Prevalence and Outcomes of Acute Hepatitis B in Okayama, Japan, 2006-2010

Hepatitis B virus (HBV) is one of the major viruses causing acute hepatitis. Recently, the incidence of acute hepatitis with genotype A has been increasing in Japan. The aim of this study was to investigate acute hepatitis B (AHB) in Okayama prefecture, with special attention to HBV genotype A. AHB patients who visited one of 12 general hospitals in Okayama prefecture between 2006 and 2010 were retrospectively analyzed. Over the course of the study period, 128 patients were diagnosed with AHB. Sexual transmission was supposed in the majority of patients (78 patients, 61%), including 59 (76%) having sex with heterosexual partners. The genotypes of HBV were assessed in 90 patients (70%), of whom 27 patients were infected with genotype A, 5 with genotype B, and 58 with genotype C. The prevalence of genotype A was significantly higher among male patients (28.7%), aged 20-29 (35.6%, p＜0.01), among men who had sex with men (100%, p＜0.005), and among patients having sex with unspecified partners (44.8%, p＜0.005). Genotype A was not a significant factor associated with delayed HBsAg disappearance. Caution should be exercised with regard to sexually transmissible diseases in order to slow the pandemic spread of AHB due to genotype A

Seismic exploration at Fuji volcano with active sources : The outline of the experiment and the arrival time data

Fuji volcano (altitude 3,776m) is the largest basaltic stratovolcano in Japan. In late August and early September 2003, seismic exploration was conducted around Fuji volcano by the detonation of 500 kg charges of dynamite to investigate the seismic structure of that area. Seismographs with an eigenfrequency of 2 Hz were used for observation, positioned along a WSW-ENE line passing through the summit of the mountain. A total of 469 seismic stations were installed at intervals of 250-500 m. The data were stored in memory on-site using data loggers. The sampling interval was 4 ms. Charges were detonated at 5 points, one at each end of the observation line and 3 along its length. The first arrival times and the later-phase arrival times at each station for each detonation were recorded as data. P-wave velocities in the surface layer were estimated from the travel time curves near the explosion points, with results of 2.5 km/s obtained for the vicinity of Fuji volcano and 4.0 km5/s elsewhere

Cytokine Gene Expression in CD4 Positive Cells of the Japanese Pufferfish, <i>Takifugu rubripes</i>

<div><p>CD4⁺ T (Th) cells are a central component of the adaptive immune response and are divided into distinct sets based on their specific cytokine production pattern. Several reports have suggested that fish possess Th subset activity similar to that of mammals. The aim of the present study was to isolate CD4⁺ T cells from the blood of Japanese pufferfish, <i>Fugu rubripes</i>, and to characterize their cytokine expression profile. We produced a specific antibody against <i>Fugu</i> CD4 and performed cell sorting with the magnetic activated cell sorting system. Sorted <i>Fugu</i> CD4⁺ cells were characterized by morphology and expression analysis of cell marker genes. <i>Fugu</i> CD4⁺ cells expressed T-cell marker genes but not macrophage or B-cell marker genes. In addition, peripheral blood lymphocytes were stimulated with lipopolysaccharide (LPS), polycytidylic acid (polyI:C), concanavalin A (ConA) prior to sorting, and then Multiplex RT-PCR was used to examine the expression of Th cytokines by the stimulated <i>Fugu</i> CD4⁺ cells. LPS and polyI:C stimulation upregulated the expression of Th1, Th17 and Treg cytokines and downregulated the expression of Th2 cytokines. ConA stimulation upregulated the expression of all Th cytokines. These results suggest that fish exhibit the same upregulation of Th-specific cytokine expression as in mammals.</p></div

Expression of cell marker genes in <i>Fugu</i> PBLs, monocytes, lymphocytes and T cells.

<p>The cell marker genes used were a monocyte marker, <i>CSF1R1</i>, <i>-2</i>, a B-cell marker, <i>IgL</i>, <i>IgM</i> and T-cell markers, <i>CD3ε</i>, <i>CD4</i>, <i>CD4REL</i>, <i>CD8α</i>, <i>TCRα</i>, <i>TCRβ</i>, <i>TCRγ</i> and <i>TCRδ</i>.</p

Reactivity of anti-<i>Fugu</i> CD4 Ab to <i>Fugu</i> CD4⁺-CHO cells.

