Epispadias and the associated embryopathies: genetic and developmental basis.

The abnormalities in the urogenital organs are frequently observed as human developmental diseases. Among such diseases, the defects in the upper part of external genitalia are rather rare named epispadias. The cleft in the dorsal part of external genitalia often reaches to the urethra. In general, the urogenital abnormalities accompany defects in the adjacent tissues and organs. The ventral body wall and bladder can also be affected in the patients with dorsal defects of the external genitalia. Therefore, such multiple malformations are often classified as bladder exstrophy and epispadias complex (BEEC). Because of the lower frequency of such birth defects and their early embryonic development, animal models are required to analyze the pathogenic mechanisms and the functions of responsible genes. Mutant mouse analyses on various signal cascades for external genitalia and body wall development are increasingly performed. The genetic interactions between growth factors such as bone morphogenetic proteins (Bmp) and transcription factors such as Msx1/2 and Isl1 have been suggested to play roles for such organogenesis. The significance of epithelial-mesenchymal interaction (EMI) is suggested during development. In this review, we describe on such local interactions and developmental regulators. We also introduce some mutant mouse models displaying external genitalia-body wall abnormalities.

Requirement of CD9 on the egg plasma membrane for fertilization.

CD9 is an integral membrane protein associated with integrins and other membrane proteins. Mice lacking CD9 were produced by homologous recombination. Both male and female CD9-/- mice were born healthy and grew normally. However, the litter size from CD9-/- females was less than 2% of that of the wild type. In vitro fertilization experiments indicated that the cause of this infertility was due to the failure of sperm-egg fusion. When sperm were injected into oocytes with assisted microfertilization techniques, however, the fertilized eggs developed to term. These results indicate that CD9 has a crucial role in sperm-egg fusion.

5α-Dihydrotestosterone negatively regulates cell proliferation of the periurethral ventral mesenchyme during urethral tube formation in the murine male genital tubercle.

Androgen is an essential factor involved in masculinization of external genitalia. Failure of the exposure to 5α-dihydrotestosterone (DHT) causes a hypoplastic penile size and urethral abnormality. The main pathology of hypospadias is defective urethral closure on the ventral side of the penis. Hormone-dependent genes are suggested as the causative factors. However, the detailed mechanisms of DHT functions on urethral tube formation remain unknown. Androgen is both a positive and negative regulator of cell proliferation. The roles of locally converted DHT in cell proliferation at the periurethral mesenchyme have not been elucidated. We revealed the expression pattern of 5α-reductase type 2 mRNA (Srd5a2) and local DHT distribution by direct measurement in this study. We also analyzed periurethral mesenchymal cell proliferation status using systematic three-dimensional (3D) reconstruction analyses. A prominent Srd5a2 expression and localized DHT distribution on the ventral side of the genital tubercle were detected. Cell proliferation was reduced in this mesenchymal region during urethral formation. The current results suggest the presence of the possible negative regulation of cell proliferation by DHT. Moreover, cell proliferation related to urethral tube formation was revealed to be DHT dose dependent. These data are expected to contribute to the understanding of the mode of regulation of cell proliferation related to urethral tube formation by DHT. These findings may also offer insight into the understanding of human hypospadias and related hormone-dependent factors.

The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants.

Gsc-1 expression marks cells with Spemann organizer, or axis-inducing, activity in the vertebrate gastrula. Gsc-1 knockouts, however, did not display phenotypes related to the early phase of expression. In this paper, additional phenotypes for the Gsc-1 mouse mutant are presented. Examination of the base of the cranium in the dorsal view revealed fusions and deletions in the midline of the prechordal chondrocranium. These defects were correlated with the sites of expression of Gsc-1 in the prechordal plate/foregut endoderm in the day 7.5/8.5 embryo. Gsc-1 expression in proximal limb buds was correlated with malformations of the shoulder and hip articulations. In addition, ribs in the seventh cervical vertebra were observed with low penetrance. The role of Gsc-1 during gastrulation and axial development is discussed in relation to possible compensatory interactions with other genes such as HNF-3beta and the recently identified Gsc-2 and Gsc-3 genes.

