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"Welcome to OBGYN World!" A novel recruitment event for medical students organized by the Japan Society of Obstetrics and Gynecology. Since 2012, the number of doctors in Japan who specialize in obstetrics and gynecology has shown a decreasing trend. To increase the number of doctors majoring in obstetrics and gynecology, the Japanese Trainees in Obstetrics and Gynecology subcommittee developed a new recruitment event called Welcome to OBGYN World! (WOW!); the aim of this event was to focus on lower grades of medical students. The present report describes the content of WOW! and the results of a post-event questionnaire administered to participating students and tutors. WOW! was held online in order to avoid the risk of Coronavirus Disease 2019 infection for participants. Sixty of the 82 medical schools nationwide (73.2%) participated in this event. Overall, there were 285 participating students, ranging from first to fourth grade in medical school, and 106 tutors were involved to teach material at the event. In the post-event questionnaire survey, 97.6% (248/254) and 100% of the participants stated they now had a high degree of interest in obstetrics and gynecology and found the specialty attractive, respectively. Furthermore, 93.6% (90/94) of the tutors stated that WOW! had helped recruitment activities in their universities. Based on this outcome, members of the Japanese Trainees of Obstetrics and Gynecology subcommittee will now try to increase the number of doctors specializing in obstetrics and gynecology by holding WOW! annually.

Objectives: To investigate the association between online ac-tivities and the number of new obstetrics and gynecology senior residents. Methods: A nationwide web-based, self-administered anon-ymous survey was conducted to investigate recruitment and clerkship activities during the coronavirus disease 2019 pan-demic. An online questionnaire was sent to 576 obstetrics and gynecology training institutions in Japan between De-cember 21, 2020, and January 31, 2021. Overall, 334 institu-tions that gave valid responses were included (response rate: 58.0%). Multivariate logistic regression analysis examined the association between online activities, including recruitment and clerkship activities, and the number of new obstetrics and gynecology senior residents in 2021. The stratified analysis by implementing face-to-face activities was conducted to clarify the association.Results: The number of new senior residents increased in 187 facilities (56.0%) and decreased in 147 facilities (44.0%). The facilities that implemented face-to-face and online activities were 185 (55.4%) and 120 (35.9%), respectively. In multivariate logistic regression analysis, an increased number of new obstetrics and gynecology senior residents was significantly associated with face-to-face activities (adjusted odds ratio (AOR)=2.58, 95% confidence interval (CI): 1.11-5.97, p

Hypophosphatasia (HPP) is a congenital skeletal disease. Impairment of bone mineralization and seizures are due to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP). Enzyme replacement therapy (ERT) is available as a highly successful treatment for pediatric-onset HPP. However, the potential for prenatal ERT has not been fully investigated to date. In this study, we assessed outcomes and maternal safety using a combinational approach with prenatal and postnatal administration of recombinant TNAP in Akp2−/− mice as a model of infantile HPP. For the prenatal ERT, we administered subcutaneous injections of recombinant TNAP to pregnant mice from embryonic day 11.5–14.5 until delivery, and then sequentially to Akp2−/− pups from birth to day 18. For the postnatal ERT, we injected Akp2−/− pups from birth until day 18. Prenatal ERT did not cause any ectopic mineralization in heterozygous maternal mice. Both prenatal and postnatal ERT preserved growth, survival rate and improved bone calcification in Akp2−/− mice. However, the effects of additional prenatal treatment to newborn mice appeared to be minimal, and the difference between prenatal and postnatal ERT was subtle. Further improvement of the prenatal ERT schedule and long-term observation will be required. The present paper sets a standard for such future studies.

This study aimed to investigate the side effects of silicone gel sheet (Lady Care®) and evaluate its prophylactic efficacy in preventing abnormal scarring. Sixty women who underwent caesarean section were recruited from September 2016 to September 2017 in this prospective study. Lady Care® was applied from the 2nd to the 6th postoperative months. Side effects of Lady Care® were evaluated through medical examinations and questionnaires. A plastic surgeon diagnosed abnormal scarring. Pruritus was diagnosed in 25 (47.2%) patients; folliculitis, four (7.5%); dry skin, four (7.5%); contact dermatitis, three (5.7%); wound infection, two (3.8%); and epidermolysis, one (1.9%), albeit with mild severity. Following Lady Care® application, no abnormal scarring and mild hypertrophic scarring was observed in 32 (64.0%) and 18 (36.0%) patients respectively. Of seven patients with pre-existing hypertrophic scars, only two showed hypertrophic scarring after Lady Care® application. Our findings support the safety and prophylactic efficacy of Lady Care®.Impact StatementWhat is already known on this subject? The incidence of abnormal scarring, i.e. keloid or hypertrophic scar formation after caesarean section (CS) is reported to be ∼41%. Abnormal or excessive scar formation can lead to functional limitations, pruritus, pain and cosmetic issues. Studies have also shown a prophylactic effect of the application of silicone materials against the development of hypertrophic and keloid scars, though prohibitive cost and lack of adhesiveness of such gel sheets are known factors limiting their usage.What the results of this study add? The new silicone gel sheet 'Lady Care®' has strong adhesive properties and is consequently not easily peeled off. Furthermore, it is easy to use and economically efficient.What the implications are of these findings for clinical practice and/or further research? This is the first clinical trial on the application of Lady Care® silicone gel sheet for the prevention of CS scarring. Our findings support the safety and prophylactic efficacy of Lady Care®.