<p>Immunofluorescence of A) control CHO cells and B) <i>Fugu</i> CD4⁺-CHO cells. Cells were incubated with anti-<i>Fugu</i> CD4 Ab as primary Ab and goat anti-rabbit IgG (PE) as secondary Ab and DAPI to mark cell nuclei. Scale bar equals 20 µm. Stained cells C) control CHO cells and D) <i>Fugu</i> CD4⁺-CHO cells were also analyzed by flow cytometry. The setting of negative gate was used with the only secondary antibody reaction to the cells analysis (gray peak).</p

Discrimination of <i>Fugu</i> PBLs, monocytes, lymphocytes and T cells.

<p>A) The morphology of purified cells was examined by microscope after May-Grünwald-Giemsa staining. G, L and M indicate granulocytes, lymphocytes and monocytes, respectively. B) Scattergram of the flow cytometric profile of purified cells. Different cell subpopulations were identified on the basis of their size and complexity, and cellular debris was excluded. Three populations (granulocytes, monocytes and lymphocytes) in PBLs were isolated by analytical gates. The monocyte, lymphocyte and T cell populations were mostly pure, and contamination with other populations was low.</p

Expression analysis of <i>Fugu</i> cytokine genes in CD4⁺ cells sorted from PBLs under immunostimulatory conditions.

<p>RNA isolated from CD4⁺ cells incubated with 20 µg/ml LPS, 20 µg/ml polyI:C or 20 µg/ml ConA for 0 (cont.), 6, 12 and 24 h. <i>Fugu</i> cytokine mRNA transcripts were determined by multiplex RT-PCR and standardized to the respective β-actin mRNA. The analysed genes were as follows: 1) <i>IL-2</i>, 2) <i>IFN-γ,</i> 3) <i>TNF-α</i>, 4) <i>IL-4/13A</i>, 5) <i>IL-4/13B</i>, 6) <i>IL-17A/F-3</i>, 7) <i>TGF-β1</i>, and 8) <i>IL-10</i>. Data are presented as mean ± S.D. of triplicate samples. *<i>P</i><0.01 as compared to the control (upregulation). The relative expression level is expressed as arbitrary units where one unit is equal to the average expression level of each cytokine gene in CD4⁺ cells from unstimulated PBLs. Gray, Blue, red and green bars indicate HBSS, LPS, polyI:C and ConA-stimulations, respectively.</p

Relative expression levels analyzed by real-time PCR.

<p>The comparative threshold cycle (Ct) method was used to determine relative transcript levels, using β-actin as a housekeeping control. The analysed genes were as follows: 1) <i>CD4</i>, 2) <i>CDREL</i>, 3) <i>CD8α</i>, 4) <i>CD28</i>, 5) <i>CD 154</i>, 6) <i>T-bet</i>, 7) <i>GATA-3</i> and 8) <i>FoxP3</i>. Data are presented as mean ± S.D. of triplicate samples.</p

The confirmation of antibody specificity using Western blotting.

<p>The recombinant CD4 was detected with A) anti-FLAG Ab and B) anti-<i>Fugu</i> CD4 Ab. C) <i>Fugu</i> CD4-CHO cells and D) <i>Fugu</i> PBLs lysates were detected with anti-<i>Fugu</i> CD4 Ab. The position of molecular weight markers is indicated to the left of each Western blot. The arrow indicates the predicted size of <i>Fugu</i> CD4 protein. Cont, translated with control plasmid. Nt, the lysate of non-transfected CHO.</p

Immunofluorescence stains of <i>Fugu</i> CD4⁺ cells in PBLs, monocytes, lymphocytes and T cells.

<p>A), B) Cells were reacted with anti-<i>Fugu</i> CD4 Ab as primary Ab and goat anti-rabbit IgG (PE) as secondary Ab and DAPI to mark cell nuclei. Scale bar equals 20 µm. C) Cells stained with anti-<i>Fugu</i> CD4 Ab were also analyzed by flow cytometry.</p