Unique roles of estrogen-dependent Pten control in epithelial cell homeostasis of mouse vagina.

Numerous studies support a role of phosphatase and tensin homolog deleted from chromosome 10 (Pten) as a tumor suppressor gene that controls epithelial cell homeostasis to prevent tumor formation. Mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen and provides a unique model for analyzing homeostasis of stratified squamous epithelia. We analyzed vaginal epithelium-specific Pten conditional knockout (CKO) mice to provide new insights into Pten/phosphoinositide-3-kinase (PI3K)/Akt function. The vaginal epithelium of ovariectomized (OVX) mice (control) was composed of 1-2 layers of cuboidal cells, whereas OVX CKO mice exhibited epithelial hyperplasia in the suprabasal cells with increased cell mass and mucin production. This is possibly due to misactivation of mammalian target of rapamycin and mitogen-activated protein kinase. Intriguingly, estrogen administration to OVX Pten CKO mice induced stratification and keratinized differentiation in the vaginal epithelium, as in estrogen-treated controls. We found that Pten is exclusively expressed in the suprabasal cells in the absence of estrogens, whereas estrogen administration induced Pten expression in the basal cells. This suggests that Pten acts to prevent excessive cell proliferation as in the case of other squamous tissues. Thus, Pten exhibits a dual role on the control of vaginal homeostasis, depending on whether estrogens are present or absent. Our results provide new insights into how Pten functions in tissue homeostasis.

Defective terminal differentiation and hypoplasia of the epidermis in mice lacking the Fgf10 gene.

Here, we characterized the skin and hair phenotype of mice lacking the fibroblast growth factor 10 gene (Fgf10), a newly identified member of the fibroblast growth factor family. Histological examination of Fgf10(-/-) newborn mouse skin revealed abnormalities in epidermal morphogenesis. The number of proliferating cells in the basal layer was decreased, the granular layer was hypoplastic and lacked distinctive keratohyaline granules and tonofibrils. The expression of loricrin, a marker of epidermal differentiation, was dramatically reduced. Despite the presence of Fgf10 transcripts in normal hair follicles, abnormalities of hair development were not observed in Fgf10(-/-) skin. These data suggest that Fgf10 is required for embryonic epidermal morphogenesis but is not essential for hair follicle development.

Loss of contractile activity of endothelin-1 induced by electrical field stimulation-generated free radicals.

1. Electrical field stimulation (EFS; 10 V, 10 Hz, 2 ms) of porcine coronary artery strips precontracted with 10 nM endothelin-1 (ET-1) for 5 min caused a biphasic response, consisting of a slight contraction during EFS and a marked and irreversible relaxation just after EFS. This irreversible relaxation after EFS has never been investigated. In the present study, we have investigated the mechanism of the relaxation after EFS. 2. The EFS-induced response was not affected by the presence or absence of endothelium and was insensitive to 10 microM tetrodotoxin (TTX). 3. In the presence of free radical scavengers (40 u ml-1 superoxide dismutase (SOD), 1200 u ml-1 catalase or 80 mM D-mannitol), the relaxation after EFS was significantly inhibited. Moreover, relaxation after EFS was not observed in porcine coronary artery strips precontracted with 20 mM KCl. 4. In a cascade experiment, EFS of Krebs-Ringer solution containing 10 nM ET-1 induced marked suppression of the contractile activity of ET-1 in porcine coronary artery strips, which was in accord with the observed decrease in release of immunoreactive ET-1 (ir-ET-1). This effect of EFS was significantly inhibited by each of the free radical scavengers, 3 mM vitamin C, 40 u ml-1 SOD, 1200 u ml-1 catalase and 80 mM D-mannitol. 5. The exchange of 95% O2/5% CO2 gas for 95% N2/5% CO2 gas significantly inhibited the EFS-induced decrease in release of ir-ET-1. 6. Neither superoxide anions generated by xanthine (10 JM) plus xanthine oxidase (0.1 micro ml-1) nor hydrogen peroxide (10 microM) exogenously added to Krebs-Ringer solution containing 10 nM ET-1 affected the level of ir-ET-1.7. Generation of hydroxyl radicals was detected in the EFS-applied Krebs-Ringer solution. The EFS-induced generation of hydroxyl radicals was dependent on the period of stimulation and 02-bubbling, and significant generation of hydroxyl radicals was detectable with stimulation of over 5 min.Moreover, hydroxyl radicals generated in 50 mM NaCl solution containing 10 nM ET-1 by H202 plus Fe2 , i.e. the Fenton reaction, significantly decreased the level of ir-ET-l.8. These findings suggest that oxygen-derived hydroxyl radicals generated by EFS of porcine coronary artery strips inactivate ET-1, probably by structural modification. Thus, porcine coronary artery strips precontracted with ET-1 are potently relaxed by EFS.