AIM To confirm that variations in cell-free fetal DNA (cffDNA) are indicators of external placental damage, we quantitatively investigated cffDNA alterations in maternal peripheral blood during external cephalic version (ECV). METHODS We recruited 48 singleton pregnant women who underwent ECV in our hospital. Before and immediately after ECV, we harvested 10 ml of maternal peripheral blood samples for cffDNA analysis. cffDNA alterations were assessed based on the fetal fraction (FF) rate. We performed ECV without epidural anesthesia but administered epidural anesthesia if ECV was disrupted due to severe pain. RESULTS The FF increased from 22.9% ± 5.7% to 27.0% ± 5.7% (p

The placenta is composed of the amnion, chorionic plate, villous and smooth chorion, decidua basalis, and umbilical cord. The amnion is a readily obtainable source of a large number of cells and cell types, including epithelium, mesenchyme, and endothelium, and is thus an allogeneic resource for regenerative medicine. Endothelial cells are obtained from large arteries and veins in the amniotic membrane as well as the umbilical cord. The amnion-derived cells exhibit transdifferentiation capabilities, including chondrogenesis and cardiomyogenesis, by introduction of transcription factors, in addition to their original and potential phenotypes. The amnion is also a source for production of induced pluripotent stem cells (AM-iPSCs). The AM-iPSCs exhibit stable phenotypes, such as multipotency and immortality, and a unique gene expression pattern. Through the use of amnion-derived cells, as well as other placenta-derived cells, preclinical proof of concept has been achieved in a mouse model of muscular dystrophy.

Amniotic fluid embolism (AFE) is a rare complication of pregnancy and its mortality rate is high. There have been few reports of AFE with presence of severe coagulopathy and incoagulable bleeding, and absence of cardiopulmonary symptoms or limited cardiopulmonary symptoms, followed by massive blood loss during delivery. Such cases have been referred to as disseminated intravascular coagulopathy-type AFE, and the characteristics of this condition have been presented previously. Here we report three cases that fulfilled the diagnostic characteristics of disseminated intravascular coagulopathy-type AFE.

We present the characterization of a case with a small supernumerary marker chromosome (sSMC) detected prenatally derived from Xq28 and 14q11.2 maternal translocation. A 33-year-old Japanese woman, primigravida, underwent amniocentesis because of fetal growth restriction and fetal structural abnormality at 30 weeks of gestation. The fetal karyotype was identified as 47,XY,+mar. Additionally, the single nucleotide polymorphism array analysis revealed copy number gains at Xq28 and 14q11.2. A male infant, weighing 1,391 g, was delivered at term by cesarean section. Maternal and paternal karyotypes were 46,X,t(X; 14)(q28; q11) and 46,XY, respectively. These findings indicated that the sSMC might have originated from chromosome disjunction at a ratio of three to one. Here we describe a case with an sSMC derived from Xq28 and 14q11.2. Our findings suggest that this sSMC is most likely pathogenic. The collection of additional cases may be required.

Lethal multiple pterygium syndrome (LMPS) is a fatal hereditary disease associated with abnormalities such as pterygium-induced congenital contractures. Fetal hydrops is present in more than half of all patients with LMPS, and all patients with LMPS are either stillborn or die in the early neonatal period. Ultrasonography findings for the prenatal diagnosis of LMPS include the detection of cutaneous webbing at multiple joints, multiple joint contractures, absent limb movement, and increased nuchal translucency. Here, we describe a patient who was diagnosed as having LMPS due to continuous fetal head flexion, despite the absence of the usual characteristics of the condition, including pterygium at the joints. Thus, continuous fetal head flexion can be a useful marker for prenatally diagnosing LMPS.

