A simplified intrathymic injection technique for mice.

Intrathymic injection is a common technique used for research concerning immunotolerance induction, gene therapy and T cell development in mice. Traditionally used protocols involve major surgery that exposes the thoracic cavity, which results in injury to the mice and increased risk of poor recovery and postsurgical complications such as infection. We introduce a simplified intrathymic injection technique that does not expose the thoracic cavity and virtually eliminates pain, distress and postoperative complications while maintaining high injection efficiency. The technique is suitable for both adult and neonatal mice.

Evaluation of the cytotoxicity of a two photon absorbing fluorescence compound on human HepG2 cells and its application to tracking human hepatic cancer cells in mice.

Small organic dyes have been applied widely in fluorescence imaging techniques for biomedical research. We investigated the cytotoxicity of a novel fluorescent dye, trans-4-(N-2-hydroxyethyl-N-ethyl amino)-4'-(dimethyl amino) stilbene (DMAHAS), on human hepatocellular carcinoma (HepG2) cells using methyl thiazolyl tetrazolium(MTT), a neutral red assay, a Coomassie brilliant blue assay, and flow cytometric analysis. Our results showed that DMAHAS had live cell permeability, stable cytosolic localization and no significant cytotoxicity to HepG2 cells. We explored its application further for tumor cell tracking in a human liver tumor xenograft mouse model. Tumor xenografts were examined by fluorescence imaging and conventional histological methods. In addition, a method based on DMAHAS release was developed for tumor-specific cytotoxicity analysis. Our study indicated that DMAHAS is a reliable probe for tumor tracking and fluorescence imaging.

[Growth inhibition of human nasopharyngeal carcinoma cell CNE-2L2 caused by suppression of 6A8 alpha-mannosidase expression].

OBJECTIVE: To study the relationship between 6A8 alpha-mannosidase expression and growth of CNE-2L2 cells, a human nasopharyngeal carcinoma cell line. METHODS: Recombinant adeno-associated virus vector (rAAV) was used as a mediator to transfer an antisense or a sense fragment of 6A8 cDNA into CNE-2L2 cells. 6A8 alpha-mannosidase expression was detected by means of mAb 6A8a staining, alpha-mannosidase activity assay and ConA binding test. Cell growth was examined by means of MTT and colony formation. Tumor growth at the inoculated site of the cells in nude mice was detected after 8 weeks. RESULTS: Transduction of antisense 6A8 could reduce the expression of 6A8 alpha-mannosidase. In comparison to those in controls, the wild type, the mock-transduced and the sense 6A8-transduced cells, the MTT value, the colony number formed and the tumor weight grown at the inoculated site of cells were significantly decreased in the antisense 6A8-transduced cells (P < 0.001). CONCLUSION: Decreased expression of 6A8 alpha-mannosidase caused an inhibition of CNE-2L2 cell growth.

Detection of varicella-zoster virus DNA by field-inversion gel electrophoresis.

A new method for detection of varicella-zoster virus (VZV) DNA using field-inversion gel electrophoresis (FIGE) was devised. VZV-genomic DNA could be differentiated from the host cell DNA of human embryonic lung (HEL) fibroblasts infected with VZV under electrophoretic conditions allowing resolution of linear and double-stranded DNAs in the 49-230 kilobase pairs (Kb) range. The detection of VZV-genomic DNA from infected HEL cells was successful regardless of whether the VZV was a laboratory strain, live vaccine strain, or fresh isolate. Under the same electrophoretic conditions, DNA of VZV-infected HEL cells could be clearly differentiated from DNA obtained from HEL cells infected with herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), or human cytomegalovirus (HCMV). Furthermore, VZV genomic DNA could be detected from as small a sample as 1.9 x 10(4) VZV-infected HEL cells. Finally, we could detect VZV genomic DNA from 10 samples of vesicle tissue (blister lids, each about 1-4 mm2) and one sample of vesicle fluid (about 5 microliters) obtained from patients diagnosed as having herpes-zoster. The results of this study indicate that FIGE is a simple and promising method for the detection of VZV from clinical materials as well as infected in vitro cultured cells.

Electron microscopic study of a herpes-type virus isolated from an infant with exanthem subitum.

Human lymphocytes and MT4 cells infected with a virus isolated from a patient with exanthem subitum were examined by transmission and scanning electron microscopy. The most striking characteristic of the ultrastructure of this herpes-type virus was that nucleocapsids located outside the nucleus were each coated distinctly with a tegument of moderate electron density. Tubular structures formed due to some mistakes in the viral assembly were also detected in the nucleus. Morphological differentiation of this virus from the other human herpesvirus was discussed. From these observations it was concluded that this virus has the same ultrastructural characteristics as HBLV (HHV-6).