Hepatitis B. Cytologic localization of virus antigens and the role of the immune response.

Antigens of the hepatitis B virus have been localized within liver tissue by various immunologic, histochemical, and electron microscopic methods. The abundance and distribution of these virus antigens are in part determined by the host immune response. This interaction of immune mechanisms with the hepatitis B virus may be related to the pathogenesis and natural history of human hepatitis B virus infection.

Centrilobular nodules correlate with air trapping in diffuse panbronchiolitis during erythromycin therapy.

BACKGROUND: Low-dose erythromycin therapy improves airflow limitation and airway inflammation in patients with diffuse panbronchiolitis (DPB). However, to our knowledge there has been no study to determine whether physiologic improvement during erythromycin therapy correlates with radiologic findings. STUDY OBJECTIVE: To clarify whether improvement in pulmonary function correlates with specific changes on chest CT. DESIGN: The relationship between five CT findings and five pulmonary function parameters was evaluated before and 3 months after low-dose erythromycin therapy in 24 patients with DPB retrospectively. RESULTS: After erythromycin therapy, the predicted percentage of vital capacity (%VC; 87.0 +/- 3.07% vs 98.9 +/- 3.39%; p = 0.00006) and 50% of the maximum midexpiratory flow rate of FVC (1.41 +/- 0.26 L/s vs 1.61 +/- 0.27 L/s; p = 0.03) significantly increased, and the residual volume/total lung capacity ratio (RV/TLC%; 44.5 +/- 1.93% vs 40.7 +/- 1.83%; p = 0.0019) significantly decreased, but the FEV(1) to FVC ratio and 25% of the maximum expiratory flow rate of FVC did not. In five CT findings, centrilobular nodules (3.7 +/- 0.4 vs 1.5 +/- 0.3; p = 0.0001), peripheral bronchiolar wall thickness (3.8 +/- 0.3 vs 2.6 +/- 0.4; p = 0.0007), and peripheral bronchiolectasis (2.8 +/- 0.3 vs 2.2 +/- 0.4; p = 0.0058) had significantly improved, whereas low attenuation area and central bronchiectasis had not. There were positive correlations of improved scores of centrilobular nodules with improved %VC (r = 0.58, p = 0.0062) and RV/TLC% (r = 0.64, p = 0.0022). CONCLUSIONS: Decreased air trapping in DPB correlates with an improvement of centrilobular nodules, which reflects the obstructive lesions of bronchioles during the erythromycin therapy.

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.

Immunohistochemical study of the distribution of intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 in chronic type B hepatitis.

The expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) in the livers of 11 patients with chronic hepatitis B was studied immunohistochemically by light and electron microscopy to clarify the role of these adhesion molecules in tissue damage in chronic hepatitis B. On hepatocytes, ICAM-1 expression was confined to the bile canalicular surface when the liver inflammation was mild. In contrast, when the liver inflammation was severe, ICAM-1 was distributed on the entire surface of the hepatocyte, including the sinusoidal and lateral membranes; lymphocytes which were mostly positive for LFA-1, were often observed invading deeply among these hepatocytes. The degree of ICAM-1 expression on the hepatocytes was also related to the expression of HLA class 1 antigen. In liver showing diffuse expression of ICAM-1 on the hepatocytes, strong expression of HLA class 1 antigen was observed, and amounts of HBV in the liver were decreased. Diffuse expression of ICAM-1 and HLA class 1 antigen was mostly observed after acute exacerbation of liver inflammation. These results suggest that the ICAM-1/LFA-1 pathway is involved in the immunological mechanism responsible for liver cell damage in chronic hepatitis B.