Dendritic cell (DC) and macrophage are the powerful antigen presenting cells. These show remarkable antigen processing function. It has been reported that antigen processing is enhanced by adjuvant. In DC and macrophage, LPS or IFNγ induces cell proliferation, cytokine production, antigen presentation to naive T cell. Among DC subset, CD8 positive DC enhances autophagy function especially in cross presentation. It is still nuclear how DC processes antigen by ubiquitin proteasome pathway or by autophagy pathway. To induce more effective immune response on vaccination or immune therapy, we study the effect of adjuvant treatment for autophagy function in DC and macrophage. Three types of different culture condition were used to establish DC subsets and compared to those function of macrophage. Then analyzed the antigen processing capacity as well as the autophagy function. We induced DC in vitro culture with GM-CSF, GM-CSF with IL-4, GM-CSF with IL-15, then stimulated DCs or macrophage by using LPS or IFNγ for up to 24 hours before antigen uptake. On antigen uptake and processing, GM-CSF induced or GM-CSF with IL-15 induced DC showed antigen processing, however GM-CSF with IL-4 induced DC showed active antigen processing within 60 min. Contrary to active antigen processing, GM-CSF with IL-14 –DC did not show any inducible effect after adjuvant treatment, but GM-CSF or GM-CSF with IL-15-DC increased the autophagy function. On the other hand, macrophage could not obtain autophagy effect thorough adjuvant treatment. DCs were studied the gene expression of cytokines, cell surface molecules such as co-stimulation molecules, mannose receptor, to evaluate antigen uptake capacity, processing pattern and autophagy.

GATA-3 is the master transcription factor for T helper 2 (Th2) cell differentiation and is critical for the expression of Th2 cytokines. Little is known, however, about the nature of the functional molecular complexes of GATA-3. We identified a high-mobility group (HMG)-box type transcription factor, lymphoid enhancer factor 1 (LEF-1), in the GATA-3 complex present in Th2 cells using a Flag-calmodulin-binding peptide (CBP)-tag based proteomics method. The interaction between GATA-3 and LEF-1 was confirmed by co-immunoprecipitation experiments using LEF-1-introduced T-cell lineage TG40 cells. The HMG-box domain of LEF-1 and two zinc finger domains of GATA-3 were found to be important for the physical association. The introduction of LEF-1 into developing Th2 cells resulted in the suppression of Th2 cytokine production. The suppression was significantly lower in the cells into which a HMG-box-deleted LEF-1 mutant was introduced. Moreover, LEF-1 inhibited the binding activity of GATA-3 to the interleukin (IL)-5 promoter. These results suggest that LEF-1 is involved in the GATA-3 complex, while also regulating the GATA-3 function, such as the induction of Th2 cytokine expression via the inhibition of the DNA-binding activity of GATA-3.

The differentiation of naive CD4 T cells into Th2 cells requires the T cell receptor-mediated activation of the ERK MAPK cascade. Little is known, however, in regard to how the ERK MAPK cascade regulates Th2 cell differentiation. We herein identified Gfi1 (growth factor independent-1) as a downstream target of the ERK MAPK cascade for Th2 cell differentiation. In the absence of Gfi1, interleukin-5 production and the change of histone modification at the interleukin-5 gene locus were severely impaired. Furthermore, the interferon γ gene showed a striking activation in the Gfi1–/– Th2 cells. An enhanced ubiquitin/proteasome-dependent degradation of GATA3 protein was observed in Gfi1–/– Th2 cells, and the overexpression of GATA3 eliminated the defect of Th2 cell function in Gfi1-deficient Th2 cells. These data suggest that the T cell receptor-mediated induction of Gfi1 controls Th2 cell differentiation through the regulation of GATA3 protein stability.

We followed a two-step surgical strategy using miniature Ommaya’s reservoirs in an early neonate with multiple large arachnoid cysts. Percutaneous fluid aspiration through the reservoirs placed during the first operation permitted the infant to develop sufficiently to withstand the second, more radical cyst wall excision.

Background Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice raised in nonair-controlled conventional circumstances spontaneously develop atopic dermatitis-like skin lesions; however, the underlying mechanisms remain unclear. Objective We wanted to identify the critical intracellular signaling molecules in T cells that induce atopic dermatitis-like skin legions in NC/Nga mice. Methods We examined the levels of signal transduction and cytokine production in T cells, particularly those in atopic dermatitis-like lesions induced by the topical injection of mite antigens in NC/Nga mice under specific pathogen-free conditions. Results In NC/Nga mice maintained under specific pathogen-free conditions, the capability of T H 1/T H 2 and T cytotoxic 1/T cytotoxic 2 (Tc1/Tc2) cell differentiation increased significantly. T-cell antigen receptor–mediated activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase cascade and nuclear factor-κB (NF-κB) signaling were enhanced in NC/Nga T cells. The expression of T H 2 cytokines (IL-4, IL-13, and IL-5) and that of GATA-binding protein 3 (GATA3), avian musculoaponeurotic fibrosarcoma (c-Maf), NF-κB, and activator protein 1 (AP1) selectively increased in draining lymph node T cells of NC/Nga mice. Moreover, the cell transfer of inhibitory NF-κB mutant-infected T H 2 cells reduced ear thickness in the mite antigen-induced skin lesion of NC/Nga mice. Conclusion Hyperresponsive T H 2 cells with an enhanced activity of NF-κB and AP1 play a crucial role in the pathogenesis of atopic dermatitis-like skin lesions in NC/Nga mice. Clinical implications These results indicate potential therapeutic usefulness of developing selective inhibitors for NF-κB in the treatment of human atopic dermatitis.

