor tissue, compared with normal mouse tissue, was demonstrated in mice carrying SW1116 xenografts. The tumor:blood ratio three days after injection was 2.64:18.5 for whole MoAb A7:F(ab')2, respectively. The tissue:blood ratios for the F(ab')2 fragments showed a value of 18.5 in tumors, whereas its was a value less than 1.0 in normal organs. The tumor accumulation of F(ab')2 fragments was also dependent on the antigenic expression of each tumor among xenografts of colon carcinoma SW1116 and WiDr, and squamous cell carcinoma KB. In kinetic experiments with whole MoAb A7 and its F(ab')2 fragments, whole MoAb A7 was lost, with a half-life of 4 days, in both blood and tumors, whereas F(ab')2 fragments were rapidly lost with a half-life of 1.5 days. These results suggested that the F(ab')2 fragments were cleared from the blood faster than was whole MoAb A7.

T cell dependency of antibody response to polyvinylpyrrolidone (PVP), sheep red blood cells (SRBC), bovine gamma globulin (BGG), and bovine serum albumin (BSA) was examined. PVP and the other three are known as a T cell-independent antigen and T cell-dependent antigens respectively. Adult mice were thymectomized, X-irradiated, reconstituted with syngeneic bone marrow cells (TxXB mice), with bone marrow cells plus thymus cells (TxXBT mice), or with bone marrow cells treated with anti-Thy-1.2 serum and complement (TxXB-theta mice) and used as experimental animals. The anti-PVP response of TxXBT mice was significantly lower than that of TxXB mice, suggesting that T cells exerted a suppressive effect on the response to PVP. Both IgM and IgG responses to SRBC and BGG occurred even in TxXB-theta mice with the aid of bacterial lipopolysaccharide (LPS). However, a significant response to BSA was not observed in TxXB mice even in the presence of LPS or several other adjuvants. These results indicate that the T cell dependency of antigens is different among so called thymus-dependent antigens, that antibody response less dependent on the helper action of T cells can be supported by LPS in the absence of T cells, and that anti-BSA response seems to be extremely T cell dependent.

The nature of the antigen recognized by the murine monoclonal antibody A7 (Mab A7) against human colorectal carcinoma was investigated using immunochemical and biochemical techniques. Binding activity of 125I-labeled Mab A7 was examined using various human cancer cell lines. Mab A7 gave the highly specific binding to colon cancer cell lines, SW1116 and WiDr, and gave only a very weak or no reactivity to gastric cancer cell lines, pancreas cell lines or lung cancer cell lines. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of the extractable antigen from SW1116 showed a single band at approximately 45,000 dalton formed by 125I-labeled Mab A7. Treatment of SW1116 with sodium periodate, pronase and ficin resulted in the loss of antigenic activity. These data strongly suggest that the antigen recognized by Mab A7 is composed of glycoprotein. Competitive binding analysis to the surface of the colon cancer cell line using polyclonal anti-CEA and Mab A7 as well as immunoblotting analysis using monoclonal anti-CEA and Mab A7 suggested that the antigen recognized by Mab A7 was different from CEA. Moreover, this antigen was also found in surgical specimens of colorectal cancer patients and its molecular property was identical to the antigen extracted from SW1116.

We investigated the effects of local administration of interferon (IFN) on 13 patients with recurrent brain tumors. Histologic diagnoses were glioblastoma (eight patients), medulloblastoma (one), ependymoma (one), ependymoblastoma (one), pontine glioma (one), and astrocytoma (one). When tumor recurrence was evident local administration of IFN was started through an Ommaya reservoir, which was placed during repeat craniotomy. No tumor regressions were seen in the patients given weekly injections of IFN; however, in two of six patients given daily injections, a decrease of tumor volume and augmentation of natural killer activity were seen.

The present study demonstrates that human peripheral blood mononuclear cells (PMC) can be stimulated in vitro to become cytotoxic T lymphocytes (CTL) by PHA. A significant cytotoxic activity of PMC was detected 48 hr after the culture initiation in the presence of 5 micrograms/ml of PHA and the peak level of the activity was obtained by culturing PMC for 72 hr. The cytotoxic cells require the presence of PHA as a cell agglutinin for the expression of their cytotoxic activity. The effector cells mediating the activity were identified as T lymphocytes by E-rosette fractionation of PMC. In this system, removal of carbonyl iron phagocytosed or attached cells from PMC did not abrogate CTL generation of PMC. In addition, human alpha-interferon did not augment CTL generation or expression of their activity. Although the target cells employed were sensitive to natural killer (NK) cells, the effector cells induced by PHA did not seem to have any relation to the NK cells. The present study may provide a useful tool to analyze for precursors of killer T cells.

