Clinical significance of a highly sensitive enzyme immunoassay of hepatitis B surface antigen using a novel electron spin resonance technique.

We developed a highly sensitive enzyme immunoassay (EIA), the p-AP/HHTIO method, that detects serum hepatitis B surface antigen (HBsAg) by measuring stabilized nitroxide radicals using a novel electron spin resonance technique [Matsuo et al. (1998) Free Radic Biol Med 25:929-935]. To demonstrate the clinical significance of this method and to reveal occult hepatitis B virus (HBV) infection in patients, we used the method to analyze serum samples of 30 patients with acute or fulminant hepatitis who were negative for HBsAg by standard EIA, and those of seven chronic HBV carriers who became negative for HBsAg during a follow-up period by standard EIA. We also examined serum HBV DNA by amplification of the HBV S gene, using the polymerase chain reaction (PCR) technique. The p-AP/HHTIO method showed that 9 of 20 (45%) patients with acute hepatitis and 2 of 10 (20%) with fulminant hepatitis were positive for HBsAg; PCR detected HBV DNA in these HBsAg-positive patients. Antibody against hepatitis B core antigen was detected in one patient with fulminant hepatitis. The p-AP/HHTIO method demonstrated prolonged seropositivity of HBsAg even after standard EIA showed a loss of HBsAg in all seven HBV carriers. Our p-AP/HHTIO method is useful for screening and diagnosing HBV infection in patients with liver diseases who are negative for conventional HBV-related serological markers.     

The three-dimensional microvascular arrangement around rat dorsal hairs as revealed by scanning electron microscopy

The three-dimensional microvascular arrangement around the dorsal hairs in vascular corrosion casts of adult Wistar rats was studied by scanning electron microscopy. Each anagen dorsal hair was surrounded by a basket-like capillary network, which was supplied by the branches of the subcutaneous artery and drained into the veins continuous with the subcutaneous vein. The capillary network surrounding the anagen dorsal hair was denser at its lower part, and became more sparse at its upper part. Transmission electron microscopy showed that the capillaries around the hair bulb possessed fenestrations. Our findings indicate that the microvascular arrangement around the anagen dorsal hair is so organized as to supply the hair bulb, which is the most important area for hair growth, with abundant blood. This study was presented at the 24th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Okayama, September 17–19, 1992.     

Graphic and movie illustrations of human prenatal development and their application to embryological education based on the human embryo specimens in the Kyoto collection.

Morphogenesis in the developing embryo takes place in three dimensions, and in addition, the dimension of time is another important factor in development. Therefore, the presentation of sequential morphological changes occurring in the embryo (4D visualization) is essential for understanding the complex morphogenetic events and the underlying mechanisms. Until recently, 3D visualization of embryonic structures was possible only by reconstruction from serial histological sections, which was tedious and time-consuming. During the past two decades, 3D imaging techniques have made significant advances thanks to the progress in imaging and computer technologies, computer graphics, and other related techniques. Such novel tools have enabled precise visualization of the 3D topology of embryonic structures and to demonstrate spatiotemporal 4D sequences of organogenesis. Here, we describe a project in which staged human embryos are imaged by the magnetic resonance (MR) microscope, and 3D images of embryos and their organs at each developmental stage were reconstructed based on the MR data, with the aid of computer graphics techniques. On the basis of the 3D models of staged human embryos, we constructed a data set of 3D images of human embryos and made movies to illustrate the sequential process of human morphogenesis. Furthermore, a computer-based self-learning program of human embryology is being developed for educational purposes, using the photographs, histological sections, MR images, and 3D models of staged human embryos.     