We used the diatom tracer method to survey the area around the Isahaya Bay to evaluate the influence of suspended particles discharged from the Isahaya reservoir. This method allowed the frustules of Skeletonema subsalsum and S. costatum, the predominant diatoms in the Isahaya reservoir and the bay, respectively, to remain even after the decomposition of cells of these phytoplanktons. We found that the greatest amounts of the suspended particles transferred within the area 1.5 km from the gates of the reservoir, at the head of the bay. We also found that most particulate organic matter in the bottom sediment originated from the primary production areas at the center and mouth of the bay. Moreover, we confirmed the reliability of the diatom tracer method from the finding that the distribution of the frustules of the diatoms in the sediment showed good agreement with that in the water column of the bay.

Differentiation of naive CD4 T cells into Th2 cells is accompanied by chromatin remodeling including hyperacetylation of histones H3 and H4 in the nucleosomes associated with the IL-4, IL-13 and IL-5 genes. A conserved GATA3 response element (CGRE) containing four GATA consensus sequences was identified 1.6 kbp upstream of the IL-13 gene, corresponding with the 5′ border of the Th2-specific histone hyperacetylation region. The CGRE was shown to bind to GATA3, histone acetyl transferase complexes including CBP/p300, and RNA polymerase II. As for the IL-5 gene locus, CD28 costimulation selectively enhanced histone hyperacetylation through NF-κB activation and subsequent upregulation of GATA3. Chromatin remodeling of type 2 cytokine gene loci occurs also in developing Tc2 cells. IL-4 production and histone hyperacetylation in IL-4-associated nucleosomes in developing Tc2 cells were significantly lower than those of Th2 cells; however, cytokine production and histone hyperacetylation of IL-5 and IL-13 genes were equivalent. Developing Tc2 cells expressed lower GATA3 levels and dramatically increased levels of repressor of GATA (ROG). A ROG response element in the IL-13 gene exon 4 displayed Tc2-specific binding of ROG, HDAC1 and HDAC2. Thus, ROG may confer CD8 T cell-specific repression of histone hyperacetylation and activation of the IL-4 gene locus.

Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.

Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor beta, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2-deficient mice, mature peripheral T cells exhibited normal anti-T cell receptor-induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2-deficient developing Th2 cells showed constitutive activation of nuclear factor kappaB (NF-kappaB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-kappaB for binding to a consensus NF-kappaB motif and inhibit NF-kappaB-driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-kappaB function.

We investigated the assembly and activation of the epidermal growth factor receptor (EGFR)–p185 c-neu heterodimer by using a sequential immunoprecipitation methodology. Using this approach we detected heterodimers and also higher-ordered oligomeric complexes. Phosphorylated EGFR–p185 c-neu heterodimeric forms were detected in the absence of EGF, but the species became highly phosphorylated after EGF stimulation. To evaluate heterodimer formation and additional transactivation by EGF, we investigated the roles of the four extracellular subdomains of p185 c-neu and the EGFR. Subdomains I–IV of the EGFR dimerized with subdomains I–IV of p185 c-neu , respectively, in a parallel manner. In addition, subdomains I–IV of the EGFR also associated with p185 c-neu subdomains III, IV, I, and II, respectively. A lack of one of the p185 c-neu cysteine-rich domains (subdomains II or IV) resulted in a loss of EGF-induced transactivation. These data suggest that two cysteine-rich domains play defining roles in ligand-dependent transactivation and that both of these cysteine-rich extracellular subdomains as well as non-cysteine-rich extracellular subdomains are involved in ligand-independent interactions with the EGFR. Our studies provide biochemical evidence of the role of the cysteine-rich domains of p185 c-neu in assembly and transactivation of erbB complexes and also indicate that these subdomains might be useful clinical targets.

The purpose of this paper is a) to make a comparative study of the existence of ikigai (reason(s) for living) in elderly people and its relevance to their family structure, physiological situation and functional capacity in both rural areas and metropolitan suburban areas, and b) position basic research into the structure of ikigai in the near future, by clarifying several related factors, from which the concept of ikigai may be defined. The meaning of the word "ikigai" in Japanese is difficult to express exactly, and specialists in gerontology have varying definitions. If ikigai were translated from Japanese into English, it could be "reason(s) for living", "self-actualization", "meaning of life" and/or "purpose in life". In this paper, ikigai is used to mean "feeling of being alive now and/or individual motivation for living". As of October 2000, we studied 1,544 people aged 65 years and over living in town Y of Niigata Prefecture (rural area), and as of January 2001, we studied 1,002 people in the same age group in town H of Saitama Prefecture (metropolitan suburban area). The above investigations revealed the following characteristics:--(a) Regarding the percentages of persons having or not having ikigai, there were no significant differences between the rural area and the metropolitan suburban area. (b) In both areas, the 3 factors of self-rated level of health, intellectual activeness and social roles, were associated with having ikigai. (c) In the rural area, the family structure was strongly associated with having ikigai, but gender or generation were irrelevant. (d) In the metropolitan suburban area, the hospitalization experience of men was strongly associated with ikigai. Furthermore, there was a strong correlation with generation. In this regard, while the contents of ikigai are seldom examined in detail, clarification of the structure of ikigai should be worked out in the next stage of the study, using covariance structure analysis. In addition the development of concrete plans to promote ikigai by municipal organs could be beneficial.