Trace element composition and histological analysis of rat bones from the space shuttle.

The physiological and pharmacobiological changes associated with space flight are of greater concern. Exposure to a weightless environment has been shown to have numerous effects on body composition and organ functions. Alterations include decreases in muscle and liver mass, changes in bone structure and integrity, and changes in cardiovascular functions. Zero-gravity in particular has been reported to inhibit several physiological processes of bone formation, retard bone growth and impair the mechanical properties of bones. This report examines the effect of 14 days of spaceflight on the bone trace element compositions of rapidly growing rats. Marked changes of bone trace element contents were found in either weight-bearing bones or non-weight-bearing bones, depending on the metal species. Histological examination revealed an irregular thickening of the endosteal surface of the cortical bone (thoracic vertebrae) of the in-flight rat, whereas it was uniform in the ground control. We suggest that the microgravity environment causes several bone alternations, such as abnormal trace element compositions and defects in vertebral maturation.

Clinical usefulness of the branched DNA assay version 2 in predicting the efficacy of Interferon treatment in a group of chronic HCV patients based on serotype.

Interferon (IFN) response depends upon pretreatment viral loads and viral genotype/serotype. This study investigated the difference in response to IFN in different viral load groups of 96 chronic hepatitis C patients and compared version 1 (bDNA1.0) and version 2 (bDNA2.0) of the branched DNA assay, according to serotype. On univariate analysis, viral load (P=0.0001, by bDNA 1.0; P=0.0020, by bDNA 2.0,), serotype (P=0.0053) and age (P=0.0073) were significant predictors of IFN response. On multivariate analysis, serotype (odds ratio, 5.44; 95% confidence interval, 1.94-15.24; P<0.01) was a stronger predictor of IFN response than age or viral load (by bDNA2.0). In very high (>6.7 log eq/ml), high (6.0 approximately 6.69 log eq/ml) and low (<6 log eq/ml) viral load groups (by bDNA2.0), complete response was 25, 55 and 92.6% in serotype 2 (sero-2), and 10, 20 and 71.4% in serotype 1 (sero-1), respectively. In sero-2, bDNA2.0 can detect high viral loads underestimated by bDNA1.0. In a low viral load group (by bDNA2.0), IFN response is dependent upon serotype; sero-2 responded better than sero-1. However, in high and very high viral load groups, sensitivities of bDNA1.0 and bDNA2.0 are not effective in clinically distinguishing CR from non-response, and aid in patient selection for IFN therapy.

Effect of a moderately energy-restricted diet on obese patients with fatty liver.

The effects of a moderately energy-restricted (25 kcal/kg) diet on liver-function tests, anthropometric measurements, mononuclear-cell phospholipid fatty acid, lymphocyte blastogenesis, and plasma prostaglandin E2 and alpha-tocopherol levels were observed at weeks 0, 8, and 24 in 14 obese patients with fatty liver. Serum aminotransferase levels were improved significantly, with decreases in the body mass index and waist circumference. Decreases in energy intake from carbohydrate and increases in intake of vitamin A, vitamin C, and vegetables were observed at week 24. In mononuclear-cell phospholipids, linoleic acid (18:2omega 6), which was significantly lower in patients than in controls at week 0, was increased at week 24. In contrast, arachidonic acid was decreased. Plasma prostaglandin E2 levels were significantly lower in patients than in controls at week 0 and increased at week 24. The mononuclear-cell response for phytohemagglutinin correlated with 18:2omega 6 in mononuclear-cell phospholipids (r = 0.692, P < 0.01). Improvement of the serum alanine-aminotransferase level correlated with an increase in the plasma alpha-tocopherol level (r = -0.667, P < 0.01) and increases in consumption of vitamin A, omega 3 polyunsaturated fatty acids, and vegetables. These findings suggest that a hypoenergetic diet rich in omega 3 polyunsaturated fatty acids and antioxidants might be beneficial for obese patients with fatty liver.

