Disruption of the temporally regulated cloaca endodermal ß-catenin signaling causes anorectal malformations.

The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. ß-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of ß-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the ß-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (Shh(CreERT2)). Both ß-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the ß-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the ß-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The Shh(CreERT2/+); ß-catenin(flox(ex3)/+); BmprIA(flox/-) mutants displayed partial restoration of URS elongation compared with the ß-catenin GOF mutants. These results indicate that some ARM phenotypes in the ß-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal ß-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.

Apnoea-hypopnoea index during rapid eye movement and non-rapid eye movement sleep in obstructive sleep apnoea.

This study investigated the differences in apnoea-hypopnoea index (AHI) during rapid eye movement (REM) sleep (AHI-REM) and AHI during non-REM (NREM) sleep (AHI-NREM) in patients with obstructive sleep apnoea (OSA). Nocturnal polysomnography was performed in 102 Japanese OSA patients and their AHI along with a variety of other factors were retrospectively evaluated. Regardless of the severity of AHI, mean apnoea duration was longer and patients' lowest recorded oxygen saturation measured by pulse oximetry was lower during REM sleep than during NREM sleep. Approximately half of the patients (n = 50) had a higher AHI-NREM than AHI-REM. In subjects with AHI >or= 60 events/h, AHI-NREM was significantly higher than AHI-REM. On multivariate logistic regression, severe AHI >or= 30 events/h was the only predictor of a higher AHI-NREM than AHI-REM. This may indicate that important, but unknown, factors related to the mechanism responsible for the severity of OSA are operative during NREM sleep.

Continued inhalation of lidocaine suppresses antigen-induced airway hyperreactivity and airway inflammation in ovalbumin-sensitized guinea pigs.

It is unclear whether inhaled lidocaine is effective against airway hyperreactivity and inflammation in asthma. The aim of this study was to investigate the effects of inhaled lidocaine on airway hyperreactivity and inflammation. Airway reactivity to inhaled histamine, cellular composition of bronchoalveolar lavage (BAL) fluid, plasma substance P (SP), and isolated lung tissue were evaluated in ovalbumin (OVA)-sensitized guinea pigs 7 days after OVA challenge. The effects of inhaled lidocaine on this model were also evaluated. Treatment with lidocaine was administered in two fashions: as single inhalation or inhalation bid for 7 consecutive days, for comparison with a saline-inhaled control group. Airway hyperreactivity to histamine, increase in number of total cells and increased proportion of eosinophils in BAL fluid, and marked eosinophil infiltration in airway walls were noted even 7 days after OVA challenge in the control group. Plasma SP level was also significantly increased. Although treatment with single lidocaine inhalation did not affect airway hyperreactivity, continued inhalation (bid for 7 days) attenuated airway hyperreactivity. Continued, but not single, inhalation of lidocaine also suppressed infiltration of eosinophils in BAL fluid and in airway walls. In addition, plasma SP levels were significantly reduced by continued but not by single inhalation. It appears possible that lidocaine when inhaled suppresses eosinophilic inflammation of the airway and SP-induced neurogenic inflammation, leading to alleviation of airway hyperreactivity.

A pen-based interface for generating graphical reports of findings in cardiac catheterization.

OBJECTIVES: This paper introduces a pen-based interface for the graphical reporting of findings in cardiac catheterization. METHODS: The user can interactively draw, erase, move, and deform coronary arteries as well as record stenoses on them. The location and degree of each stenosis is represented visually and the doctor can record various treatments such as bypasses and stents on the diagram. In addition, the system automatically extracts semantic information from the graphical representation and stores it in XML format. The system can also generate a table in the format specified by the American Heart Association. RESULTS: Our current implementation is a research prototype and is not yet being used in clinical practice. However, we have already demonstrated it to medical professionals and confirmed the following benefits. CONCLUSIONS: This system is useful not only as a tool for efficiently generating reports of findings but also as an effective explanation tool for patients.