Mitsubishi Materials Corporation (MMC) and the Kansai Electric Power Co., Inc. (KEPCO) are jointly developing intermediate temperature SOFCs that use doped lanthanum gallate electrolyte. The primary target of the collaboration is the development of modular SOFC systems for on-site power generation. Manufacturing technology of electrochemically active cells composed of (La,Sr)(Ga,Mg,Co)O 3 electrolyte, Ni-(Ce,Sm)O 2 anode and (Sm,Sr)CoO 3 cathode was established using tape-casting and screen-printing methods. Seal-less planar-type stack design was employed for manufacturing kilowatt-scale modules. The first generation module successfully provided the output power of 1 kW with thermal self-sustainability below 800°C. Maximum electrical efficiency obtained with this module was 43% [LHV] together with the corresponding fuel utilization of 78%. Dynamic performance tests demonstrated the capability of output power alteration from 0.6 to 1 kW while maintaining high electrical conversion efficiency. Further testing and modification of the module for methane fuel utilization are in progress.

Background: Cognitive impairment is a major health issue, but epidemiological data on mild cognitive decline have been almost absent in Japan. Methods: Of all residents aged 65 years and over living in Yoita town, Niigata Prefecture, Japan in the year 2000 (n = 1673), 1544 participated in the interview survey held at community halls or at home (92.3% response). They underwent the Mini-Mental State Examination (MMSE) for assessment of cognitive function and answered questionnaires comprising socio-demographic, psychological, physical and medical, and social activity items. Higher-level functional capacities were evaluated with the Tokyo Metropolitan Index of Competence (TMIG-Index of Competence). According to subject’s age and MMSE score, all subjects were classified into 3 groups: control (MMSE > 1 SD below age-specific means), mild cognitive decline (MMSE ≥ 21 and ≤ 1 SD below age-specific means), and severe cognitive decline (MMSE ≤ 20), and compared various characteristics among these groups. Results: Mean MMSE score of the subjects showed a linear decline with advancing age. Among the participants, 232 (15.2%) were classified as mild cognitive decline. Compared with the controls, the subjects with mild cognitive decline reported poorer subjective health, more depressive moods, more history of stroke, more prevalence of basic activity of daily living (BADL) disability, and lower higher-level functional capacity, even after controlling for possible confounding factors. They also reported a low level of social activities: both participating in group activities and enjoying hobbies were less frequent. Their food intake pattern tended to be monotonous. Conclusions: Older persons with mild cognitive decline comprised a substantial proportion (15.2%) of the community-dwelling older population. In addition to lower cognitive function, they had lower levels of functional capacity and social activity.

Problem to date: Similar in nature to the knapsack problem and the indivisible investment problem, there exists a static optimal problem the variables of which have both discrete and continuous values as the optimum variables: that is, a mixed-integer programming problem as one optimal problem of the mixed programming problems. A relaxed method or a group method etc. has been used for these problems hitherto. Just like the dynamical indivisible investment problem and the resource allotment problem/the disposition of personnel problem through several periods, there are few optimal control methods for the mixed-quantized dynamical optimal control problem which has both discrete and continuous values. The proposed method in this paper: For the mixed-quantized optimal control problem in which the state equation is linear, the control problem is given by the formulation of Halkin’s discrete time optimal control problem. The mixed-quantized discrete maximum principle is given as the algorithm for this control problem. Where, for the maximization of Hamiltonian in each discrete time, the relaxed method—which improved branch and bound method—is used. Effects obtained in this paper: As the applications in this control problem, the above dynamical investment and the allotment problem through the several periods etc. are considered. In this paper, the solution to the discrete time mixed-quantized optimal control problem is given, and the efficiency of this method (mixed-quantized discrete maximum principle)—which is applied infrequently to this field—is shown, along with a numerical example.

