Well-Differentiated Papillary Mesothelioma of the Tunica Vaginalis: A Case Study and Review of the Literature

Well-differentiated papillary mesothelioma is an uncommon tumor of the testes that usually presents as a hydrocele. Here, we present the case of one patient who did not have a history of asbestos exposure. The tumor was localized in the tunica vaginalis and was composed of three pedunculated masses macroscopically. Microscopically, branching papillary structures with focal coagulative necrosis were present. In addition to immunohistochemistry, simian virus 40 DNA was also tested by polymerase chain reaction. This report presents one case of this rare entity, its clinical and macroscopic features, and follow-up results.

No Detection of Simian Virus 40 in Malignant Mesothelioma in Korea

BACKGROUND: Simian virus 40 (SV40), a polyomavirus, was discovered as a contaminant of a human polio vaccine in the 1960s. It is known that malignant mesothelioma (MM) is associated with SV40, and that the virus works as a cofactor to the carcinogenetic effects of asbestos. However, the reports about the correlation between SV40 and MM have not been consistent. The purpose of this study is to identify SV40 in MM tissue in Korea through detection of SV40 protein and DNA. METHODS: We analyzed 62 cases of available paraffin-blocks enrolled through the Korean Malignant Mesothelioma Surveillance System and performed immunohistochemistry for SV40 protein and real-time polymerase chain reaction (PCR) for SV40 DNA. RESULTS: Of 62 total cases, 40 had disease involving the pleura (64.5%), and 29 (46.8%) were found to be of the epithelioid subtype. Immunostaining demonstrated that all examined tissues were negative for SV40 protein. Sufficient DNA was extracted for real-time PCR analysis from 36 cases. Quantitative PCR of these samples showed no increase in SV40 transcript compared to the negative controls. CONCLUSIONS: SV40 is not associated with the development of MM in Korea.

Detection of SV40 Large T Antigen in Malignant Lymphomas

BACKGROUND: The association of simian virus 40 (SV40) with certain types of human cancers, including malignant lymphomas, has been a topic of interest for some time. Although the virus is distributed worldwide, its incidences vary according to the specific types of tumors, and the epidemiological areas. The aim of this study was to investigate the frequency of SV40 in malignant lymphomas among Korean patients. METHODS: One hundred seventy three cases of malignant lymphomas were evaluated by immunohistochemical staining for SV40 large T antigen (TAg), using an extremely sensitive, tyramide based, catalyzed signal amplification method. RESULTS: From 158 non-Hodgkin's lymphomas, including 115 diffuse large B-cell lymphomas, and 15 Hodgkin's lymphomas, none of the cases were positive for SV40 TAg. CONCLUSIONS: SV40 does not appear to be related to the pathogenesis of malignant lymphomas among Koreans.

Immortalization of human precartilaginous stern cells by transfecting SV40Tag / 中华创伤骨科杂志

Objective To establish the strain of immortalized human precartilaginous stem cells (PSCs) which can be a stable cell resource for study of the molecular mechanism of gene targeting on differ-entiation of PSCs. Methods Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs using lipofeetin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Immunohistochemistry, RT-PCR and Southern blot were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. Results The positive colonies were isolated and subcultured, named as immortalized precartilaginous stem cells (IPSCs), which were confirmed as positive to fibrnblast growth factor receptor-3 (FGFR-3). The existence of SV40Tag cDNA was detected by Southern blot and the expression of SV40Tag mRNA and protein by RT-PCR and immunohistochemistry. Conclusion IPSCs strain with SV40Tag can be constructed successfully.

Establishment and further identification of immortalization human hepatocyte lines / 中华传染病杂志

Objective To establish immortalized human hepatocyte lines for studies of bioartificial liver,hepatocyte transplantation,and drug metabolism in vitro.Methods Primary human hepatocytes were isolated by 4-step perfusion technique with collagenase and transfected with recombinant retrovirus containing Simian virus 40 large T antigen(SV40 LT).Subsequently,immortalized human hepatocytes were evaluated by analysis of gene expression and functional characteristics in vitro.Results Two immortalized human hepatocyte lines,HepLi2 and HepLi3,were obtained after primary human hepatocytes being infected by SV40 LT containing recombinant retrovirus for 3-4 weeks.The immortalized human hepatocytes showed classical appearance of hepatocyte observed by phase contrast microscope.The protein expression of SV40 LT in HepLi2 and HepLi3 cells were detected by Western blotting.The mRNA expressions of albumin(Alb),glutathione S-transferase(GST-p),human blood coagulation factor X(HBCF-X)and β-actin in HepLi2 and HepLi3 cells were detected by reverse transcription polymerase chain reaction(RT-PCR),and the mRNA expressions of cytochrome(CY)450 subtypes(CYP3A5,CYP2E1,CYP2C8-19 and CYP3A4)in HepLi2 and HepLi3 cells were also observed by RT-PCR.Levels of alanine transaminase (ALT),lactate dehydrogenase(LDH)and Alb were detected in the supernatant of immortalized human hepatoeyte culture.Conclusions The immortalized human hepatocyte lines have the biological characteristics of primary human hepatocytes and have the CYP450 functions of hepatocytes,which may be heIDful for the studies of bioartificial liver,heoatocvte transplantation and drug metabolism in vitro.