An electron microscopic and immunohistochemical study of Merkel cell carcinoma

Two cases of Merkel cell carcinoma (MCC) were examined by electron microscopy and immunohistochemistry. Histologically, tumor cells, which extended from the dermis into the subcutis, showed anastomosing bands with partial trabecular pattern. The ultrastructural study showed tumor cells in case 1 with numerous neurosecretory granules. The number of granules in case 2, however, was smaller compared with that in case 1. Perinuclear bundles of filaments were present in case 2, but few bundles were observed in case 1. By immunohistochemistry, cytokeratin (CK)-8, -18, -19, and -20 and epithelial membrane antigen were stained positively within tumor cells in both cases. It was interesting that staining patterns of chromogranin A and of neuron-specific enolase were different in the two cases. These data indicated that CK-20 is a useful marker for diagnosing MCC and that ultrastructural and immunohistochemical differences in both cases were the result of phenotypic variation.     

Etiological Study of Diarrheal Patients in Vientiane, Lao People’s Democratic Republic

The etiological agents of diarrhea in Vientiane, Lao People’s Democratic Republic (Lao PDR), were studied in the period from October 1996 to August 1997. A total of 880 patients with diarrhea visiting medical facilities were examined for Shigella, Salmonella, diarrheagenic Escherichia coli, Vibrio, Aeromonas, Campylobacter, and rotavirus. Shigella spp., heat-stable enterotoxin (ST)-producing E. coli, and serogroup-based enteropathogenic E. coli were found to be the main organisms causing diarrhea in Vientiane, with frequencies of 16.8% (148 of 880), 17.2% (111 of 645), and 11.0% (97 of 880), respectively. Relatively low incidences were observed in the cases of Salmonella spp., (0.6%; 5 of 880), Campylobacter spp. (4.4%; 39 of 880), and rotavirus (6.1%; 9 of 148), and no isolates of V. cholerae O1 or O139 or Aeromonas were recovered. An analysis of the incidences of enteropathogens with respect to age and seasonal variations demonstrated that the frequencies of isolation of Shigella spp. and heat-labile enterotoxin-producing E. coli were significantly higher in those aged 1 to 5 years than in those younger than 1 year of age and those older than 5 years of age (P < 0.0001 and P < 0.05, respectively) and that the frequencies of isolation of Shigella spp. and ST-producing E. coli were significantly higher in the rainy season than in the dry season (P < 0.005 and P < 0.001, respectively). Almost all strains of Shigella spp. tested were resistant to ampicillin, tetracycline, and erythromycin and were susceptible to cefdinir and ofloxacin. This is the first intensive and longitudinal study to define the etiologic agents of diarrheal diseases in Lao PDR.     

Mutation analysis of two Japanese patients with Fanconi-Bickel syndrome

Fanconi-Bickel syndrome (FBS), or glycogen storage disease type XI, is a rare autosomal recessive disorder characterized by hepatorenal glycogen accumulation, Fanconi nephropathy, and impaired utilization of glucose and galactose. Recently, this disease was elucidated to link mutations in the glucose transporter 2 (GLUT2) gene. Only three mutations in three FBS families have been reported. Therefore, it is important to elucidate mutations in the GLUT2 gene in FBS by answering the question of whether the syndrome is a single gene disease. In this report, we describe two patients in two unrelated families clinically diagnosed with FBS. No mutation in the entire protein coding region of the GLUT2 gene was detected in patient 1, which suggested that no mutation existed in the GLUT 2 gene, or that some mutations had affected the expression of the GLUT 2 gene. In patient 2, a novel homozygous nonsense mutation (W420X, Trp at codon 420 to stop codon) was detected. These results support the correlation between GLTU2 gene mutation and FBS syndrome. However, many patients must be analyzed to determine whether other genes are involved in FBS. Received: July 16, 1999 / Accepted: September 3, 1999     

Neutralizing activity of the antibodies against two kinds of envelope glycoproteins of Sendai virus