Regulatory mechanisms of T cell autoreactivity to MHC class II molecules were studied in transgenic (Tg) mice with auto-I-Ak-reactive TCR alphabeta transgenes (designated as MS Tg mice). Our previous study revealed that the T cell tolerance established in autoreactive MS Tg mice was not due to either clonal deletion in the thymus, anergy or an active suppression in the periphery. We proposed a novel form of self tolerance termed 'clonal insufficiency', where autoreactive T cells were conditionally rendered unresponsiveness to self antigen in vivo, although retaining full potential reactivity in in vitro conditions. Here, we investigated the role of co-stimulatory molecules for the induction of self tolerance with 'clonal insufficiency'. MS Tg mice were mated with CD80 (B7-1) Tg mice in which B cells exclusively and constitutively expressed CD80 molecules. Both MS Tg mice and CD80 Tg mice by themselves showed no evidence for activation of T cells and B cells, whereas MS x CD80 double-Tg mice with a H-2k background revealed an abnormal increase in the number of splenocytes and in the expression of activation markers (CD69 and CD25) on CD4 T cells in the spleen. These results indicated that the self tolerance established in MS Tg mice involved a down-regulation of CD80 molecules on B cells in vivo, resulting in a failure of sufficient T-B interactions. In addition, the serum concentration of IL-10, one of the down-regulators of CD80 expression, was found to be increased significantly in MS Tg mice. The autoreactivity of MS Tg T cells detected in vitro was significantly blocked by recombinant IL-10. Thus, IL-10-mediated down-regulation of CD80 on B cells was suggested to be involved in the clonal insufficiency in MS Tg mice in vivo.

An enzyme-linked immunosorbent assay (ELISA) was developed for direct quantitation of microcys- tins (MCs), a group of freshwater cyanobacterial toxins. An anti-MC monoclonal antibody exhibiting broad cross-reactivity to major MC derivatives was used. The detection limit and linear range of the ELISA standard curve with microcystin-(leucine-ar-ginine) (MCLR), a variant of MCs, were 20 and 20–500 pg/mL, respectively. For analysis of MC released from cyanobacterial cells, water sample filtered through a glass fiber filter was applied directly to ELISA. For analysis of total MC (released MC plus intracellular MC), intracellular toxin was extracted by freeze-thawing twice before filtration. Mean recovery of MCLR added to tap water and toxin-free environmental water was 101%, with a coefficient of variation (CV) of 7.3% at toxin levels of 20–500 pg/mL. Mean recovery of MCLR added to toxin-free cyanobacterial extracts was 93%, with a CV of 12.5% at toxin levels of 50–500 pg/mL. At 20 pg/mL, an increasing matrix effect on assay variance was observed; therefore, both released MC and total MC were measured in the range 50–500 pg/ mL. Comparative studies with a liquid chromatographic (LC) method showed that the ELISA gives a reliable correlation with LC for analysis of MC in water extracts of natural blooms and cultured cyanobacterial cells (r = 0.98). The ELISA was applied to water samples collected from lakes and ponds in Japan. In 4 of 13 and 12 of 17 samples, 81–800 pg released MC/mL and 64–94 000 pg total MC/mL were detected, respectively. By LC separation followed by the ELISA analysis, the presence of MCLR, microcystin-arginine-arginine, and micro-cystin-tyrosine-arginine were confirmed in 4 ELISA-positive samples selected randomly. The newly developed ELISA is a reliable and powerful method for mass monitoring of MC levels in environmental water.

CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates suggests that it plays a role in the pathogenesis of inflammatory diseases. In this study, we used CD69-deficient (CD69 KO) mice to assess the role of CD69 in the pathogenesis of dextran sulphate sodium (DSS)-induced acute and chronic colitis. The severity of colitis was assessed by the survival rate, clinical signs, colon length, histological examination and the expression of cytokines and chemokines in the large intestines. Both acute and chronic colitis were attenuated in the CD69 KO mice, as reflected by the lower lethality, weight loss, clinical signs, and improved histological findings. CD69(+) cells infiltrated extensively into the inflamed mucosa of the colon in WT mice after DSS treatment. Experiments with the transfer of WT CD4 T cells into CD69 KO mice restored the induction of colitis. The administration of an anti-CD69 antibody also inhibited the induction of the DSS-induced colitis. These results indicate that CD69 expressed on CD4 T cells plays an important role in the pathogenesis of DSS-induced acute and chronic colitis, and that CD69 could be a possible therapeutic target for colitis.

Gluconacetobacter xylinus is involved in the industrial production of cellulose. We have determined the genome sequence of G. xylinus NBRC 3288, a cellulose-nonproducing strain. Comparative analysis of genomes of G. xylinus NBRC 3288 with those of the cellulose-producing strains clarified the genes important for cellulose production in Gluconacetobacter .

