Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment

<p>Abstract</p> <p>Background</p> <p>It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV.</p> <p>Results</p> <p>We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-α stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1) and U1 cells (latently infected U937 cells with HIV-1) displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-α, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane.</p> <p>Conclusion</p> <p>Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute infection. Our data also suggest that HIV expression may lead to the upregulation of certain host cell molecules that are recruited to sites of particle assembly, possibly coordinating particle production.</p

Analysis of Morphology and Infectivity of Measles Virus Particles

The measles virus (MeV) shows polymorphisms in morphology and viral particle size, however, the localization of viral proteins and infectivity in viral particles of different sizes have not been well characterized. To determine the localization of viral proteins and the infectivity of viral particles of different sizes, MeV-infected cells and their culture supernatant were analyzed by electron microscopy and membrane filter fractionation. The sizes of MeV-like particles were distributed between 50 and 1000 nm and the major distribution peak was found for particles with diameters of 350-400 nm. The MeV M protein was lined under the envelope of all MeV-like particles and membrane-filter-fractionated MeVs of all sizes showed infectivity. These findings suggest that MeV particles, particularly large particles, can be used as a vaccine by designing a chimera virus containing antigens or genome of other virus species

Unique Enhancement of Multinuclear Giant Cell Formation in AGS Cell Line Infected with Helicobacter pylori

Helicobacter pylori (H. pylori) causes pathological changes of gastric epithelial cells induced by pathogenic factors such as CagA and VacA, namely hummingbird cells (HBC) formation and vacuolization, respectively, in cultured cell lines. Cytopathic effects of other pathogenic factors produced by H. pylori have not been reported. In this study, we examined whether H. pylori induces unique morphological changes other than HBC formation and vacuolization, and we established a new marker of the bacterial infection in vitro. The cytotoxicity of H. pylori was examined in the AGS cell line, and a new morphological change, namely multinuclear giant cells (MNGC) formation, was observed in this cell line. The enhancement of MNGC formation was observed following H. pylori infection but was not associated with CagA, which causes HBC formation. We characterized the factor causing MNGC formation enhancement as heat-stable and water-soluble, and finally considered the factor to be H. pylori lipopolysaccharide (LPS). We considered that H. pylori LPS enhances MNGC formation in vitro. The cytopathic effect may provide an important marker that may clarify the mechanism of H. pylori pathogenesis in human gastric epithelial cells

Willingness to Work and the Working Environment of Japanese Dental Hygienists

Japanese dental hygienists’ employment rate is low. The environment factors and daily job contents that contribute to willingness to work of Japanese dental hygienists and their structures were investigated. A cross-sectional survey was conducted using a self-administered postal questionnaire distributed for one thousand and twenty-three members of Japan Dental Hygienist Association registered in Iwate prefecture affiliation. Three items concerning willingness to work, satisfaction for the 9 items about working environment, anxiety for work, and 106 daily job contents were used for analysis. Structural equation modeling, decision analysis, and correspondence analysis were carried out. The present study found that working environment such as interpersonal relationship was more important than social environment such as salary for the regular employee of Japanese dental hygienist working at private dental office. However, salary was only the determinant for the dental hygienist who strongly disliked their work. And daily job contents affected the willingness to work. Especially, jobs concerned with prosthodontic treatments were of major concern. Improving the working environment and avoiding assignment of tasks that require lower level of skill may improve dental hygienists’ willingness to work and may assist to improve the employment rate of dental hygienist in Japan

Frequent Establishment of Long-term-cultured Myofibroblast Cell Lines Derived from Dupuytren’s Nodules, Which Are Implantable into Nude Mice

The purpose of this study is to establish myofibroblast cell lines derived from Dupuytren’s nodules. Cells were obtained from 6 patients with Dupuytren’s contracture, and those from 4 out of the 6 patients were serially passaged. The morphologic characteristics were observed by light and electron microscopies. Each karyotype was analyzed by a trypsin-Giemsa banding technique. The production of α smooth muscle actin (αSMA) was detected by immunological methods and reverse transcription polymerase chain reaction. Transforming growth factor β1 (TGFβ1) was measured by enzyme immunoassay. Xenografting was performed by inoculating the cultured cells into 5-weekold nude mice (BALB/c nu). The cell lines could be subcultured for more than 1 year, therefore, we considered them to be established. Spindle-shaped cells were arranged irregularly, and showed myofibroblast-specific ultrastructural characteristics. The karyotype analysis did not reveal any deletion of the chromosomes where αSMA and TGFβ1 genes were localized. The cell lines showed electron-dense bundles and maintained the production of αSMA, its messenger RNA, and TGFβ1. The implantability into nude mice was 33-100%. Our results indicated that the established cell lines were myofibroblastic cell lines. We could implant the cell lines into nude mice and they may have beneficial applications in animal models of contractile diseases