Isolation of the SV40 induced tumor (“T”) antigen from transformed hamster kidney cells

Summary A procedure for the isolation and partial purification of tumor (T) antigen induced by SV 40 virus in transformed hamster kidney cells is described. The method involves differential precipitation with ammonium sulfate, fractionation on a hydroxylapatite column and concentration by ultrafiltration. There is very little loss in activity as measured in total CF units and the degree of purification is about one hundredfold. Analysis of the purified preparation indicates four peaks of CF activity ranging in molecular weight from 70,000–400,000.In partial fulfillment of the requirements for the degree of Doctor of Philosophy of the Hebrew University, Jerusalem, Israel.     

Molecular characterisation of a putative endogenous retrovirus cDNA isolated from human placental tissue

The human genome comprises of abundant DNA sequences related to endogenous retroviruses (ERV) and a variety of solitary long terminal repeats (LTRs). Substantial numbers of intact retroviral particles have been detected by electron microscopy in normal human placental villous tissue particularly in syncytiotrophoblast. Understanding the molecular structure, organisation and distribution of these ERV sequences may lead to elucidation of their possible dual function at the foetal-maternal interface; proliferation and differentiation of cytotrophoblast and induction of local pregnancy-associated immune suppression thus allowing survival of the foetal allograft. In this study, antibody probes were used to screen a human placental expression library and cDNA clones isolated were characterized by polymerase chain reaction, Southern blot hybridisation, DNA cloning and partial nucleotide sequencing. A specific 1.7kb-cDNA clone was isolated from a human placental expression library. Further characterisation showed this clone represents a single copy gene, approximately 9-10kb and did not hybridise to the env region of ERV3 human endogenous retrovirus. The 1.7kb-cDNA clone may represent a provirus co-expressed with cellular sequences.     

Efficient isolation of endogenous rhesus retrovirus from trophoblast

An examination of various rhesus monkey (Macaca mulatta) organs has shown a preference for type C viral antigen expression in the placenta. Separate cocultivations of isolated trophoblasts from 10 rhesus monkey placentas with cell lines from heterologous mammalian species led to rapid isolation of type C rhesus retrovirus in 4 of 10 cases. These four retrovirus isolates have been designated MMC-2 through MMC-5. Five of the remaining six sets of cocultivations grew simian foamy virus and were discontinued. Distinction of these viral isolates from the initial rhesus isolate (MMC-1) and the previous isolate from the stumptail monkey, Macaca arctoides (MAC-1), could be made by liquid DNA hybridization, although not by limited restriction endonuclease digestion. Both MAC-1 and MMC-1 were obtained in single long-term cocultivation experiments (over 7 months). The present isolates MMC-2 through MMC-5 were detected in 2 to 5 weeks. Consequently, primary trophoblast cells represent a useful differentiated cell type for isolation of infectious retrovirus from this primate species.     

Characterization of defective SV40 isolated from SV40-transformed cells.

Defective SV40 viruses were isolated from SV40-transformed monkey, human and hamster cells after Sendai virus-mediated fusion of the transformed cells with TC7 cells, a stable line of African green monkey kidney cells. Viral isolates were concentrated and purified and the defective viruses examined by electron microscopy. The buoyant densities in CsCl of the defective viruses ranged between 1.32 and 1.33 g/cc. DNA isolated from defective viruses was characterized by dye-buoyant density centrifugation and by velocity sedimentation in neutral CsCl. The DNA was heterogeneous in size and contained some covalently closed double-stranded circular molecules.     

Hamster tumors induced by type I avian adenovirus (CELO)

Summary Hamster tumors induced either by CELO virus or by transplants of CELO virus-induced tumors were studied grossly, histologically and by immunofluorescence. Regardless of the material used for induction, the histological pictures were similar. All tumors were fibrosarcomas showing varying degrees of differentiation. However, slight variations were noted between virus-induced and transplanted tumors. These included increased cellular pleomorphism with prominent bizarre giant cells in the virus-induced tumors, and increased mitotic activity in the transplants. In both types of tumors, immunofluorescence occurred within the cytoplasm at the nuclear envelope. Viral antigen was not demonstrable by immunofluorescence.Contribution Nr. 1267 of the Rhode Island Agricultural Experiment Station.     

Molecular Analysis of SV-40-CAL,a New Slow Growing SV-40 Strain from the Kidney of a Caged New World Monkey with Fatal Renal Disease

A decline of the Callimico goeldii population in American zoos is presently occurring due to glomerulonephritis of unknown etiology. We hypothesized that this emerging idiopathic fatal renal disease (IFRD) was caused by a virus. We therefore attempted to isolate virus from the kidneys three C. goeldi in Illinois that had IFRD. Along with other viruses, Simian virus 40 (SV-40) strain CAL was isolated. SV-40-CAL is currently the slowest-growing natural isolate of SV-40 in CV-1 cells. Inefficient SV-40-CAL growth in CV-1 cells stems from two features: a suboptimal protoarchetypal regulatory region, and a Large tumor antigen gene sequence like that of SV-40 strain T302, previously considered the slow-growing natural isolate of SV-40. To our knowledge, this is the first documented isolation of SV-40 from a New World monkey outside of a laboratory setting. Though SV-40 is renaltropic, the role of SV-40-CAL in IFRD is uncertain. Transmission of SV-40 to C. goeldii through anthropogenic activity is suspected.     

SV-40 large-T immortalization of embryonic bone cells: establishment of osteoblastic clonal cell lines

Cells derived from embryonic rat calvariae were immortalized by retroviral delivery of cDNA for the SV-40 large T antigen and the bacterial neomycin resistance gene. After selection with G418, cells were cloned by limiting dilution and screened for expression of osteoblast characteristics. One clone (RCT-3), derived from cells collected during the third period of enzymatic digestion, showed high constitutive expression of alkaline phosphatase (ALP), synthesized type I collagen in the virtual absence of type III and exhibited a parathyroid hormone (PTH)-responsive adenylate cyclase (EC50, 10 nM). Messenger RNAs for osteonectin and osteopontin were present in RCT-3 cells and osteopontin mRNA was enhanced by 1,25 (OH)2 vitamin D3 treatment. The other cell line (RCT-1), derived from cells released during the first 10 min of digestion, expressed osteoblast features only after 3 d treatment with 1 microM retinoic acid (RA). ALP activity increased from 0.003 to 0.25 mumole/min/mg protein, there was a substantial increase in the steady-state level of type I collagen mRNA and a dose-dependent and saturable response to PTH was induced (EC50, 10 nM). Osteopontin mRNA was induced by 1,25 (OH)2D3. This study has provided two new cell lines which may be useful models for studies of differentiation-related gene expression in bone cells.     

Possible pathways of circulation of human endogenous retrovirus similar to mouse mammary tumor virus

It is shown that polypeptides which are immunologically related to gp52 mammary tumor virus are found in T and B peripheral blood lymphocytes in all breast cancer patients, in children with B-cell lymphosarcomas, and in B lymphocytes of some healthy donors. These proteins are not found in patients with tumors of other sites. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 11, pp.554–556, November, 1995 Presented by Yu. N. Solov'ev, Member of the Russian Academy of Medical Sciences     

阻断CD40-CD40配体共刺激信号诱导的移植免疫耐受对树突状细胞的影响

目的:探讨阻断CD40-CD40配体共刺激信号诱导移植免疫耐受对树突状细胞功能的影响。方法:将抗CD40配体抗体(MR-1)应用于小鼠心脏移植受体以诱导移植免疫耐受,使用磁珠细胞提取装置从排斥及免疫耐受受体中提取树突状细胞。使用流式细胞仪测定树突状细胞表面分子CD40、CD80及CD86的表达。于体外以LPS再刺激树突状细胞,并使用ELISA法测定上清中的细胞因子IL-10和IL-12的水平。同时采用混合淋巴细胞反应(MLR)测定了树突状细胞的刺激性及免疫调节能力。结果:免疫耐受受体的树突状细胞表达低水平的共刺激分子CD40、CD80及CD86,同时分泌高水平的IL-10和低水平的IL-12。而且这些树突状细胞具有较弱的刺激性,并能抑制脾细胞的增殖。结论:阻断CD40-CD40配体共刺激信号可以产生免疫耐受状态,在这样的免疫耐受受体中诱导出未成熟表型的IL-10highIL-12low树突状细胞,这些细胞具有免疫调节活性。     

Absence of SV-40 large T antigen (Tag) in malignant mesothelioma effusions: an immunocytochemical study

Simian Virus 40 (SV 40) was recently implicated in the pathogenesis of malignant mesothelioma. The oncogenic capacity of SV-40 is a function of a nuclear protein, the large T antigen (Tag). SV-40 Tag DNA sequences are detected by the polymerase chain reaction in 40-80% of malignant mesothelial proliferations. However, the role of immunohistochemistry (IHC) in demonstrating the nuclear localization of Tag is controversial. We sought to determine the clinical utility of SV-40 Tag IHC in pleural effusion cytology as an ancillary tool in the cytologic diagnosis of malignant mesothelioma (MM). Formalin-fixed, paraffin-embedded cell block sections from 100 pleural effusions (32 MMs, 25 benign reactive, 43 metastatic adenocarcinomas) were immunostained for the SV-40 anti-Tag, using two primary monoclonal SV-40 Tag antibodies: clone Pab 416 and clone Pab 101. Despite strong and consistent immunoreactivity in positive controls, no nuclear immunostaining was observed in any case. We believe the small sample size in cytology cell block sections, the low viral copy number in infected cells, and the effect of formalin fixation may have resulted in absence of immunoreactivity. The role of SV-40 Tag IHC in diagnostic cytopathology remains unclear unless further studies reliably show its detection.     

Hamster brain tumor cells persistently infected with measles-subacute sclerosing panencephalitis virus

Summary A persistent infection was established in a cell line derived from a hamster brain tumor (HBT) with the HBS strain of measles-subacute sclerosing panencephalitis (SSPE) virus. The persistently infected cells (HBT-M) were studied with regard to their growthin vitro and their transplantabilityin vivo. Although the growth of the HBT-M cells paralleled that of the HBT cellsin vitro their transplantability was decreased in weanling hamsters. Hydrocortisone treatment of the hamsters abrogated the lowered transplantability restoring the tumorproducing capacity to levels comparable to the HBT cells. The decreased cell growth of the HBT-M cellsin vivo was attributed to the acquisition of measles virus (MV) antigens and the host immune response directed against these new antigens.With 1 Figure