Inhibition of herpes simplex virus type 1 replication in fibroblast cultures by human blood mononuclear cells.

An assay was developed to test the effect of human blood mononuclear cells (MNCs) on herpes simplex virus (HSV) replication. In this assay, human fibroblast monolayers were inoculated with HSV and then cultured with or without blood MNCs. Fewer HSV-infected cells were recovered from human fibroblasts cultured in the presence than in the absence of blood MNCs. This inhibition of viral replication by MNCs was independent of HLA matching between the MNCs and fibroblasts and persisted even when T cells were depleted by antibody and complement. However, depletion of Leu11+ MNCs either by panning or with antibody and complement reduced the ability of the cells to suppress HSV infection, whereas enrichment of Leu11+ cells by fluorescence-activated cell sorting increased the viral suppression. Depletion of OKM1+ MNCs also reduced the viral suppression. After coculturing of MNCs and HSV-infected fibroblasts for 3 days, alpha interferon (IFN) and gamma IFN were detected in the supernatants. Predepletion of Leu11+ MNCs reduced the amount of gamma IFN produced in these cultures. Incubation of the MNCs and HSV-infected fibroblasts with antibody specific for either alpha or gamma IFN resulted in reduced viral suppression. Preincubation of MNCs for 18 h with either interleukin 2 or alpha IFN or for 7 days with antigen increased the suppression of HSV infection. These results suggest that natural killer cells with the Leu11+ phenotype participate in the recognition of HSV-infected cells at a point sufficiently early to interfere with the spread of infection in vitro and may inhibit viral replication by natural killer cell cytotoxicity, by generation of interferon, and by lymphokine-activated killing.     

Comparison of aldolase isozymes in placenta, HeLa cells, and human fibroblast cultures

The aldolase specific activity of the human carcinoma cell line, HeLa, against fructose 1,6-diphosphate as substrate is 4- to 5-fold greater than the specific activity of diploid human fibroblast cultures derived from skin and lung. HeLa aldolase is isozyme is predominantly the A type and its substrate preferences resemble human placenta. These findings provide further support for the oncofetal enzyme consitution of HeLa cells.     

Detection of phosphohexose isomerase deficiency in human fibroblast cultures

Summary Fibroblasts cultured from two patients afflicted with nonspherocytic hemolytic anemia due to phosphohexose isomerase (PHI) deficiency show on the average 53% of the normal PHI-activity. The presence of the defective enzyme in cells derived from the heterozygous relatives of the patients is revealed by an intermediate average specific activity; the wide range of PHI-activities observed in these cells, however, precludes the detection of heteozygotes. The PHI-genotypes of the patients and of their heterozygous and normal relatives respectively, can be distinguished by starch gel electrophoresis and by heat-inactivation studies with fibroblast-homogenates. These latter experiments confirm the results obtained with hemolysates (Tariverdian et al., 1970).D-Glucose-6-phosphate-ketol-isomerase, E.C.5.3.1.9.Supported by the Deutsche Forschungsgemeinschaft.Supported by the Stiftung Volkswagenwerk.     

Variegated translocation mosaicism in human skin fibroblast cultures.

The term "variegated translocation mosaicism" is used to describe the repeated occurrence, within cultures of human skin fibroblasts, of a multiplicity of chromosomal rearrangements. With respect to the frequencies of such cytogenetically aberrant clones we found that they (1) were not detectable in routine diagnostic skin fibroblast cultures from 29 subjects with a wide variety of indications for biopsy; (2) were not detectable during in vitro aging of diploid strains with four normal individuals; (3) could be detected after rescue from bacterial contamination of a culture from an otherwise normal diploid male; (4) occurred with high frequencies in independent cultures from another apparently normal subject; (5) occurred with high frequencies in multiple biopsies obtained at autopsy from a patient with Werner's syndrome who died of sepsis; (6) were of pseudodiploid nature; and (7) involved a different spectrum of chromosomes in different individuals. A consistent association with mycoplasma contamination could not be made.     

Fibronectin matrix: antibody-induced reorganization in human fibroblast cultures

Fibronectin, a major pericellular glycoprotein of adherent cells, was predominantly present in fibrillar structures in human fibroblast cultures as shown by indirect immunofluorescence. In conventional "patching experiments" where one day old cells were exposed to anti-fibronectin IgG in the cold, washed, and reincubated at 37 degrees no redistribution was seen. However, continuous exposure of the cultures to IgG at 37 degrees resulted in redistribution. The fibrillar structures were lost and fibronectin aggregates (patches) were found. Fab-fragments had no such effect. These results support the findings that fibronectin is predominantly a matrix protein and show that matrix components may be redistributed in cell culture conditions.     

Cultured human fibroblast enzymes. III. Enzyme activity in a triploid strain]

Complex investigation of 5 enzymes was carried out in a cell strain with triploidy 69, XXY, derived from a human spontaneous abortus. The activity of 3 enzymes (acid phosphatase, lactate and malate dehydrogenases) in triploid cells proved to be significantly increased as compared to those of 3 diploid strains, whereas the activity of alkaline phosphatase was decreased. The activity of glutamate-oxalacetate transaminase did not change. The absence of the pronounced genetic dose effect and different alteration of the activities of the enzymes studied may be considered as an expression of a disbalance of enzymes in cytogenetically defective cells.     

