Susceptibility of cell lines of primate and nonprimate origin to certain human viruses

A comparative study was made of the susceptibility of 11 cell lines of human and animal origin, the WI-38 cell strain and fresh cultures of human thyroid, monkey kidney and hamster embryo tissues to certain human viruses. The animal cell lines were derived from monkey, rabbit, mouse, pig and calf tissues. The viruses used were strains of influenza A₂ and B viruses, parainfluenza viruses types 1, 2 and 3, RS virus, adenoviruses types 3, 4 and 21, poliovirus type 1 and Coxsackie A type 21 and Coxsackie B type 3 viruses. Cell lines derived from nonprimate tissues were generally less susceptible than cell cultures of human and simian origin. The combined use of fresh cultures of human thyroid and monkey kidney tissues and of a human cell line seems to provide a satisfactory indicator system for the viruses employed in this study.     

Decreased cAMP levels in human diploid cells exposed to cholinergic stimuli.

Carbachol at a concentration of 1 micron caused very significant decreases in PGE1 stimulated cAMP levels in three human diploid lung fibroblasts, WI-38, MRC-5, and IMR-90. Detailed studies with WI-38 cells showed that carbachol acted at low concentrations and very rapidly, cAMP levels being reduced by 50% in about 1 minute. The effect was moderately reduced but still very significant in Ca++ free medium containing 10(-5)M EGTA. Acetylcholine (in the presence of physostigmine), pilocarpine, and muscarine lowered cAMP levels at concentrations of 1 or 10 micron. The effects of carbachol were exquisitely sensitive to atropine but were unaffected by hexomethonium or d-tubocurarine. These data suggested that the cholinergic response of WI-38 cells was of the muscarinic type.     

Sensitivity of influenza a viruses to human interferons in human diploid cells

Abstract The sensitivity of influenza virus to the action of natural human interferon (IFN)-α+β and -γ, and to the action of highly purified recombinant HuIFN-αB, -αD, and -αF, has been investigated. A plaque assay for the fowl-plague strain of influenza A virus has been established using human embryonic foreskin (HEF) cells. The sensitivity of influenza virus to all IFNs tested in this assay was comparable to that shown by vesicular stomatitis virus (VSV) which was taken as the reference standard. The high sensitivity to IFN action found for the fowl-plague strain was confirmed for the WSN strain of human origin in a yield reduction assay.     

Transformation of dog embryo kidney cells by human herpesviruses

The infection of dog embryo kidney (DEK) cells with herpes simplex virus type 2 (HSV-2) or human cytomegalovirus (HCMV) led to the development of transformed cell lines. Rapidly dividing DEK cells with unlimited division potential exhibited growth in 2% serum, contained nuclear virus antigens, and formed small (+/- 0.2 mm) colonies in 0.3% agarose. Immortal cell lines showing the same transformation properties were also obtained after transfection with purified HSV-2 or HCMV DNA. These results confirm the transforming capacity of both herpesviruses as well as the usefulness of this different type of mammalian cells in transformation studies.     

gamma-Glutamyltransferase in human diploid fibroblasts and other mammalian cells.

gamma-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S3 and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that gamma-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme.     

The export of glutathione from human diploid cells in culture.

The export of glutathione from cultured human diploid fibroblasts into the surrounding medium was found by isotopic labeling experiments using [35S]cystine and by enzymatic measurements. The major part of the glutathione exported from the cells was found in normal culture medium as mixed disulfide of glutathione and cysteine. Radioactivity of 35S, mostly derived from cellular glutathione, was mainly located in glutathione moiety, not in cysteine moiety, of the mixed disulfide. Export of free glutathione was found when cystine-free medium was used. It was, therefore, concluded that mixed disulfide of glutathione and cysteine was formed in the medium by exported glutathione and medium cystine via sulfhydryl-disulfide exchange reaction. Amount of total glutathione exported from the cells was measured by enzymatic method and it was found that more than 10% of normal cellular glutathione was exported within 2 h. Apparent concentration of glutathione in the medium after a day of culture reached 3 to 4 micrometer, which was comparable to that observed in normal plasma by the same enzymatic method.     

Use of human diploid cells in vaccine production

An informal meeting was held at the National Institute for Biological Standards and Control (NIBSC), Potters Bar, U.K. on 24 August 1989 between representatives from industry, the World Health Organisation (WHO) and NIBSC to discuss issues related to the use of human diploid cells (HDC) in virus vaccine production. This record of the discussions and concensus views which emerged are provided to stimulate consideration by a wider audience. A list of participants is given in the Appendix.     

Genome structure and virion polypeptides of the primate herpesviruses Herpesvirus aotus types 1 and 3: comparison with human cytomegalovirus.

Two serologically distinguishable primate herpesviruses, Herpesvirus aotus type 1 and type 3, were examined with regard to their genomes and structural polypeptides. The duplex DNA genomes of these two viruses were found to be essentially identical in molecular weight (Mr approximately equal to 145 X 10(6)) and guanine plus cytosine composition (55%). Both contained unique and inverted repeat nucleotide sequences of the same size and arrangement, which, as judged by DNA-DNA hybridization and restriction enzyme analyses, were at least 95% homologous. In addition, no differences were observed in electrophoretic profiles of virion polypeptides. Because of their great similarity with respect to these criteria, the two viruses ought to be considered independent isolates (or strains) of a single virus, which should be designated H. aotus type 1. The elevated molecular weight and presence of two sets of inverted repeat sequences closely resemble the structure of the human cytomegalovirus genome. However, no sequence homology (less than 5%) nor similarity in virion polypeptides was detected between H. aotus type 1 and human cytomegalovirus.     

Quantitation of in situ hybridization of ribosomal ribonucleic acids to human diploid cells

The hybridization of 5S and 28S ribosomal RNAs to human fibroblast and leukocyte cells was used as a model system to quantitate the technique of in situ hybridization for human diploid cell types. Quantitation consisted of counting (scoring) the number of grains formed over both interphase nuclei and metaphase chromosomes on slides after various hybridization procedures. The average number of grains/nucleus per slide was then used to determine hybridization percentages. As with nitrocellulose filter hybridizations the kinetics of in situ hybridizations can be fit with a single first-order rate constant. However, the in situ hybridization rate was approximately 10 times slower than the corresponding filter hybridization rate. The efficiency of in situ hybridization was found to range between 5 and 15% for both leukocyte and fibroblast cell types and for both metaphase and interphase nuclei. Determination of the parameters of the in situ hybridization reaction of ribosomal RNAs to diploid chromosomes define the experimental conditions needed for the localization of single copy genes to diploid chromosomes.     

Susceptibility of human T cells, T-cell subsets, and B cells to cryopreservation

We have studied the number of viable and functionally active T and B lymphocytes obtainable after cryopreservation to determine the best and most practical way to recover the maximal number of viable and functionally active cells. Assays were done on purified populations of human T and B cells recovered after cryopreservation. The results were compared to those obtained from similar types of cells fractionated from fresh and from cryopreserved mononuclear cells. The number of viable T cells recovered after cryopreservation was significantly lower than the number of viable T cells obtained from either fresh or cryopreserved mononuclear cells. The residual viable T cells recovered after cryopreservation showed significantly reduced blastogenic activity in response to pokeweed mitogen (PWM) stimulation. This occurred despite their normal blastogenic response to phytohemagglutinin and their normal ability to help B cells in the production of immunoglobulins following PWM stimulation. The reduction in the blastogenic responses of these T cells to PWM stimulation is attributed to the loss of a portion of the PWM responding subset of T cells. The loss in this subset of T cells was related to the exposure of cells to ammonium chloride prior to cryopreservation. The viability and functional abilities of B cells were not affected regardless of whether purification was done before or after cryopreservation. These findings indicate that extrinsic membrane damage to T cells induced prior to cryopreservation can affect the viability and responsiveness of a certain population of normal T cells. The damage can be minimized by reversing the sequence of T-cell isolation and freezing so that isolation of T cells is done after, rather than before, freezing. These results could be important in the study of T cells from patients with T-cell abnormalities, since the patients' cells could have an intrinsic membrane defect which would make them sensitive to freezing similar to that induced by extrinsic damage.     

Polyadenylic acid sequences in rhinovirus RNA species from infected human diploid cells.

Polyadenylic acid sequences were shown to be present in rhinovirus virion RNA. Virus-specified RNA from human embryo lung cells infected with rhinovirus also contained polyadenylic acid but did not contain any polyuridylic acid sequences.