Production of virions with retrovirus morphology by human embryonal carcinoma cells in vitro.

Embryonal carcinoma (EC) cell lines established from human testicular germ-cell tumours produce, at low frequency, virions morphologically identical to type C retroviruses that have been observed by other workers in human placental tissues. The virus particles are formed while budding from the cell surface, and their numbers are increased by inducing the EC cells with 5-iodo-2'-deoxyuridine and dexamethasone. Assays for RNA-dependent DNA polymerase (reverse transcriptase) associated with purified virions suggested a low level of activity. In addition, another type of virus is occasionally produced by induced cells of three EC lines. These particles also form during the process of budding from the cell surface, but they have surface projections (spikes). Extracellular spiked virions frequently are pleomorphic, with a condensed, eccentric nucleoid, and thus morphologically resemble type B retroviruses. No virions of either type were detected with or without induction in cultures of differentiated EC cells or in cultures of yolk sac carcinoma or teratoma cells, both of which are considered malignant but differentiated derivatives of EC cells. The lack of virion production by these differentiated cells suggests developmental regulation of virus replication.

Replication of lactate dehydrogenase-elevating virus in macrophages. 1. Evidence for cytocidal replication.

Cultures of starch-elicited peritoneal mouse macrophages in medium containing macrophage growth factor (MGF) were infected with lactate dehydrogenase-elevating virus (LDV) and, after various times in culture, LDV production was monitored as a function of time by infectivity titrations in mice, by measuring [3H]uridine incorporation into LDV RNA and extracellular LDV, by autoradiographic analysis of the proportion of productively infected cells and by electron microscopy. Regardless of the age of the cultures when infected with LDV, only a small proportion of the macrophages (generally between 3 and 20% of the total) became productively infected after a primary infection; maximum virus RNA synthesis and virus production occurred during the first 24 h after infection and then decreased precipitously. Productively infected macrophages could be readily recognized in electron micrographs of 24-h infected macrophage cultures and in sections of spleens from 24-h infected mice by characteristic morphological alterations. These consisted of formation of clusters of double-membrane vesicles with a diameter of 100 to 300 mumol, budding of nucleocapsids into vesicles with single membranes and accumulation of mature virions in these vesicles. One to 4 days later, however, such cells were no longer found in infected cultures or spleens of infected mice and superinfection did not restimulate LDV replication. Cultures established with macrophages from 1-day LDV-infected mice also did not support LDV replication. We conclude that LDV replication in cultures or mice is limited to a single cycle in a subpopulation of macrophages and that infection leads to cell death and rapid phagocytosis of the dead cells by the resistant, uninfected macrophages.

Relationship of Treponema pallidum to acidic mucopolysaccharides.

Attempts were made to relate Treponema pallidum to the acidic mucopolysaccharides that occur in vivo within host ground substance and in vitro on the surface of cultured testicular cells. Infected testicular tissue was fixed and processed for transmission electron microscopy in the presence of ruthenium red. The use of this inorganic dye demonstrated the large quantity of mucopolysaccharide within testicular tissue and the intimate association of treponemes with this material. Wheat germ agglutinin and soybean agglutinin agglutinated freshly harvested trypsinized testicular cells and trypsinized cultured cells derived from normal rabbit testes (NRT). When stained with toluidine blue, both cell preparations were metachromatic. Prior treatment of cultured NRT cells with hyaluronidase slightly decreased their sensitivity to agglutination by wheat germ agglutinin and soybean agglutinin. Lectin agglutination, metachromasia, and hyaluronidase susceptibility indicated that freshly harvested testicular cells and NRT cells have surface-associated acidic mucopolysaccharides that are probably hyaluronic acid and chondroitin sulfate. A rabbit erythrocyte "sandwich" technique was devised to show that hyaluronidase removed wheat germ agglutinin receptors from the cultured NRT cells. Prior incubation of NRT cells with hyaluronidase, followed by the addition of T. pallidum, resulted in a reduction in numbers of treponemes attached to the NRT cells. The attachment of T. pallidum appears to be mediated through the acidic mucopolysaccharides on the surface of NRT cells. The findings are discussed in terms of the importance of host ground substance mucopolysaccharide to the syphilitic infective process.

Morphologic evidence for retrovirus production by epithelial cells derived from a human testicular tumor metastasis.

Ultrastructural examination of primary and subcultured epithelial cells established in vitro from an abdominal metastasis of a human testicular tumor revealed particles with the morphology of retroviruses. These structures, found only after extensive scanning of the cells, were observed budding from microvilli and from the outer cell membrane and as extracellular particles. Production of these virus particles was stimulated by the incubation of cells in culture medium containing 5-iodo-2'-deoxyuridine and dexamethasone.

Outer envelope of virulent Treponema pallidum.

Ultrathin sections of virulent Treponema pallidum (Nichols strain) were examined with the electron microscope, and the presence of an outer cell envelope was documented.

Treponeme outer cell envelope: solubilization and reaggregation.

Exposure of the avirulent Kazan 5 treponeme to low concentrations of sodium dodecyl sulfate (1.4 mm) results in the solubilization of the outer envelope. Maximal reaggregation of the outer envelope preparation requires cations, and cation concentration affects both the yield and structure of the aggregates. The reaggregated outer envelope preparation can be resolubilized with the chelating agent, ethylenediaminetetraacetic acid, or with sodium dodecyl sulfate.

Isolation of the outer sheath of Leptospira and its immunogenic properties in hamsters.

Spherical forms of Leptospira interrogans serotype canicola Hond Utrecht IV were induced with 1 m NaCl. Electron micrographs of these salt-altered cells (SAC) revealed that the outer envelope or sheath had pulled away from the protoplasmic cylinder. Treatment of SAC with 0.02% sodium lauryl sulfate solubilized the sheath and released the protoplasmic cylinder. Further processing of the solubilized sheath yielded a pellet which displayed a membrane structure in electron micrographs. The released protoplasmic cylinder showed loss of intracellular organization and the outer envelope present in normal cells. Immunization of hamsters with whole formalized cells, SAC, or sheath in doses as low as 10 mug/animal protected them from death upon challenge with virulent canicola 27.