Molecular genetic cascades for external genitalia formation: an emerging organogenesis program.

External genitalia are anatomical structures located at the posterior embryonic region as part of several urogenital/reproductive organs. The embryonic anlage of the external genitalia, the genital tubercle (GT) develops as a bud-shaped structure with an initial urethral plate and later urethra. Embryonic external genitalia are considered to be one of the appendages. Recent experiments suggest that essential regulatory genes possess similar functions for the outgrowth regulation of the GT and limb appendages. The transient embryonic epithelia located in the distal GT are called the distal urethral epithelium (DUE) regulating, at least in part, the (distal) GT development. This review covers the available data about early patterning of GT and discusses the molecular developmental similarities and points of divergence between the different appendages. Development of the male and female external genitalia is also reviewed.

Unique functions of Sonic hedgehog signaling during external genitalia development.

Coordinated growth and differentiation of external genitalia generates a proximodistally elongated structure suitable for copulation and efficient fertilization. The differentiation of external genitalia incorporates a unique process, i.e. the formation of the urethral plate and the urethral tube. Despite significant progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes for external genitalia. The sonic hedgehog (Shh) gene and its signaling genes have been found to be dynamically expressed during murine external genitalia development. Functional analysis by organ culture revealed that Shh could regulate mesenchymally expressed genes, patched 1 (Ptch1), bone morphogenetic protein 4 (Bmp4), Hoxd13 and fibroblast growth factor 10 (Fgf10), in the anlage: the genital tubercle (GT). Activities of Shh for both GT outgrowth and differentiation were also demonstrated. Shh(-/-) mice displayed complete GT agenesis, which is compatible with such observations. Furthermore, the regulation of apoptosis during GT formation was revealed for the first time. Increased cell death and reduced cell proliferation of the Shh(-/-) mice GT were shown. A search for alterations of Shh downstream gene expression identified a dramatic shift of Bmp4 gene expression from the mesenchyme to the epithelium of the Shh mutant before GT outgrowth. Regulation of mesenchymal Fgf10 gene expression by the epithelial Shh was indicated during late GT development. These results suggest a dual mode of Shh function, first by the regulation of initiating GT outgrowth, and second, by subsequent GT differentiation.

Effects of suplatast tosilate (IPD Capsules) on the production of active oxygen by neutrophils and of IL-8 by mononuclear cells.

In bronchial asthma, eosinophils and neutrophils are activated, so that the production of active oxygen species increases, causing airway epithelial injury. Suplatast tosilate (IPD Capsules) is a novel immunomodulating antiallergic drug that acts against bronchial asthma through a new mechanism. To evaluate the effects of suplatast tosilate on mononuclear cell-mediated IL-8 production, and neutrophil-mediated active oxygen species production at sites of inflammation, we collected peripheral blood from healthy subjects and separated the neutrophils as well as mononuclear cells. Suplatast tosilate was added at a concentration of 1 x 10(-6), 1 x 10(-7) or 1 x 10(-8) M, and cells were incubated for 10 min at 37 degrees C. Then, the neutrophils were stimulated with fMLP, and luminol-dependent chemiluminescence (LDCL) was measured, while IL-8 production was determined with an ELISA kit. Suplatast tosilate (1 x 10(-6) M) inhibited neutrophil-mediated active oxygen species production by 12.4% in terms of the peak, and by 16% in terms of the integral value. Moreover, it significantly inhibited mononuclear cell-mediated IL-8 production at concentrations of 1 x 10(-6), 1 x 10(-7) and 1 x 10(-8) M, in a concentration-dependent manner. This study indicated that suplatast tosilate may inhibit neutrophil infiltration by suppressing monocyte-mediated IL-8 production, and it may also inhibit the activation of neutrophils at sites of inflammation. These results suggest the possibility that suplatast tosilate may not only be of benefit for asthma, but may also prevent or control pulmonary fibrosis or emphysema, for which no effective treatment is presently available.