Comparative metabolic effects of fructose and glucose in human fibroblast cultures

Summary The comparative metabolic effects of fructose and glucose were determined in human fibroblast cultures. Cells were grown in four different media containing 5.5 and 27.5 mM of glucose and fructose, respectively. For these two hexoses, we compared their uptake, consumption, and conversion into¹⁴CO₂ and¹⁴C-lipids. D-Fructose was taken up in fibroblasts by an unsaturable process and its consumption was much smaller than that ofD-glucose. Whatever the experimental procedure, the glycogen content of cells grown in fructose media was significantly lower than of those grown in glucose media. Labeling of fructose and glucose with¹⁴C showed that more carbon from fructose than from glucose was incorporated into CO₂ and glycerolipids. The relative distribution of¹⁴C in the different lipid fractions was similar for both hexoses. These results indicated that the pathways of intermediary metabolism in fibroblast cultures were influenced by the nature of the carbohydrate present in the culture medium and that fructose was a better lipogenic substrate than glucose in human fibroblast cultures. This work was supported by grants for the Institut National de la Santé et de la Recherche Medicale (ATP 82-79-114).     

Herpes simplex virus infection in normal and mutant human fibroblast cultures

Reproductive efficiency of herpes simplex virus type 1 has been determined in several normal and mutant human skin fibroblast lines. The mutant cell lines were derived from individuals diagnosed as having Duchenne muscular dystrophy, a disease thought to involve genetically determined membrane defects. Yields of infectious virus, determined by plaque assay on rabbit kidney cell monolayers, were consistently lower from dystrophic cells than from normal cells. The yield from dystrophic lines was 3–20% of the normal. The time course of production of infectious particles did not appear to vary between dystrophic and normal host cells. Also, the initiation of replication of the viral genome did not appear to be altered in the dystrophic lines. It is proposed that a late maturation function is involved in the lower virus productivity in dystrophic cells.     

Staining of the spindle apparatus in human lymphocyte and fibroblast cultures

Summary The spindle apparatus of human mitoses can be preferentially stained with Giemsa if the cells are fixed with methanol-acetic acid and treated for about 15 min with 0.1xSSC at 92°C and for 18 h with 2xSSC at 60°C.     

Environmental pH modulation of collagenase in normal human fibroblast cultures

Normal human skin fibroblast cultures have been used to assess the effects of relatively minor changes in environmental pH on collagenase, a major extracellular gene product. Collagenase accumulation in the culture medium, measured both as enzyme activity and immunoreactive material, was 2- to 10-fold greater at pH 7.6–8.2 than at pH 6.8–7.2. The pH-associated increase in collagenase was parallel by an increase in general protein synthesis. Nevertheless, prototypic lysosomal and cytoplasmic enzymes changed very little under identical culturing conditions. Although substantial intracellular protein degradation occurred at all pH values, the small differences either in general protein degradation or in specific collagenase degradation in the medium were of insufficient magnitude to account for the increased accumulation of collagenase.     

Increased nuclear sizes in senescent human diploid fibroblast cultures.

Measurement of nuclear size in cultured human diploid fibroblasts (WI38) reveals a shift to larger cell nuclei as afunction of in vitro passage. Examination of nuclear size distribution on the basis of replicative potential reveals that at all levels of in vitro passage, the rapidly replicating cell population have smaller nuclear sizes than comparable populations of slow or non-replicating cells. While the nuclear size distribution of the rapidly replicating cell population remains relatively constant, there is a marked shift in the nuclear size distribution of the slow or non-replicating cell population to larger sizes with increasing in vitro passage.     

Low-serum medium development for human diploid fibroblast microcarrier cultures

A new medium supplement mixture, PPRF92, has been developed to enable the serial subculture of human diploid fibroblasts (MRC-5 cells) on microcarriers. Furthermore, the PPRF92 supplements enable cell growth at serum levels as low as 1%. Through an optimization programme, the PPRF92 supplements have evolved into a simple mixture with the concentrations of key components at a level that makes the overall cost very competitive with medium containing 10% foetal bovine serum (FBS). Furthermore, the PPRF92 supplement mixture is most efficacious when FBS is replaced with the cheaper, and more widely available, adult bovine serum (ABS). Although medium exchange with serum is necessary in order to achieve confluence on microcarriers, the PPRF92 mixture is only necessary at the initiation of each passage. Using the medium replinishment protocol that has been developed in our laboratory, MRC-5 cells were successfully serially passaged through 13 bead-to-bead transfers on microcarriers in DMEM/F12 medium enriched with the PPRF92 supplement mixture reported here, and 1% ABS.Correspondence to: L. A. Behie     

Isolation of the pericellular matrix of human fibroblast cultures

The pericellular matrix of human fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. The matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the major glycosaminoglycan components and fibronectin and procollagen as major polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix.