FL Cell Cultures Research Articles

Reaction Pattern in Vertebrate Cells (RiV): May the anti‐RiV ELISA Detect a Pathologic Cell Proliferation? (First Results)

AbstractRiV is mainly represented by characteristic exosome‐like particles with a diameter of 30 to 70 nm. RiV particle preparations (RiV‐PP) contain identified proteins. In the present paper, the authors started an evaluation of the hypothesis, that RiV‐reactive antibodies, detected in patient sera by an indirect ELISA, may be used for the diagnosis of pathologic cell proliferations. On the basis of the frequency distribution curve of about 500 blood donor sera, cut‐off values for RiV‐reactive IgG and IgM antibodies could be defined. Additionally it was found that 1.6 % of blood donor sera reacted strongly with an RiV‐PP (of calf kidney cells) for IgG antibodies. 29 % of cancer patient sera (n = 48), 17 % of 53 patients with endoprothetics and with lung disorders, 44.3 % of 115 patient sera of a doctor's practice and 28 % of 97 sera of 10 to 11 years old schoolchildren were positive in the anti‐RiV ELISA with IgG antibodies. About half of the selected group of schoolchildren contained different autoantibodies and some of these children displayed already insulin dependent diabetes mellitus (as shown by others). A correlation of our ELISA results with the other autoimmune antibody results was not found. Using RiV‐PP as an antigen complex, isolated from calf kidney cell cultures or from human FL cell cultures and HeLa cell cultures, results with small differences between human and animal RiV origin were obtained. Using the human RiV, the number of positive sera was not significantly lower. Generally, the anti‐RiV ELISA, using RiV‐PP from human cell cultures, detect autoantibodies against RiV‐specific proteins. This assay may detect, but not diagnose pathologic cell proliferation. The possibilities of RiV ELISA for diagnostics of defined autoimmune diseases, as well as further studies and the more meaningful RiV immunoblot are discussed.

DNA polymerase activities in friend cells during the differentiation process

DNA synthesis and DNA polymerase activities were followed in FL cell cultures (clone 5.86) at different stages of differentiation. A temporary block of growth and DNA synthesis was observed after the addition of the inducers (DMSO or HMBA). This delay in the growth and in the DNA synthesis initiation is not observed in cultures of DMSO-resistant variants after treatment with DMSO. In both uninduced and induced cultures, during growth, the DNA γ-polymerase activity is constant and the α-polymerase activity is strictly dependent on the DNA synthesis rate. On the other hand, a different behaviour between induced and uninduced cultures is observed for the DNA polymerase β: its activity is constant in uninduced cultures, whereas it changes in induced cultures at various stages of differentiation, dropping to lower values at early times and rising to values similar to those observed in uninduced cultures at later times. This behaviour is not observed in cultures of a DMSO-resistant variant clone: in this case the β-polymerase activity is constant in both the absence and the presence of DMSO or HMBA.

Propagation of human hepatitis A virus in conventional cell lines.

Fecal extracts of hepatitis A (HA) patients were selected for the presence of hepatitis A virus (HAV) by radioimmunoassay (RIA) and immune electron microscopy (IEM). When FL and Vero cells were inoculated with fecal extracts containing HAV, development of hepatitis A antigen (HAAg) was evident in the cytoplasm of the two cell lines by the indirect immunofluorescence (IF) test. The antigen was detectable in the cells 12 hr postinoculation (pi), and reached a plateau within two days pi. FL cell cultures inoculated with a specimen containing HAV were harvested and passaged four times. During the passages, efficient production of HAAg was confirmed in the infected cultures by three different serological tests: The indirect IF test, RIA using fixed cells, and RIA by the sandwich method. At the second and fourth passages, HAV particles were recovered in abundance from infected FL cell cultures by IEM. Throughout these experiments, no cytopathic effect (CPE) was discernible in the cultures.