Role of chemical mediators in airway hyperresponsiveness in an asthmatic model.

BACKGROUND: Airway hyperresponsiveness (AHR) is one of the characteristic features of human asthma. The presence of AHR and the precise mechanisms immediately after establishment of sensitization in guinea pigs are unclear, although there are many reports showing allergen exposure that causes an increase in bronchial responsiveness associated with eosinophil influx into the airway in sensitized guinea pigs. OBJECTIVE: We investigated the inhibitory effects on AHR to histamine of ONO-1078, a leukotriene antagonist; indomethacin, a cyclooxygenase inhibitor; S-145, a thromboxane A(2) (TXA(2)) antagonist, and Y-24180, a platelet-activating factor (PAF) antagonist, to assess the involvement of chemical mediators in AHR employing ovalbumin (OA) sensitized guinea pig models. METHODS: Male Hartley guinea pigs were used. Each group comprised 4-7 animals. The animals were sensitized to OA, injecting intraperitoneally 30 mg of cyclophosphamide and 2,000 microg of OA together with 100 mg of aluminum hydroxide as the adjuvant. The guinea pigs were artificially ventilated via a cannula using a small-animal respirator after intraperitoneal anesthesia with pentobarbital sodium for tracheotomy. The pressure at the airway opening (PAO) was measured using a differential pressure transducer, and a differential pressure of peak PAO (peak DeltaPAO) at inspiratory phase as an overall index of bronchial response to bronchoactive agents was used. While being artificially ventilated, the animals were exposed to physiological saline solution containing various concentrations of histamine (4.9, 9.8, 20, 39, 78, and 156 microg/ml) by inhalation for 30 s at 3-min intervals. Determinations were made at 1 min after each inhalation. The chemical mediators were each (30 mg/kg of ONO-1078, 3 mg/kg of S-1452, and 1 mg/kg of Y-24180) administered orally to sensitized guinea pigs, and the airway response to histamine was assessed. Each group comprised 4-7 animals. RESULTS: The airway response to histamine was significantly greater in the sensitized group than in the nonsensitized group at histamine concentrations of 36 (p < 0.05), 78, and 156 mg/ml (p < 0.01). Leukotrienes C(4) and D(4): 30 mg/kg of ONO-178 did not show any inhibitory effect on airway response to inhaled histamine. Cyclooxygenase: 5 mg/kg of indomethacin did not show any inhibitory effect on the airway response to inhaled histamine. TXA(2): the AHR to inhaled histamine at doses of 9.8, 39, 78, and 156 microg/ml was significantly inhibited by prior administration of 3 mg/kg of S-1452. PAF: the AHR to inhaled histamine at doses of 9.8, 39, and 78 microg/ml was significantly inhibited by prior administration of 1 mg/kg of Y-24180. CONCLUSIONS: S-1452 (3 mg/kg) and Y-24180 (1 mg/kg) significantly inhibited AHR to histamine, while ONO-108 (30 mg/kg) and indomethacin (5 mg/kg) did not. The results suggest that TXA(2) and PAF are involved in AHR in OA-sensitized guinea pigs.

External genitalia formation: role of fibroblast growth factor, retinoic acid signaling, and distal urethral epithelium.

The process of fetal external genitalia development might be divided into two processes. The first process accomplishes the initial outgrowth of the anlage, genital tubercle (GT). Previous analysis suggests that the distal urethral epithelium (DUE) of the GT, the Fgf8-expressing region, regulates the outgrowth of the GT. The second process eventually generates the sexually dimorphic development of the external genitalia, which is dependent on the action of steroid hormones. Several key genes, for example, RARs, RXRs, RALDH2, and CYP26, were dynamically expressed during GT development. The teratogenic dose of RA at 9.0 d.p.c. induced a drastic malformation of the urethral plate during GT formation, but did not show gross abnormalities in its outgrowth. In RA-treated embryos, Fgf8 expression was still detected in the distal GT regions. Possible regulatory roles of the FGF and RA signaling systems in external genitalia formation are discussed.

