Cytopathic Cell Research Articles

Toxoplasma Cystozoite Antigenic Change in Tissue Culture

Antigenic differences between the endozoite and cystozoite of Toxoplasma gondii were revealed by fluorescent antibody staining (Lunde and Jacobs, 1983, Journal of Parasitology 69: 806-808). Antisera against the cystozoite reacted only against the cystozoite while anti-endozoite sera reacted partially against the cystozoite as well as against the endozoite. Absorption of sera with endozoites removed only positive reactions with endozoites. It was of interest to determine whether or not cystozoite antigens persisted on the surface of organisms derived from tissue cultures infected with cystozoites. Bovine embryo skeletal muscle cell cultures were infected with cystozoites derived from Toxoplasma strain ME 49 cysts separated from triturated mouse brain by centrifugation at 5,000 g for 30 min in 17% Ficoll-PBS (pH 7.2). The cystozoites were obtained by digestion with pepsin-HCl (Jacobs et al., 1960, Journal of Parasitology 46: 11-21) for 10 min and were removed from the digestion medium by 2 washings and centrifugations and resuspended in Eagle's complete medium with glutamine and 10% fetal calf serum. Approximately 100 x 103 cystozoites were added to each of 2 tissue cultures in 35 x 100 mm disposable culture dishes and incubated at 37 C with 5% CO2. The tissue cultures were confluent when inoculated. Gentamicin, 50 ,g/ml, was added to the culture medium to inhibit possible bacteria contaminants. On day 3 the cultures showed cytopathic effect, cell lysis and rounding, and were examined. Viable zoites were observed. Cells were mechanically removed from the culture dishes, centrifuged at 1,000 g and washed once with PBS pH 7.2. Portions of the sedimented zoites were then placed within circumscribed areas on microslides. Toxoplasma endozoites obtained from bovine embryo skeletal muscle cells infected with endozoites, RH strain, were also added to similar slides. The zoites were air dried and fixed briefly in absolute methanol. Fluorescein-labeled rabbit antiserum against either endozoites or cystozoites (Lunde and Jacobs, 1983, loc. cit.) was then added to the slides. After 60 min at 37 C the slides were washed and examined for immunofluorescence. Toxoplasma derived from tissue cultures infected with cystozoites or with endozoites reacted identically with the fluorescein-tagged anti-sera. The organisms all reacted positively with anti-endozoite antibody and showed no reaction with anti-cystozoite antibody. It appears, therefore, that only a few generations [we estimate about seven (Kaufman et al., 1958, American Journal of Ophthalmology 46: 255-260)] of the parasites within cultured cells results in the loss of the specific surface determinants of the cystozoites. The fact that this transformation occurs suggests that it probably takes place at the first division of the cystozoites within the cultured cells, because new membranes are formed in the division process. It was difficult to obtain enough cystozoites to infect the cultures, and recover and identify adequate numbers of individual parasites to determine their surface antigens. Because of this, a series of experiments to determine precisely when this change occurred was not done.

Natural resistance of mice to mouse hepatitis virus type 3 infection is expressed in embryonic fibroblast cells.

Mouse hepatitis virus 3 (MHV3) infection in mice varies according to the mouse strain used; they may show resistance, semi-susceptibility (paralysis) or full susceptibility (lethal acute hepatitis). In order to study the mechanism of inborn resistance, viral infection was carried out in primary cultures of embryonic fibroblasts originating from various mouse strains exhibiting different sensitivities to MHV3 infection. Virus-induced cytopathic effects and cell membrane antigens as well as virus replication and interferon synthesis were studied. Persistent infection was induced in four out of six primary embryonic fibroblast cultures and in six of six secondary cultures. Two primary embryonic fibroblast cultures from susceptible strains underwent total lysis. A high yield of virus was obtained, as tested by viral titres and cell membrane antigen detection. No significant difference in virus production or in interferon synthesis was observed in fibroblast cultures of the various mouse strains tested. Cytopathic effects characterized by cell lysis were related, however, to phenotypes in vivo. This effect was lost after a single trypsinization of the culture. In addition, resistance to virus-induced cell lysis was inhibited by actinomycin D treatment. These results indicate that the intrinsic resistance of fibroblasts is effective not against virus replication but against virus-induced cell lysis. Such a cellular control mechanism may be an important factor for resistance in vivo.