Considerations on Structure-Activity Relationships with Mengovirus of Substituted 5-Amino-4-Cyanopyrazoles

In a screening program using the agar diffusion-plaque inhibition test, 22 out of 27 substituted aminopyrazoles tested showed marked antiviral activity against mengovirus in FL cell cultures. The efficacy of the different derivatives was compared by determining their effect on the yield of infectious virus after a one-step growth cycle. The antiviral activity against mengovirus correlated with the chain length of the substituents and increased or decreased according to the position of the side chain.

Antitrachoma activity of rifamycin B and 8-O-acetylrifamycin S.

IT has been found that rifampicin1 inhibits2–4 irreversibly5,6 the development of trachoma agent, and that the effect of rifampicin on trachoma is caused by the inhibition of the agent's DNA dependent RNA polymerase present in the trachoma elementary bodies7,8 and in the developing inclusion bodies. Thus, rifampicin inhibits trachoma agent and prokaryotic cells by the same mechanism9. Studies have shown4 that the presence of the hydrazone side chain in the rifamycin SV molecule enhances the antitrachoma activity of the antibiotic. We investigated the antitrachoma activity of rifamycin B. a natural fermentation product of Streptomyces mediterranei, and two other rifamycin derivatives which lack anti-RNA polymerase activity10: compound VIII (23-dehydroxy-27-demethoxy-23,27-epoxyrifamycin SV) and 8-O-acetylrifamycin S. We found that only 8-O-acetylrifamycin S effectively inhibited the development of trachoma agent in FL cell cultures.

Studies on the formation of hyperplastic focus by variola virus in human cell cultures: I. In vitro quantitation of variola virus by focus counting in HeLa and FL cell cultures

Conditions under which variola virus, both major and minor, can be quantitated by counting hyperplastic foci in HeLa and FL cell cultures were studied. Foci were countable as red spots of 0.3–0.5 mm diameter when infected cultures were stained with neutral red at 4–6 days after inoculation. Focus-forming activity was inactivated by antiserum against vaccinia virus, but was insensitive to antibody after an initial adsorption period. Some physicochemical treatments inactivated focus-forming activity in parallel with loss of pock-forming activity. It was shown by density gradient centrifugation in sucrose that the peak of pock-forming and focus-forming activities coincided. Fluorescent antibody studies revealed that all cells in a focus contained specific viral antigen. These results are considered to substantiate that foci are formed by variola virus.

Effects of pleuropneumonia-like organisms on cultured human cells

A strain of PPLO isolated from contaminated tissue cultures exhibited the typical characteristics of mycoplasma by agar cultivation and electron microscopy and was sensitive to Aureomycin. Among many types of cultured cells studied, human amnion cells of the FL line showed the most pronounced morphological changes (cytopathic effect) and cell destruction as a result of infection with this strain. The mycoplasma, as observed by the direct microscopic demonstration method in association with FL cells, had an average diameter of 308 mμ. The titers in the tissue culture fluid approximated 10 7 colony-forming units (CFU) per ml. This PPLO strain, and many other strains, propagated to considerable titers (approximately 10 6 CFU/ml) also in two freshly prepared cell-free tissue culture media (slightly better in yeast extract-containing medium, LY medium). In infected FL cell cultures, after an initial period of normal division, the FL cell population decreased due to extensive cell destruction. Subsequently, a population of cells increased at a reduced rate. The latter population, which has been carried up to twelve months in serial transfer in the presence of high PPLO titers, is morphologically different from uninfected FL cells. The deleterious cell effects of this PPLO strain were not diminished by an increase in the l-arginine concentration in the culture medium.

HUMAN INFECTION EXPERIMENTS WITH THREE CELL-CULTURED TRACHOMA AGENTS.

In recent years many strains of trachoma have been isolated and grown in the yolk sacs of fertilized chicken eggs. Infection of human volunteers with egg-grown agents have repeatedly and successfully been carried out (reviewed by Bernkopf 1 ). A small number of egg-grown strains have also been adapted to growth in cell culture. 2-4 The question arose whether these strains retained their virulence for the human host after repeated passage in vitro. The present paper reports the results of infection experiments in three blind children with three strains of trachoma adapted to growth in FL cell monolayers. Material and Methods 1. Trachoma Strains. —The origin and laboratory history of the three trachoma strains employed in the present experiments are shown in Table 1. The T'ang strain of trachoma (TE 55 ) 5 was originally received from Dr. L. H. Collier, London. Its adaptation to, and growth in, FL cell cultures has

Isolation and properties of a human enterovirus, non-pathogenic for monkey kidney cell cultures and suckling mice.

SummaryA virus was isolated in human amnion FL cell cultures on April 14, 1959 from the rectal swab obtained from a 9-monthold male child admitted to the Children's Hospital, University of Pittsbur...

Susceptibility of a trachoma agent grown in FL cell cultures to antibiotics and a sulfa drug.

FL cell cultures infected with the T'ang strain (TE55) of trachoma have been used to evaluate the effect of a number of antibiotics and a sulfa drug. Complete inhibition was observed by doses of 0.1 μg/ml of tetra- and oxytetracycline. Chloramphenicol, erythromycin and chlortetracycline were effective in 10–100 times higher doses. Penicillin prevented the appearance of infective particles in doses of 0.1 unit/ml. Lower doses of the antibiotics showed a partial inhibiting effect. Both penicillin and tetracycline also inhibited the agent when added in the later stages of its developmental cycle. The sulfa drug sulfanilamido-3phenyl-2pyrazole prevented the appearance of infective particles in doses of 100 μg/ml. Its inhibitory effect was limited to the early developmental stages of the agent, and could be reversed by PABA, folinic and folic acid. Aminopterin, like the sulfa drug, inhibited the agent at an early stage of development. No inhibition was observed when large doses of streptomycin, bacitracin and neomycin were employed.

The Growth Cycle of a Trachoma Agent in FL Cell Cultures

Summary FL cell cultures were infected with a Chinese strain of trachoma, and the development of infectivity during a single growth cycle was studied. A noninfective phase lasting at least 24 hr after infection was demonstrated. Infectivity reappeared at 28 hr and reached a maximum at 48 to 72 hr. These findings agree well with the morphological findings reported before, according to which particles of elementary body size changed their appearance shortly after infection, could again be demonstrated at 28 hr, and then rapidly increased in number.

Correlation between morphological and biochemical changes and the appearance of infectivity in FL cell cultures infected with trachoma agent.

Correlation between morphological and biochemical changes and the appearance of infectivity in FL cell cultures infected with trachoma agent.

Use of egg white in maintenance Medium for FL cell cultures

Use of egg white in maintenance Medium for FL cell cultures

Biochemical changes in FL cell-cultures infected with a trachoma agent.

THE multiplication of a trachom aagent in FL cell-cultures has recently been reported1. In an investigation of the growth cycle in FL cell-cultures2 it was found that the agent passes through a non-infective phase, lasting 24 hr. Newly formed infective particles were first demonstrated after 28 hr. and then they increased rapidly in number. In the present work, examinations for deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and a high polymer carbohydrate in cell-cultures were carried out and related to the appearance of infectivity.

Mutation of polioviruses with respect to size of plaque: I. Selection of minute plaque mutants of three types of polioviruses in tissue cultures

Minute plaques ( m) of the three types of polioviruses have appeared in the course of their serial propagation in HeLa and FL cell cultures. The m mutants cause tiny plaques on monolayers which do not enlarge with time. No significant differences were found between the m mutant and m + wild type virus either in cytopathic effect or in growth rate. It is quite obvious that m mutants constitute a class different than any other so far described. The property responsible for the minute-plaque formation is obscure.

Preparation of high potency poliovirus in FL cell cultures at room temperature.

Rate of formation and release of poliovirus Type 1 were studied at 32° and 37°C in FL cell cultures. Virus accumulated almost as rapidly and reached practically the same intracellular levels at 32°C as at 37°C. However, release of virus was much slower at 32° than at 37°C and as a result, at 32°C the intracellular concentration of virus remained at or near peak levels for 4 to 6 hours. From these observations, highly potent virus for use in immunological studies was prepared by mechanical disruption of infected cells maintained at room temperature (26–32°C) for 18–20 hours.