Summary Murine monoclonal antibodies against the fusion (F) and hemagglutininneuraminidase (HN) proteins of Sendai virus (SV) were prepared and studied on their antiviral activities, particularly on the neutralization of infectivity. On the analysis with solid phase competitive ELISA, 26 anti-HN antibodies were divided into at least four groups (HN-I, -II, -III and -IV). Antigenic sites recognized by the HN-I, -II, and -III group antibodies topographically separate from each other. Sites recognized by the HN-IV group antibodies overlaps partially with ones recognized by the HN-I, HN-II and -III group antibodies. The antibodies belonging to the HN-III group highly neutralize the infectivity of SV and weakly or not at all inhibit the hemagglutination (HA). In contrast, the HN-IV group antibodies strongly inhibit HA, but weakly neutralize the infectivity. Adsorption of SV to chicken red blood cells or L cells is inhibited by the HN-IV antibodies, but scarcely by the HN-III antibodies. On the other hand, incubation with HN-III antibodies of HeLa cells that have been preadsorbed with SV at 4° C, followed by culture at 37° C, causes inhibition of infection, but the HN-IV antibodies do not effectively interfere with such infection.The competitive ELISA showed that 17 anti-F antibodies were divided into two groups (F-I and -II). Two antigenic sites recognized by the antibodies, however, seem to be near to each other because a certain competition is observed between the antibodies of both groups. Two of the seven antibodies belonging to the F-II group inhibit the hemolysis activity and also neutralize the infectivity of SV, but the other five F-II antibodies do not. One of the anti-F antibodies has a low HI activity, and, in competition tests, competes with one of the anti-HN antibodies (HN-IV).With 2 Figures     

IL-6-STAT3 controls intracellular MHC class II alphabeta dimer level through cathepsin S activity in dendritic cells

We found IL-6-STAT3 pathway suppresses MHC class II (MHCII) expression on dendritic cells (DCs) and attenuates T cell activation. Here, we showed that IL-6-STAT3 signaling reduced intracellular MHCII alphabeta dimmer, Ii, and H2-DM levels in DCs. IL-6-mediated STAT3 activation decreased cystatin C level, an endogenous inhibitor of cathepsins, and enhanced cathepsin activities. Importantly, cathepsin S inhibitors blocked reduction of MHCII alphabeta dimer, Ii, and H2-DM in the IL-6-treated DCs. Overexpression of cystatin C suppressed IL-6-STAT3-mediated increase of cathepsin S activity and reduction of MHCII alphabeta dimer, Ii, and H2-DM levels in DCs. Cathepsin S overexpression in DCs decreased intracellular MHCII alphabeta dimer, Ii, and H2-DM levels, LPS-mediated surface expression of MHCII and suppressed CD4(+) T cell activation. IL-6-gp130-STAT3 signaling in vivo decreased cystatin C expression and MHCII alphabeta dimer level in DCs. Thus, IL-6-STAT3-mediated increase of cathepsin S activity reduces the MHCII alphabeta dimer, Ii, and H2-DM levels in DCs, and suppresses CD4(+) T cell-mediated immune responses.     

C and V proteins of Sendai virus target signaling pathways leading to IRF-3 activation for the negative regulation of interferon-beta production

We here report a molecular basis for downregulation of interferon (IFN)-beta production by V and C proteins of Sendai virus (SeV). The infection of HeLa cells with SeV poorly induced IFN-beta even if the expression of C/C' was disrupted. In contrast, when the expression of C/C'/Y1/Y2 or V/W was disrupted, SeV infection strongly induced IFN-beta production and significantly activated the interferon regulatory factor (IRF)-3 pathway. The independent expression of C or V inhibited the double-stranded (ds) RNA- or Newcastle disease virus (NDV)-induced activation of IRF-3 and NF-kappa B, as well as the IFN-beta promoter. This inhibitory effect was also observed when Y1, Y2, or a C-terminal half fragment (aa 85-204) of C was independently expressed. Phosphorylation and homodimer formation of IRF-3 were suppressed not only in cells infected with SeV capable of expressing both C/C'/Y1/Y2 (or Y1/Y2) and V/W, but also in HeLa cells constitutively expressing Y1. These results suggest that C, Y1, Y2, and V block signaling pathways leading to IRF-3 activation to downregulate IFN-beta production.