Dual-phase response model for bronchial asthma.

The authors have successfully developed an animal model of dual-phase bronchial responses and very high IgG titer by sensitizing Hartley-strain male guinea pigs. Specific airway resistance, which was determined in a two-chamber body plethysmograph, was elevated to sevenfold during immediate response, followed by a late phase response with a smaller but marked elevation in resistance. Furthermore, hematological and histological examinations revealed that the total cell count increased in BAL obtained during both immediate and late bronchial responses as compared to pre-OVA challenges. A significant increase in BAL eosinophils was present only for the late bronchial samples, and this finding was supported by histological examination.

Molecular analysis of external genitalia formation: the role of fibroblast growth factor (Fgf) genes during genital tubercle formation.

The molecular mechanisms underlying the development of the external genitalia in mammals have been very little examined. Recent gene knockout studies have suggested that the developmental processes of its anlage, the genital tubercle (GT), have much in common with those of limb buds. The Fgf genes have been postulated as regulating several downstream genes during organogenesis. Fgf8 was expressed in the distal urethral plate epithelium of the genital tubercle (GT) together with other markers such as the Msx1, Fgf10, Hoxd13 and Bmp4 expressed in the mesenchyme. To analyze the role of the FGF system during GT formation, an in vitro organ culture system was utilized. It is suggested that the distal urethral plate epithelium of GT, the Fgf8-expressing region, regulates the outgrowth of GT. Ectopic application of FGF8 beads to the murine GT induced mesenchymal gene expression, and also promoted the outgrowth of the GT. Experiments utilizing anti-FGF neutralizing antibody suggested a growth-promoting role for FGF protein(s) in GT outgrowth. In contrast, despite its vital role during limb-bud formation, Fgf10 appears not to be primarily essential for initial outgrowth of GT, as extrapolated from Fgf10(-/-) GTs. However, the abnormal external genitalia development of Fgf10(-/-) perinatal mice suggested the importance of Fgf10 in the development of the glans penis and the glans clitoridis. These results suggest that the FGF system is a key element in orchestrating GT development.

Effects of ONO-1078 (pranlukast) on cytokine production in peripheral blood mononuclear cells of patients with bronchial asthma.

BACKGROUND: ONO-1078 (pranlukast) is a leukotriene receptor antagonist developed in Japan. This drug has been shown to be useful in oral treatment of bronchial asthma. The present study was undertaken to assess the effects of this drug on the production of cytokines in the peripheral blood mononuclear cells of patients with asthma under stimulation with specific antigens. METHODS: Peripheral blood mononuclear cells from mite antigen-positive asthmatic patients (immunoglobulin E RAST score > 3) were incubated for 72 h in the presence of mite antigen (10 microg/mL). The supernatant of the culture was subjected to enzyme-linked immunosorbent assay (ELISA) to quantify interleukin (IL) -4, IL-3, IL-5, and granulocyte macrophage-colony stimulating factor (GM-CSF). Other peripheral blood mononuclear cells from the same patients were incubated for 72 h in the presence of both mite antigen (10 microg/mL) and ONO-1078 (0.5, 1, or 10 microg/mL), followed by ELISA of the supernatant to quantify the cytokines. RESULTS: Production of IL-4, IL-5, and GM-CSF by mononuclear cells under stimulation with mite antigen was markedly suppressed when they were exposed to ONO-1078 at a concentration of 10 microg/mL. CONCLUSION: The results suggest that ONO-1078 acts directly on peripheral blood mononuclear cells and that blockade of leukotriene receptors on blood mononuclear cells by the cysteinyl-leukotriene receptor antagonist (LTRA) pranlukast (ONO-1078) can dose-dependently inhibit release of immunoreactive TH2-type cytokines (IL-3, IL-4, GM-CSF, and possibly IL-5), but not of the TH1-type cytokine IL-2, when stimulated by mite allergen in vitro. The data may provide clues to the mechanism by which a number of LTRA including zafirulukast and montelukast can reduce airway, sputum and blood eosinophil counts in clinical asthma. It supports animal studies showing that anti-IL-5 antibodies partially block cys-LT-induced airway eosinophilia, suggesting that cys-LTs may cause secondary release of IL-5 from an unknown cell-type. These findings indicate that ONO-1078 suppresses the production of IL-4 (a cytokine that affects IgG antibody production), IL-5, and GM-CSF (cytokines that affect eosinophil activation) by peripheral blood mononuclear cells under stimulation with specific antigens in patients with bronchial asthma. Because of its anti-inflammatory effects, ONO-1078 should be useful in the treatment of bronchial asthma.

