Amnion Cultures Research Articles

Rubella Interferon and Factors Influencing the Indirect Neutralization Test for Rubella Antibody

Summary Resistance of primary human amnion cultures to Sindbis virus was found to correlate well with the presence of specific rubella cytopathic effects. Certain exceptions to this relationship were noted. Sindbis virus interfering activity present in rubella infected cultures was related to interferon on the basis of chemical, physical and biologic properties. With the indirect interference neutralization test for rubella, using amnion cultures and challenge with Sindbis virus, rubella cytopathology was found to be a useful criterion for determining the optimum time for Sindbis virus challenge. The incorporation of normal rabbit serum enhanced neutralization of rubella virus. Important factors contributing to reliability of indirect neutralization results include the use of 10 or more TCID50 of rubella virus, and the use of more than two cultures per serum dilution tested. It is concluded that the results of interference neutralization tests may reflect a variety of factors in addition to the specific neutralization of rubella virus.

Loss of radioactivity from labeled DNA of primary human amnion cells.

Primary human amnion cultures labeled with glycine-2-C(14) or thymidine-2-C(14) released more radioactivity into the media than could be accounted for by cell replacement. This is interpreted as degradation of acid-insoluble polydeoxynucleotides in a living human cell.

Metabolic alterations with senescence of human cells. Some observations in vitro.

This report describes experiments designed to study changes in metabolic patterns associated with the senescence of human amnion cells in vitro. Senescence has been defined as the increase in the chronological age of a population of cultured cells which are predominantly nondividing. This definition is based on the concept formulated by Comfort and Shock. 1 The general scheme of these experiments consists of the determination of radioactivity in various cell components obtained from cultures previously labeled with C14 substrates. Materials and Methods Amnion Culture.— Cell suspensions were prepared from amniotic membranes carefully dissected from placentae obtained at normal full-term deliveries. 2 Approximately 5 million cells suspended in 10 ml. of nutrient medium, consisting of 10% human and 5% calf or 20% horse serum in Eagle's basal medium, 3 were implanted in milk dilution bottles of 200 ml. capacity. The media were renewed on the 4th and 7th days. On the

Propagation in Tissue Culture of Cytopathic Agents from Patients with Rubella-Like Illness.

SummaryViruses, apparently not heretofore described, have been isolated from the urine or blood of 4 patients with rubella-like illnesses. These agents were serially propagated in primary human amnion cultures and produced unique cytopathic changes characterized by the aggregation of nuclear material and the presence of inclusion bodies. Other attributes demonstrated included: filterability, failure to grow in embryonated eggs or produce disease in newborn mice, lack of hemadsorption or hemagglutination, interference with Sindbis virus, relative stability on storage, and destruction by ether. By inhibition of specific cytopathic activity, low but consistent rises in levels of neutralizing antibody were demonstrated in paired sera from cases of rubella. Moreover, patients from 5 epidemics of rubella, widely separated in time, exhibited serologic evidence of infection with the agents studied. These findings suggest that the viruses herein described may be responsible for a significant proportion of illnesse...

The maturation and release of infectious polio and Coxsackie viruses in individual tissue cultured cells.

Examination of the intracellular and extracellular virus yields from individual FL and primary human amnion cells of uniform size, infected at a high multiplicity and incubated for specified time intervals, showed that the time of the onset of the appearance of mature infectious virus and the amount of accumulated infectious virus were variable from cell to cell. The duration of the major accumulation of mature virus by individual cells was found to be from 4 to 6 hours. The cells in the FL population, infected with Type 1 poliovirus, fell into two classes in regard to the accumulation and release of virus. The “early releasers” released some virus during the period of accumulation, yet they continued to accumulate additional virus which they subsequently released. The “late releasers”, represented by the majority of the cells, released virus at some time after accumulation had ceased. All cells in the primary human amnion cultures, infected with Coxsackie A-9 virus studied fell into the “late releasing” class.

Effect of aging and rotation on human amnion cell response to polio and Sindbis viruses.

