Rapid detection of human herpesvirus 8 DNA using loop-mediated isothermal amplification

The reliability of a loop-mediated isothermal amplification (LAMP) method for the detection of human herpesvirus 8 (HHV-8) DNA was evaluated. Although LAMP products were produced with the DNA sample extracted from BCP-1 cells, LAMP products were not produced with the DNAs from seven other human herpesviruses. The detection limit of the HHV-8 LAMP method was 100 copies of target sequence/tube. To determine whether the HHV-8 LAMP method could be used to quantify viral DNA, threshold times, which are defined as the time (in s) it takes to reach the threshold turbidity level (0.1), were measured for the amplification of serial dilutions of a DNA plasmid containing the target sequence. The standard curve possessed a correlation coefficient of 0.9428 with a slope of -84.079 and y-intercept value of 1936.2. Additionally, an attempt was made to detect viral DNA in 17 specimens collected from Kaposi's sarcomas and two cell lines obtained from primary effusion lymphomas. HHV-8 DNA was detected in 14 of the 17 Kaposi's sarcoma tissue samples and both of the primary effusion lymphoma cell lines. Viral DNA was not detected in HHV-8 LAMP-negative samples using the real-time PCR method.     

Ability of herpes simplex virus vectors to boost immune responses to DNA vectors and to protect against challenge by simian immunodeficiency virus

The immunogenicity and protective capacity of replication-defective herpes simplex virus (HSV) vector-based vaccines were examined in rhesus macaques. Three macaques were inoculated with recombinant HSV vectors expressing Gag, Env, and a Tat-Rev-Nef fusion protein of simian immunodeficiency virus (SIV). Three other macaques were primed with recombinant DNA vectors expressing Gag, Env, and a Pol-Tat-Nef-Vif fusion protein prior to boosting with the HSV vectors. Robust anti-Gag and anti-Env cellular responses were detected in all six macaques. Following intravenous challenge with wild-type, cloned SIV239, peak and 12-week plasma viremia levels were significantly lower in vaccinated compared to control macaques. Plasma SIV RNA in vaccinated macaques was inversely correlated with anti-Rev ELISPOT responses on the day of challenge (P value<0.05), anti-Tat ELISPOT responses at 2 weeks post challenge (P value <0.05) and peak neutralizing antibody titers pre-challenge (P value 0.06). These findings support continued study of recombinant herpesviruses as a vaccine approach for AIDS.     

Cloning and characterization of a TNF-like protein of Plecoglossus altivelis (ayu fish)

Ayu TNF cDNA contains an open reading frame of 708bp encoding 235 amino acids. Poly adeniration (A) signal and eight AU-rich sequences were present in 858bp 3' UTR. Southern blot analysis indicated that ayu TNF is single-copy gene. The genomic DNA sequence of ayu TNF, consisting of four exons and three introns, was shown to be conserved well throughout evolution from fish to mammals. The amino acid sequence of ayu TNF was shown to have 32-41% of amino acid identity to other known fish TNF, and about 30% of amino acid identity to mammalian TNFs. A phylogenetic analysis based on the amino acid sequence of TNF indicated that ayu has a distinctive evolutionary path. Also, two residues of cysteine important for the formation of the three-dimensional structure were conserved in ayu TNF. For the functional analysis, ayu TNF was inserted into expression vector pCold/TF, transferred into Chaperone Competent Cells BL21 (pKJE7); this produced soluble mature ayu recombinant TNF. Ayu recombinant TNF was shown to induce respiratory burst activity from ayu kidney. The above results indicate that ayu TNF plays an important role in phylaxis, as it does in mammals.     

Insecticide resistance in potential vector mosquitoes for West Nile virus in Japan

Culex pipiens complex is the significant vector mosquito of West Nile virus. To take stock of the current situation of insecticide susceptibilities and design an ideal mosquito control strategy, we collected Culex pipiens pallens Coquillet, Culex pipiens form molestus Forskal, and Culex quinquefasciatus Say from fields in Japan and conducted bioassays for five larvicides (fenitrothion, temephos, etofenprox, diflubenzuron, and pyriproxyfen) by using a larval dipping method. Among five insecticides tested, obvious reduced susceptibilities were observed for etofenprox, which is the only pyrethroid compound registered as a larvicide in Japan. Twenty-two of 56 colonies exhibited a >10% survival rate at the etofenprox concentration of 5.7 microg/ml, which is a 10 times higher concentration of the working solution. The LC50 of a colony collected from Fukuoka prefecture for etofenprox exceeded 60 microg/ml (resistance ratio >2,307), and this colony also exhibited cross-resistance to other pyrethroids, permethrin (299-fold) and phenothrin (1,200-fold). The insect growth regulators diflubenzuron and pyriproxyfen were found to be sufficiently effective enough to control Culex larvae present, but decreased sensitivities to these insecticides were slightly detected in some colonies of Cx. p. form molestus collected from urban areas. Several etofenprox-resistant colonies of Cx. p. form molestus exhibited simultaneously decreased susceptibilities to other insecticides, including temephos, diflubenzuron, and pyriproxyfen.     