Middle ear defects associated with the double knock out mutation of murine goosecoid and Msx1 genes.

A number of developmental regulatory genes, including homeobox genes, are dynamically expressed in the mammalian cephalic ectomesenchyme during craniofacial morphogenesis. Owing to the vast amount of gene knock out experiments, functions of such genes are now being revealed in the mammalian skeletal patterning process. The murine goosecoid (Gsc) and Msx1 genes are expressed during craniofacial development and each mutant mouse displays intriguing facial abnormalities including those of middle ear ossicles, suggesting that both genes play roles in spatial programming of craniofacial regions. In order to examine whether these genes could function in concert to direct particular craniofacial morphogenesis, double knock out mice were analyzed. The phenotype of the double mutant mice was restricted to the first arch derivatives and was apparently additive of the single gene mutant mice, implying region specific genetic interactions of these homeobox genes expressed in overlapping regions of middle ear forming ectomesenchyme. Our results also suggested that the patterning of distal portions of the malleus depends on the tympanic membrane, for which normal expressions of both the genes are prerequisite.

Effects of saiboku-to on dual-phase bronchoconstriction in asthmatic guinea pigs.

In recent years, bronchial asthma has come to be regarded pathologically as a chronic inflammatory disease of the respiratory tract. Inhalational steroids and antiinflammatory drugs are recognized as being effective against bronchial asthma. In this study, the effects of Saiboku-to, a Chinese herbal (Kampo) formulation, were investigated on asthmatic guinea pigs sensitized to ovalbumin (OA). Following 7-day administration of Saiboku-to (500 micrograms/kg), the late asthmatic response (LAR) to an antigen challenge was found to be inhibited. The number of eosinophils in fluid obtained by bronchoalveolar lavage (BAL) 4 h after antigen challenge was decreased while the infiltration of eosinophils and T-lymphocytes into the lung parenchyma was inhibited. These findings suggest that Saiboku-to has the potential to become a useful drug in the treatment of bronchial asthma.

Effects of saiboku-to on the survival of human eosinophils.

In recent years, bronchial asthma has come to be regarded as a chronic inflammatory disease of the respiratory tract, with mast cells, lymphocytes and eosinophils playing important roles in its pathogenesis. Proteins contained in eosinophil granules, especially major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPO), can cause tissue injury. When stimulated, eosinophils release mediators such as leukotriene C4 (LTC4) and platelet activating factors (PAF). Thus, they are recognized as effector cells that are actively involved in the development of allergic inflammation. In this study, eosinophils from healthy volunteers were used to investigate the effects of Saiboku-to on eosinophils whose survival had been prolonged through stimulation with eosinophil-activating cytokines such as interleukin (IL)-3, IL-5 and granulocyte macrophage colony stimulating factors (GM-CSF). As a result, the cytokine-enhanced survival of eosinophils was significantly shortened by the addition of Saiboku-to. These findings suggest that Saiboku-to has the potential to inhibit allergic responses by directly affecting eosinophils which are related to allergic inflammation.

Roles of histamine receptor in a guinea pig asthma model.