Cytotoxicity of mercurial preservatives in cell culture.

The cytotoxicity of two kinds of mercurial preservatives, thimerosal and phenylmercuric acetate, was studied using Chang's human conjunctival epithelia in cell culture. The cultured cells were exposed for 5 s, 2 min and 24 h to each of the two preservatives at various concentrations, obtained by serial dilution. The cytopathic effect and cell desquamation from the wall of culture flask were observed with an inverted microscope and LD50 was calculated by Van der Waerden's method. The LD50 values of thimerosal were 291.6, 47.4 and 2.2 micrograms/ml at exposure times of 5 s, 2 min and 24 h, respectively. Those of phenylmercuric acetate were 1,120.2, 227.5 and 2.6 microgram/ml at exposure times of 5 s, 2 min and 24 h, respectively. Cytotoxicity can be expressed quantitatively and precisely by LD50. LD50 is lower than the concentrations that are necessary to induce observable morphological changes. The results may help to choose the least toxic concentration of mercurial preservatives for addition to ophthalmic solutions.

Interaction of influenza virus with mouse macrophages.

Mouse peritoneal and alveolar macrophages differed substantially in their response to influenza in vitro. Immunofluorescent and infectious-center techniques showed that viral proteins were produced in only a small subpopulation (17%) of peritoneal macrophages and that these infected cells were removed from culture by 3 days postinfection. In contrast, alveolar macrophages were highly susceptible to influenza, and viral antigens were produced in all cells. This was accompanied by a cytopathic effect and cell death. However, no infectious virus was released and the infection was considered abortive. With mouse cytomegalovirus, however, both alveolar and peritoneal macrophages were equally restrictive, and viral antigens were produced in only 1 to 5% of either cell population. No significant differences were observed between mouse-virulent and -avirulent strains of influenza in their interaction with macrophages either in vitro or in vivo. In vivo, both strains induced an influx of cells to the alveolar spaces by 3 to 4 days postinfection, and this was reflected by a 5- to 10-fold increase in the number of "macrophages" in harvest fluids at this time. Many of these cells had an altered morphology compared with alveolar macrophages from uninfected mice, and the cell population as a whole was not susceptible to influenza. However, this resistance was lost by 7 days of in vitro culture.

Cell fusion induced by a plant virus

Cytopathic effects of a plant rhabdovirus on established cultures of leafhopper cell monolayers were studied. When potato yellow dwarf virus inoculum with multiplicities of about 800 infectious units per cell was used to inoculate Aceratagallia sanguinolenta vector-cell monolayers, damage to the cells could be observed under a phase contrast microscope as early as 30 min after the start of inoculations. Subsequent development of damage caused gradual detachment of cells from the surface of culture flasks. However, surviving infected cells began to exhibit some morphological changes 8 to 10 hr after inoculation. The distinctness of the perimeter of the cells began to disappear. A reduction in cytoplasm and a congregation of nearby nuclei occurred 20 to 24 hr after inoculation. Extensive cell fusion was observed by phase contrast microscopy 40 hr after inoculation and was also evident by fluorescence microscopy when infected cells were stained with fluorescein-conjugated antibodies to the virus. Similar cytopathic effects and cell fusion were also observed in Dalbulus elimatus non-vector-cell monolayers inoculated with concentrated virus inoculum. Virus inoculum rendered noninfectious by exposing it to ultraviolet light still caused cell fusion, and the longer the uv irradiation, the greater the capacity of the preparation to cause cell fusion.

Protein synthesis and cell surface proteins in molluscum contagiosum virus infected cells.

The proteins synthesized in molluscum contagiosum virus (MCV) infected cells during one hour pulse-labelling experiments with14C amino acids were separated by sodium dodecyl sulphate acrylamide gel electrophoresis and analysed by autoradiography. In addition, the surface proteins of infected cells were labelled with125I and then analysed with the same procedure in order to detect differences in the spatial arrangement of proteins during the cytopathic effect (c.p.e.) and compared with the controls. In both types of experiments the electrophoretic pattern of human embryonic cells did not show qualitative differences between infected cells and control cells. This indicated that MCV fails to express, among the “early” events, the function(s) necessary to switch off host protein synthesis. The iodination of the cell surface showed that the cytopathic cell alteration did not correspond with a variation in the protein distribution on the cell surface.