Immortalization of Human Precartilaginous Stem Cells by Transfecting SV40Tag / 华中科技大学学报（医学）（英德文版）

Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs.Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection.Colonies were isolated by puromycin selection and expanded by multiple passages,lmmunohistochemistry,RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines.The positive colonies were isolated and subcultured,designated immortalized precartilaginous stem cells (IPSCs),which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR.SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting,and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR.These findings suggested that IPSCs strain with SV40Tag was constructed successfully.

Endometrial Cell Culture: Isolation, Characterization, and Immortalization / 대한불임학회지

No abstract available.

Construction of immortalized rat astrocyte strain by transfection of simian virus 40 large T antigen gene / 中华麻醉学杂志

Objective To immortalize rat astrocytes which could be used as cell carriers for transgenic cellular analgesia. Methods Astrocytes were isolated from cerebral cortex of newborn SD rats by trypsin digestion and cultured according to the method of differential cell adhesiveness and transfected with plasmid pCMVSV40T/ PUR containing the simian virus 40 large tumor antigen (SV40Tag) gene. The positive colonies were isolated by puromycin selection and expanded by many passages. The integration and expression of large T antigen gene were detected and the immuno-reactivity of glial fibrillary acidic protein (GFAP) was determined by PCR, RT-PCR and immuno-cytochemistry. Results Rat astrocytes were successfully cultured in vitro and positively stained for the astrocytic marker GFAP. The positive colonies were isolated and subcultured for 50 passages. PCR and RT-PCR products were analyzed using 1.5% agarose gel electrophoresis. The size of amplifacation product of target gene was identical to that of the positive control (558 bp) . There was no PCR and RT-PCR product from non-transfected cells. DNA sequencing and BLAST showed that 558 bp nucleotides were identical to the SV40Tag gene of the Genbank (100%). The transfected cells were positively stained for the SV40Tag and GFAP. Conclusion Immortalized rat astrocyte strain with SV40 tag gene is constructed successfully.

Construction of immortalized rat neural progenitor cell strain by transfection with simian virus 40 large T antigene gene / 中华麻醉学杂志

Objective To establish immortalized neural progenitor cell strain and provide stable cell resource for cell-transplantation and gene therapies. Methods Plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigen gene (SV40Tag) were transfected into the primary cultured neural progenitor cells (NPCs) of newborn rat using lipofectin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Anti-nestin antibodies were used to identify the cultured cells. The specific molecular marker of neurons and astrocytes were detected using immunocytochemistry method to investigate the capability of differentiation of the transfected cells. The expression of SV40Tag in expanded cell lines was identified by RT-PCR, Southern blot and immunocytochemistry method.Results One anti-puromycin cell clone was obtained, which was microtubule-associated protein-2 (MAP-2) positive cells with the capability of proliferation and could differentiate into MAP-2 or glial fibrillary acidic protein positive cells. The existence of SV40Tag cDNA and the expression of mRNA and protein of SV40Tag were confirmed in transfected cells. The transfected cells were expanded to immortalized cell strain maintained for more than 50 passages, named as immortalized neural progenitor cell (INPC) . INPCs were elliptical or triangular cells with two or three short axons. The population doubling time of INPC was (22.9?2.7)h, subculture, freezing and recovering had no effect on cellular shape and proliferation of INPC. Conclusion Immortalized neural progenitor cell strain was established successfully. It may provide stable cell resource for the basic researches and cell-transplantation therapies with NPC.

Transfection of normal human melanocytes with SV40 T antigen gene / 中华皮肤科杂志

Objective To investigate the immortalization of human melanocytes by transfection with SV40 T antigen ( SV40T ). Methods By using SofastTM,a gene transfection reagent, the reconstructed eukaryotic expression vector SV40T-pEGFP was stably transfected into cultured primary human melanocytes, then the positive cells were selected with G418. After the positive cells were expanded in culture, the expression of SV40T gene was detected by RT-PCR and PCR, and the protein expression of SV40T by Western blotting. Results The genome DNA and total RNA were isolated from the positive cell clones, and a 288 bp fragment, which was specific for the SV40T antigen gene, was amplified. The results of immunohis-tochemistry and Western blotting confirmed the expression of SV40T protein in transfected cells. Conclusion SV40T antigen gene can successfully induce the immortalization of human melanocytes.

Biological character of immortalized rat astrocyte strain by transferction of simian large T antigen gents / 中华麻醉学杂志

Objective To study the biological character of rat astrocyte strain immortalized by simian virus 40 large T antigen gene(SV40Tag) and explore the feasibility of using it as cell vehicle for transgenic cellular analgesia.Methods Rat cerebral cortical astrocytes (AST) and immortalized rat astrocyte strain (IAST) were cultured in vitro. Morphology and growth features of AST and IAST were examined and compared after subculture, freezing and recovery. The ultrastructure of the cells was observed by transmission electron microscopy. The glial fibrillary acidic protein (GFAP) in these cells was detected by immuno-cytochemistry. The cell proliferation rate and cell cycle were determined by bromodeoxyuridine labelling and flow cytometry. AST and IAST were cultured in soft agar and inoculated in nude mice to investigate the tumorigenesis of IAST. Results LAST could be subcultured successively. The cells remained monolayer, anchorage dependent and the growth was attachment-inhibited. When AST was subcultured for only 10 passages, replicative senescense began. Subculture, freezing and recovery did not influence the shape and proliferation of IAST (94%?5%) , but decreased the vitality rate of AST (54%?4% ) (P