Summary1) The cytopathic response of primary human amnion cell cultures to chick embryo adapted Type II poliovirus was absent in stationary cultures inoculated at 5–10 days of age. In stationary cultures inoculated when 30 or more days of age, complete destruction of cells followed. Multiplication of virus in young cultures occurred in the absence of recognizable cytopathic change. Cytopathic response of cultures of approximately 14 days was markedly enhanced by rolling after inoculation. The effect of rolling was not clearly apparent when other strains of poliovirus including attenuated strains were tested. 2) Frequently the cytopathic response of amnion cultures to Sindbis virus was reduced or absent in young as compared with old cultures, but response of young cultures was variable. No effect of rolling on cytopathic response could be demonstrated. Viral multiplication was greater in older cultures.

Cultivation of measles virus in stable line of human amnion cells.

SummaryCytopathogenic changes induced by measles virus propagated in a stable line of human amnion cells is described. The prominent change is necrosis in contrast to formation of “giant multinuclear cells” occurring in primary human amnion cultures infected with measles virus. Preliminary studies indicate the general utility of this line of cells for investigations on measles virus.

Further tissue culture studies of non-gonococcal urethritis and Reiter's syndrome.

Materials and Methods A new series of male patients with urethritis following sexual intercourse was investigated. Cases were selected for study when urethral smears and cultures revealed no gonococci, and a visible purulent or semi-purulent discharge showed, on stained smear, a moderate to large number of pus cells, but no predominant organism in significant numbers. Table I (overleaf) gives the relevant clinical data of the 24 cases. In the present study the urethra was irrigated with 5 to 10 ml. culture medium, instead of Hanks' solution, so that the washings could be added to the cells directly, or at a 50 per cent. concentration, thus avoiding the 1 in 10 dilution previously used. The tissue culture methods were similar to those employed for the Hela cells, except that the 2 per cent. calf serum medium was prepared with inactivated serum heated to 560 C. for 30 minutes, and that medium ML 192/2 (Waymouth, 1956), in which 0 05 per cent. bovine albumin replaces the serum component, was used for most of the amnion cultures. The Chang conjunctivai cells, obtained originally from Microbiological Associates, proved less satisfactory than the Hela strain for prolonged observation, because of a tendency to become rounded and peel off the glass. Moreover, their behaviour in the 2 per cent. calf serum medium was variable; on occasions the cells remained healthy for up to 18 days, whereas at other times non-specific degenerative changes were visible in control cultures after 9 days. Cases 9 to 16 were observed twice for cytopathogenic effects against Chang cells; initially, when fresh washings were used, the control

Fluorescent antibody studies with herpes simplex virus in unfixed preparations of trypsinized tissue cultures.

Summary: The classical strain of herpes simplex virus (Burnet & Lush, 1939) and a recently isolated strain (Melbourne 1/56) were propagated in tissue culture of human amnion and infant-mouse kidney ‘epithelial’ cells. Following the inoculation of 103·25 TCCD50 (tissue culture cytopathogenic dose) of the Melbourne 1/56 herpes simplex virus into amnion cell culture, the titre of virus dropped to 100·50 TCCD50 at 12 hr. and the maximum level of virus (103·50 TCCD50) was detected at 48 hr. after inoculation. The earliest morphological changes were observed in the tube-cultures c. 18 hr. after inoculation of the virus and eosinophilic intranuclear inclusion bodies were observed in the amnion cells c. 24 hr. after virus injection. Cell degeneration was prominent at 36 hr. when most cells were highly retractile and rounded. Cell suspensions, harvested from infected and uninfected cultures of amnion and mouse- kidney epithelium, were treated with immune herpes serum followed by fluorescent rabbit anti-human γ-globulin, and specific peripheral fluorescence was observed in the unfixed preparations of the infected tissue cultures. The limited surface staining was ascribed to the impermeability of the unfixed cytoplasmic membranes to globulins.

Cytopathic changes associated with poliomyelitis infections in human amnion cells

The cellular changes associated with polio virus infections in human amnion cultures differ from previously studied cells (HeLa, monkey kidney, and human fetal cells) in that (1) the release of newly formed virus occurs 4–8 hours later, (2) the nucleolus becomes indistinguishable before other gross cellular changes occur, (3) spherical projections, or nodules, appear over the free surface of cells, (4) the time of appearance of such nodular cells is coincidental with the time of virus release, and (5) occasional cultures of amnion undergo different terminal degenerative changes in which the cells become very highly vacuolated.