Graft rejection is one of the major obstacles to successful bone marrow transplantation (BMT). If resistance to marrow grafting could be avoided, BMT could be used widely in treatment of hematological and immunological disorders. There has been evidence that natural killer (NK) cells play a major role in genetic resistance to BMT and that macrophages are also involved in genetic resistance. Agents toxic to macrophages such as silica and carrageenan have been found to have a suppressive effect on genetic resistance to BMT. Parenteral fat emulsions are known to accumulate in macrophages and to impair various functions of macrophages and those of the reticuloendothelial system. We show here that the administration of a fat emulsion, Intralipos 20%, to recipient mice can suppress genetic resistance to bone marrow grafts and NK cell activity probably through the impairment of the macrophage function. The administration of the fat emulsion might be a new tactic in conditioning protocols for human BMT in the future.

One hundred and eighteen patients with 169 bladder tumors were treated transurethrally by Nd:YAG laser surgery. In relation to the tumor staging, 79 of 103 patients of T1 (77%) underwent laser operation only, but most of the T2 cases (87%) required laser therapy with electroresection. With regard to the tumor size, 93 of the 97 small tumors (96%) were treated with laser irradiation alone, and 25 of 44 middle-sized tumors (57%) were eradicated by laser therapy, During and after the operation we never experience severe complications such as perforation of either the bladder or the intestine. Uncontrollable bleeding did appear in four cases. Tumor recurrence occurred in 25% for one year and 50% for three years. Therefore, laser surgery was limited to T1 tumor. But the indication of laser surgery would be magnified with the progress of the instrument and the technique.

Mice of strain MRL/Mp-lpr/lpr develop resistance against tolerance induction with aggregate-freed rabbit gamma-globulin as early as 6 weeks of age and show very complete resistance when they are 10 weeks old; congenic mice MRL-Mp+/+ of 6 and 10 weeks can be rendered tolerant. By reconstitution experiments, it was shown that T cells from MRL/Mp-lpr/lpr are responsible for the resistance to tolerance induction. A cell which is not colchicine-sensitive contributes to tolerance of MRL/Mp-+/+, but not to MRL/Mp-lpr/lpr mice. Resistance of T cells against tolerance depends on expression of the lpr gene.

Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells, the generation of donor-derived T cells being observed in two out of 14 recipients transferred with as few as 1.5 X 10(4) cells. The stem cell activity of spleen cells was estimated to be about 1% of that of bone marrow cells, and no activity was found in thymus cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. Spleen cells showed a markedly high level of activity 7 days after the reconstitution, followed by a decline, whereas the activity of bone marrow cells was very low on day 7 and increased crosswise. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells. Such patterns of compartmentalization of stem cells in the spleen and bone marrow of irradiated recipients completely conform to the general scheme of the relationship between restricted stem cells and less mature stem cells, including pluripotent stem cells, which became evident in other systems such as in the differentiation of spleen colony-forming cells or of stem cells for B cell lineage.

Mice depleted of T cells by adult thymectomy, X-irradiation and reconstitution with syngeneic bone marrow cells, either untreated or treated with anti-Thy-1 serum and complement, were immunized intensively with alum-precipitated bovine serum albumin (AP-BSA) along with or without bacterial lipopolysaccharide (LPS), but no significant anti-BSA antibody response was detected. Priming of the T-cell depleted mice, however, either by a single injection of AP-BSA plus LPS or by multiple injections of AP-BSA without LPS, resulted in the generation of immunological memory. A single injection of AP-BSA without LPS was ineffective. The memory required the aid of syngeneic T cells to be recalled by the challenge with AP-BSA plus LPS. On the other hand, multiple injections of AP-BSA plus LPS did not cause the generation of memory and the response of these mice to the challenge was lower than that of unprimed control mice. These results suggest that (1) the anti-BSA response is highly dependent on the helper function of T cells, (2) the degree of T-cell requirement for the memory generation is very low, and (3) priming with too much strong stimulation in the absence of functional T cells leads to the suppression or abortion of previously generated immunological memory.

Age-dependent changes in immune response and in tolerance occur at different rates in inbred mice of different genetic backgrounds. The change in tolerizability is connected with the change in the balance of the suppressor helper circuit; the change in B-cells, at least in C57BL/6 mice, appears to be also due to a regulatory change. Changes in immune responsiveness and tolerance inducibility appear to vary independently of one another.