The comparison of cancer cell seeding, deformation and viability in the lung, muscle and liver of nude mice in real-time is reported here. The mice were intubated to support ventilation with positive end-respiratory pressure (PEEP) for imaging on the lung. Human fibrosarcoma cells with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (dual-color HT-1080 cells) were injected into the tail vein for lung imaging, the portal vein for liver imaging or the abdominal aorta for muscle imaging which was performed with an Olympus OV100 Small Animal Imaging System. The length of the cytoplasm and nuclei in 20 seeded cancer cells were measured. A large number of cells initially arrested in the lung capillaries and many cells formed aggregates. The cell number decreased rapidly at 6 and 24 h. There was no significant difference in cancer cell survival when immunocompetent C57BL/6 mice were used in place of the nude mice, suggesting that T cell reaction is not very important in the first 24 h after seeding of cancer cells in the lung. In the lung and liver, little cancer cell deformation occurred. In contrast in the muscle, the cytoplasm and nuclei of the seeded cells were highly deformed and many fragmented cells were observed. The rate of cancer cell death was highest in the lung and lowest in the muscle. In each organ, single disseminated cells tended to die earlier than aggregated cells. The results of this study suggest that the early steps of metastasis are different in the lung, liver and muscle.

Among three newly prepared monoclonal anti‐aflatoxin M1 antibodies, named AM.1, AM.2 and AM.3, both AM.1 and AM.3 possessed high specificity and sensitivity towards aflatoxin M1, while AM.2 exhibited lower specificity. Employing AM.3 and horseradish peroxidase‐labelled second antibody, an improved ELISA was developed with a detection limit of 1.0 pg ml‐1 of aflatoxin M1 in solution. The contents of aflatoxin M1 in powdered milks suspended in water were assayed by the indirect ELISA. The detection limit was 5 pg g‐1dry weight, and no clean‐up procedures were required. The reliability of the present ELISA with monoclonal antibody AM.3 was confirmed with reference powdered milks for aflatoxin M1. A limited ELISA survey showed the presence of aflatoxin M1 in commercial milk powders sampled in France, the US and Thailand at levels of 30–418 pg g‐1, and it was confirmed by an improved HPLC analysis.

CD69 is a type II membrane protein expressed as a homodimer composed of heavily glycosylated subunits. CD69 is known as an early activation marker antigen of lymphocytes,and its selective expression in inflammatory infiltrates suggests that it plays a role in the pathogenesis of inflammatory diseases. In order to address the role of CD69 in the pathogenesis of arthritis and allergic airway inflammation, we established CD69-deficient mice. CD69-deficient mice displayed a markedly attenuated arthritic inflammatory response and airway inflammation. The administration of anti-CD69 antibody inhibited the induction of the antigen-induced airway inflammation and hyperresponsiveness. These results indicate that CD69 plays a crucial role in the pathogenesis of arthritis and allergic airway inflammation and that CD69 could be a possible therapeutic target for arthritis and asthma in human patients.

A scanning electron microscope (SEM) was utilized for electron beam direct lithography. The scanning electron microscope was modified by installing a blanking unit, thus modifying the deflection coil and condenser coil and adding an external signal line for secondary electron beam detection. Without modifying the microscope tube and the internal circuit of the SEM, lithography of a layout pattern was made possible. By implementing a lithography system on a Bitmap-IV CAD system designed for layout pattern designing, both circuit design and lithography were carried out. The designed layout pattern data were translated to the lithography data described by electron beam direct lithography language (EBDL). Using the algorithm taking into account the characteristic of electron beams in SEM, the minimum pattern dimension of 4 μm with an alignment tolerance of ±2 μm on a chip area of 3.2 mm × 3.2 mm was achieved in patterning circuits in CMOS. LSIs and micromachine layouts. The system was totally automated in operations including positioning.

Many coating methods of hydroxyapatite (HAp) on mechanically strong materials as substrates have been studied for increasing the mechanical strength of bioactive HAp. In this paper, coating of HAp on 3mol% Y2O3 partially stabilized zirconia (PSZ) substrate, a bioinert and highly strong and fracture-resistant material was studied utilizing a new plasma spraying method of a radio-frequency (r. -f.) thermal plasma. High contents of HAp in the coated layer were obtained by using larger HAp powders at smaller plasma powers and at lower substrate temperatures. The decomposition of the HAp phase was suppressed below the substrate temperature of about 1500°C. Adhesive strength of the coated HAp layer to the substrate was measured to be 5-14MPa. The surface of the coated layer had many pores. Thus, a possibility of coating HAp on the PSZ utilizing a r. -f. thermal plasma process was indicated. The HAp coated layer on the PSZ can be used as a mechanically strengthened and biocompatible material.