Different element ratios of red cosmetics excavated from ancient burials of Japan.

Marker elements of red cosmetics, collected from ancient burials of Matsuyama, Tokushima and Nara Japan, were determined by emission spectrometry (ICP/AES). The mass ratios of Hg, Fe, Cu, and Zn were different between samples. Element levels were compared with reference to relative amounts of sulfur. Of the possible contaminants from the bone and sand of burials, the relative amounts of Hg and Fe to S were most commonly available to evaluate the difference between the cosmetics. The cosmetics were divided into four groups; type I (high Hg with less Fe), type II (both moderate Hg and Fe), type III (moderate Hg with high Fe) and type IV (less Hg with high Fe). The main constituents of cosmetics are mercury sulfide (cinnabar) or ferric oxide mixed with trace metals. Zinc contents differ between the Fe and Hg amounts for the three areas. Cosmetic compositions varied with each burial site, suggesting that they were derived from different mines of ancient Japan.

Immunological mechanism of chronic liver injury in viral hepatitis.

Chronic hepatitis B and chronic hepatitis C centering on immunohistochemical studies of the liver were investigated as possible mechanisms of the chronicity of viral hepatitis. Although liver cell injury was presumed to be caused by CTL in both chronic hepatitis B and C, other mechanisms of cell injury were also presumed with chronic hepatitis, type C, including autoimmune mechanisms.

Malignant pheochromocytoma with hepatic metastasis diagnosed 20 years after resection of the primary adrenal lesion.

A 59-year-old woman developed multiple hepatic tumors 20 years after resection of pheochromocytoma of the left adrenal gland. Thin-needle aspiration biopsy of the tumor at segment 2 of the liver under ultrasound control showed histology compatible to pheochromocytoma. Then, on the basis of the diagnosis of hepatic metastasis of malignant pheochromocytoma, transcatheter arterial embolization (TAE) was performed for the purpose of the treatment of hepatic metastasis. After TAE, the size of the hepatic metastatic lesions was decreased. The present case suggests the necessity of long-term follow-up in pheochromocytoma cases.

Use of bronchopulmonary lavage for eliminating inhaled fume particles from a patient with arc welder's lung.

A 42-year-old man, who had worked as a welder for 20 years, was admitted to our hospital complaining of a dry cough. A chest radiograph showed diffuse small nodular shadows and chest computed tomography revealed small patchy opacities. A transbronchial lung biopsy specimen showed welding fume particles mainly located in alveolar space with mild fibrosis of alveolar septa. In order to prevent further fibrosis, bronchopulmonary lavage (BPL) was performed to eliminate the fume particles. The amount of iron particles derived from the total lavage fluid was 911.7 mg.

Evaluation of antibody against AR142, a synthetic peptide derived from the core sequence of hepatitis C virus, in the diagnosis of non-A, non-B chronic liver disease.

We evaluated serum anti-hepatitis C virus (HCV) using a synthetic peptide (AR142) which includes an epitope in the core region of HCV. The incidence of anti-AR142 in 98 patients with type non-A, non-B chronic liver diseases (NANB-CLD) was 89.8%, while all the 28 patients with non-type C chronic liver diseases were negative for anti-AR142. Among 98 NANB-CLD patients, 74 were positive for both anti-AR142 and anti-C100-3, 23 showed discordant results, and one was positive for neither. Eighty-one NANB-CLD patients underwent reverse transcription-polymerase chain reaction assay to detect viremia and 76 (93.8%) had a detectable level of HCV-RNA. Titers of anti-AR142 were not different among groups of different disease activities, genotypes of HCV, nor amount of serum HCV-RNA. These observations suggest that anti-AR142 could be a useful marker for chronic HCV infection.