Neutrophil activation and arteritis induced by C. albicans water-soluble mannoprotein-beta-glucan complex (CAWS)

We have established a mouse model which shows the symptoms of coronary arteritis after consecutive injections of CAWS, which is released from Candida albicans. In this study, we examined neutrophil activation in the initial period after CAWS injection intraperitoneally. During 10 min to 16 h after the injection, blood profiles and neutrophil functions were determined. At the same time, levels of inflammatory cytokines and chemokines in plasma were measured. Furthermore, level of ICAM-1 as a marker of lesion in arterial endothelial cells was measured. Counts of the peripheral leukocytes increased immediately after CAWS injection, especially involving neutrophil. In vitro sensitivity of neutrophils to stimuli was enhanced. Moreover, proinflammatory cytokines (IL-1beta, IL-12 and IL-6) increased in plasma initially followed by an increase in IL-10, G-CSF, MIP-2 and soluble ICAM-1. Locally, ICAM-1 message in arterial walls was significantly increased 16 h after CAWS injection. A decrease in C3 levels was observed in plasma, suggesting complement activation and consumption. In summary, neutrophil activation occurred after CAWS injection, followed by complement activation, and production of proinflammatory cytokines chemokines and G-CSF which may be involved in development of coronary arteritis.     

Determination of Nonspherical Morphology of Doxorubicin-Loaded Liposomes by Atomic Force Microscopy

The 3-D morphology of doxorubicin (DOX)–loaded liposomes with a size of circa 100 nm was characterized by atomic force microscopy in an aqueous environment. Prolate liposomes appear in accordance with linear expansion of DOX fiber bundles precipitated inside liposomes. Oblate and concave liposomes were simultaneously observed with increased DOX concentrations; however, their morphologies were not readily determined by 2-D cryo-TEM imaging. Precise data analysis of the 3-D parameters of each liposome allowed semiquantitative evaluation of the transformation of spherical liposomes into nonspherical—prolate, oblate, and concave liposomes. In addition, nonspherical liposomes became spherical on the replacement of the liposomal outer phase consisting of a sucrose solution, with water and subsequent water influx. All spherical liposomes transformed into oblate and concave liposomes with a return to hyperosmotic conditions, when transferred from water to sucrose solution. Furthermore, the concave liposomes did not appear under DOX incubation conditions (65°C), which could be due to the amorphous and supersaturated DOX inside the liposomes that restrained liposomal shrinkage. As atomic force microscopy has improved our ability to image 3-D morphologies of liposomes in various conditions, it is an alternative analytical tool to cryo-TEM and may have future applications in regulatory tests for quality control and assurance.     

Lifestyle factors and social ties associated with the frequency of laughter after the Great East Japan Earthquake: Fukushima Health Management Survey

Purpose

Although mental health problems such as depression after disasters have been reported, positive psychological factors after disasters have not been examined. Recently, the importance of positive affect to our health has been recognised. We therefore investigated the frequency of laughter and its related factors among residents of evacuation zones after the Great East Japan Earthquake of 2011.

Methods

In a cross-sectional study on 52,320 participants aged 20 years and older who were included in the Fukushima Health Management Survey in Japan’s fiscal year 2012, associations of the frequency of laughter with changes in lifestyle after the disaster, such as a changed work situation, the number of family members, and the number of address changes, and other sociodemographic, psychological, and lifestyle factors were examined using logistic regression analysis. The frequency of laughter was assessed using a single-item question: “How often do you laugh out loud?”

Results

The proportion of those who laugh almost every day was 27.1%. Multivariable models adjusted for sociodemographic, psychological, and lifestyle factors demonstrated that an increase in the number of family members and fewer changes of address were significantly associated with a high frequency of laughter. Mental health, regular exercise, and participation in recreational activities were also associated with a high frequency of laughter.

Conclusion

Changes in lifestyle factors after the disaster were associated with the frequency of laughter in the evacuation zone. Future longitudinal studies are needed to examine what factors can increase the frequency of laughter.