Histamine plays an important role in bronchoconstriction mediated by histamine receptors which provoke bronchial asthma attack. In this study, we measured H1 and H2 receptors in the guinea pig lung membrane fraction and obtained the following results. The maximum binding (Bmax) of H1 receptors in the guinea pig lung membrane fraction was significantly higher in the OA-sensitized group than that in the non-sensitized group, but affinity (Kd) did not differ between the groups. Otherwise, the maximum binding (Bmax) of H2 receptors in the guinea pig lung membrane fraction was significantly lower in the OA-sensitized group than that in the non-sensitized group. But affinity (Kd) did not differ between the groups. These findings suggest a close association of Histamine receptors both H1 and H2 in the pathology of asthma.

Microphthalmia (mi) mice display an aberrant bone trace element composition.

It has been recognized that bone trace element composition analysis provides clues when analyzing bone-related physiological conditions. Increasing numbers of bone-related genetic diseases have been identified recently. In this study, we have analyzed bone trace element composition in a genetic mutant animal model. Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types, including melanocytes, mast cells, and osteoclasts. Previous studies have shown that different alleles of the mi locus show osteopetrosis. In order to gain insights into the effects of a particular genetic defect on bone trace element composition and bone structure, we performed bone trace element composition analysis using inductively coupled plasma atomic emissions spectrometry (ICP-AES). Marked changes in bone trace element levels were found in vertebrate bones of mi mutant mice. The implications and possible applications of bone trace element analysis will be discussed in this article.

Aberrant regulation of bone trace elements in motheaten and osteopetrosis mutant mice.

Increasing numbers of genetic diseases involving bone development and models for these diseases have been identified recently. Analysis of these bone diseases have revealed that regulated action of multiple growth factors and subsequent signal transduction are essential for normal bone formation. In this paper, two murine mutant mice viable motheaten and osteopetrosis are analyzed. Mice with the recessive 'viable motheaten' mutation express a severe immunodeficiency syndrome and bone defects. Mutations at the motheaten locus were shown to be the result of aberrant splicing of the gene encoding hematopoietic cell phosphatase (Hcph). Mice homozygous for the osteopetrosis mutation develop congenital osteopetrosis due to a severe deficiency of osteoclasts. It has been recognized that bone trace element composition analysis helps to define bone-related physiological conditions. We have analyzed bone trace element composition in viable motheaten and osteopetrosis mutant animal models in this study. In order to gain insights into the effects of particular genetic defects on bone trace element composition, inductively coupled plasma atomic emissions spectrometry (ICP-AES) analysis was performed. Marked changes in bone trace element levels were found in limb bones of viable motheaten and osteopetrosis mutant mice. An assessment of these trace element spectrum in the two mutant models with respect to each genetic defects are discussed in this paper.

Dysregulation of trace element composition in ovariectomized cynomolgus monkey bones.

One of the challenging issues in modern biomedical science is the increasing number of osteoporosis patients due to the expansion of elderly populations. Among aging-related pathogenic changes, alterations in bone function and skeletal pathogenesis is a particularly important issue of concern. Osteoporosis is one of the most serious bone-related pathogenic states, as it causes serious loss of quality of life. Alterations in estrogen levels in accordance with aging are one of the key risk factors for osteoporosis. Complexed estrogen actions on bones can be traced by analyzing bone mineral components, as those elements accumulate as mineral complexes, reflecting the context of multiple cellular reactions such as bone resorption/osteogenesis. We have analyzed bone trace element composition in ovariectomized (OVX-treated) Cynomolgus monkey models in this study. In order to gain insights into the effects of such defects on bone trace element composition, inductively coupled plasma atomic emissions spectrometry (ICP-AES) analysis was performed. Marked changes in bone trace element levels were found in vertebral bones of OVX-treated Cynomolgus monkeys. An assessment of these trace element spectra in OVX model animals is discussed. These results could provide useful markers for understanding the physiological states of bones in postmenopausal women.