Airway inflammation and airway hyperresponsiveness are central issues in the pathogenesis of asthma. CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates suggests that it plays a role in the pathogenesis of inflammatory diseases. In CD69-deficient mice, OVA-induced eosinophilic airway inflammation, mucus hyperproduction, and airway hyperresponsiveness were attenuated. Cell transfer of Ag-primed wild-type but not CD69-deficient CD4 T cells restored the induction of allergic inflammation in CD69-deficient mice, indicating a critical role of CD69 expressed on CD4 T cells. Th2 responses induced by CD69-deficient CD4 T cells in the lung were attenuated, and the migration of CD4 T cells into the asthmatic lung was severely compromised. The expression of VCAM-1 was also substantially altered, suggesting the involvement of VCAM-1 in the CD69-dependent migration of Th2 cells into the asthmatic lung. Interestingly, the administration of anti-CD69 Ab inhibited the induction of the OVA-induced airway inflammation and hyperresponsiveness. This inhibitory effect induced by the CD69 mAb was observed even after the airway challenge with OVA. These results indicate that CD69 plays a crucial role in the pathogenesis of allergen-induced eosinophilic airway inflammation and hyperresponsiveness and that CD69 could be a possible therapeutic target for asthmatic patients.

Mycobacterium tuberculosis (tubercle bacilli) and the related acid-fast bacteria including Mycobacterium bovis Bacille Calmett-Guerin (BCG) have a characteristic cell wall (CW) containing various lipoglycans and glycolipids. Such lipoglycans have been reported to activate type-I inflammatory responses via dendritic cells (DCs) through Toll-like receptor 2. In this study, lipoglycans, lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinositol mannoside (PIM), were purified from the CW fractions of M. bovis BCG Tokyo-172, and the effect on the differentiation of human peripheral blood naive CD4 T cells into T h 1 and T h 2 was examined. LAM/LM molecules enhanced T h 1 differentiation under both T h 1 and T h 2 conditions, whereas some other glycolipids and phospholipid enhanced T h 2 differentiation under T h 2 conditions. Other components had little effect under the given conditions. Even in highly purified CD4 T cell cultures, LAM/LM enhanced T h 1 generation only under T h 1 culture conditions. These results indicate that LAM/LM possesses a potent augmenting activity in T h 1 differentiation in human CD4 T cells. LAM/LM appeared to act directly on naive CD4 T cells to enhance T h 1 differentiation under T h 1 culture conditions, while acting indirectly to up-regulate the generation of T h 1 cells via IL-12/DCs under T h 1 and T h 2 conditions. Therefore, these results provide the first evidence indicating that LAM/LM from M. bovis BCG may possess a potent modulating activity in the human system, and thus supporting the strategy for the use of BCG components in the vaccine development for such T h 2 diseases as allergic asthma and rhinitis.

The maintenance of memory T cells is central to the establishment of immunological memory, although molecular details of the process are poorly understood. In the absence of the polycomb group (PcG) gene Bmi1 , the number of memory CD4 + T helper (Th)1/Th2 cells was reduced significantly. Enhanced cell death of Bmi1 −/− memory Th2 cells was observed both in vivo and in vitro. Among various proapoptotic genes that are regulated by Bmi1, the expression of proapoptotic BH3-only protein Noxa was increased in Bmi1 −/− effector Th1/Th2 cells. The generation of memory Th2 cells was restored by the deletion of Noxa , but not by Ink4a and Arf . Direct binding of Bmi1 to the Noxa gene locus was accompanied by histone H3-K27 methylation. The recruitment of other PcG gene products and Dnmt1 to the Noxa gene was highly dependent on the expression of Bmi1. In addition, Bmi1 was required for DNA CpG methylation of the Noxa gene. Moreover, memory Th2-dependent airway inflammation was attenuated substantially in the absence of Bmi1. Thus, Bmi1 controls memory CD4 + Th1/Th2 cell survival and function through the direct repression of the Noxa gene.

Background Studies of human asthma and of animal models of allergic inflammation/asthma highlight a crucial role for T H 2 cells in the pathogenesis of allergic asthma. Repressor of GATA (ROG) is a POZ (BTB) domain–containing Kruppel-type zinc finger family (or POK family) repressor. A repressive function to GATA3, a master transcription factor for T H 2 cell differentiation, is indicated. Objective The aim of this study was to clarify the regulatory roles of ROG in the pathogenesis of T H 2-driven allergic diseases, such as allergic asthma. Methods We examined allergic airway inflammation and airway hyperresponsiveness (AHR) in 3 different mouse models, which use either ROG-deficient ( ROG −/− ) mice, ROG transgenic mice, or adoptive transfer of cells. Results In ROG −/− mice T H 2 cell differentiation, T H 2 responses, eosinophilic airway inflammation, and AHR were enhanced. In ROG transgenic mice the levels of eosinophilic airway inflammation and AHR were dramatically reduced. Furthermore, adoptive transfer of T H 2 cells with increased or decreased levels of ROG expression into the asthmatic mice resulted in reduced or enhanced airway inflammation, respectively. Conclusion These results indicate that ROG regulates allergic airway inflammation and AHR in a negative manner, and thus ROG might represent another potential therapeutic target for the treatment of asthmatic patients.