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PubMed1995

Differentiation of a culture-adapted mutant bloodstream form of Trypanosoma brucei into the procyclic form results in growth arrest of the cells.

The bloodstream forms of Trypanosoma brucei monomorphic strain 427 serially passaged in rats can differentiate in vitro equally well in HMI-9, HMI-10, SDM-79 or Cunningham's medium into the insect (procyclic) forms by a simple temperature shift from 37 to 26 degrees C in the presence of citrate and cis-aconitate. The procyclic forms thus generated can also continue to multiply at 26 degrees C without replacing the culture medium. The same strain of T. brucei pre-adapted to grow as bloodstream forms in HMI-10 medium at 37 degrees C is also capable of differentiating showing a similar rate of variant surface glycoprotein (VSG) disappearance and appearance of phosphoenolpyruvate carboxykinase (PEPCK) under the same experimental conditions. However, appearance of both procyclin mRNA and procyclin protein is much delayed and the resulting procyclic forms cannot multiply. The culture-adapted bloodstream forms are capable of infecting rats, and the cells thus harvested from the rats can differentiate but cannot multiply in the same manner as the original culture-adapted bloodstream forms. Apparently, a certain variant has been selected during the adaptation of T. brucei bloodstream forms from rat blood to the culture medium. This variant could be a useful tool for identifying the genes involved in differentiation of T. brucei and multiplication of the procyclic forms. Comparison of the protein profiles between the wild-type and the variant during differentiation showed that a major protein band of about 70 kDa remained in the non-dividing variant procyclic forms but vanished in the rapidly dividing wild type procyclic forms. N-terminal determinations indicated that the 70-kDa protein band consists of bovine serum albumin and fetuin. Presumably these two serum proteins are actively taken up by the bloodstream forms via endocytosis. Since the procyclic forms are incapable of endocytosis, the serum proteins may be rapidly diluted in the growing wild type procyclic cells but remain unchanged in the non-dividing procyclic cells of the variant. Further studies are underway in trying to identify the key distinctions between these two lines of cells at the molecular level.

Animals↗

PubMed1984

Replication and morphogenesis of avian coronavirus in Vero cells and their inhibition by monensin.

Avian infectious bronchitis virus (IBV) was adapted to Vero cells by serial passage. No significant inhibition of IBV replication was observed when infected Vero cells were treated with alpha-amanitin or actinomycin D. In thin sections of infected cells, assembly of IBV was observed at the rough endoplasmic reticulum (RER), and mature IBV particles were located in dilated cisternae of the RER as well as in smooth cytoplasmic vesicles. In addition to typical IBV particles, enveloped particles containing numerous ribosomes were identified at later times postinfection. Monensin, a sodium ionophore which blocks glycoprotein transport to plasma membranes at the level of the Golgi complex, was found to inhibit the formation of infectious IBV. In thin sections of infected Vero cells treated with the ionophore, IBV particles were located in dilated cytoplasmic vesicles, but fewer particles were found when compared to controls. A similar pattern of virus-specific proteins was detected in control or monensin-treated IBV-infected cells, which included two glycoproteins (170 000 and 24 000 daltons) and a polypeptide of 52 000 daltons. These results suggest that the ionophore inhibits assembly of a virus which matures at intracellular membranes.

Animals↗

PubMed1987

Cold-adaptation of human rotavirus.

A human rotavirus strain was cold-adapted for possible future use as a live vaccine. The original strain was isolated in 1980 in primary cynomolgus monkey kidney cells and has a serotype I and subgroup II antigenicity. The virus was serially passaged in African green monkey kidney cells; it was cultivated at 37 degrees C at the first stage of passages, and the cultivation temperature was then shifted down stepwise by 3 degrees C per each 10 passages. Finally the virus was passaged 10 times at 25 degrees C (total passage number of 55). The virus formed small-size plaques with irregular shaped borders at 31 degrees C. Growth at 25 degrees C of the cold-adapted virus was higher than that of the original virus. There was no difference between the migration patterns of 11 dsRNA segments in polyacrylamide gel electrophoresis of the original and the cold-adapted viruses.

Adaptation, Physiological↗

PubMed1988

Current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses.

Vaccines against parainfluenza (PIV) and respiratory syncytial viruses (RSV) that are currently being developed include both live and subunit vaccines. Candidate live PIV vaccines that have been found to be attenuated and efficacious in rodents or primate models are (1) cold-adapted, temperature-sensitive mutants of PIV-type 3 that have been serially passaged at low temperature (20 degrees C) in simian kidney tissue culture; (2) protease-activation mutants (PIV-1-Sendai), which have mutations that decrease the cleavability of their F glycoprotein by host cell protease; (3) an animal virus, bovine PIV-3 virus, which is antigenically related to the human PIV-3 virus, and (4) vaccinia recombinant viruses bearing RSV or PIV-3 glycoproteins. Subunit RSV and PIV-3 viruses are being produced and evaluated as immunogens. A major concern with these vaccines is the possibility of disease potentiation following virus infection as occurred previously with formalin-inactivated measles and RSV vaccines. Studies indicate that PIV-3 and RSV glycoprotein vaccines are immunogenic and efficacious in animals but insufficient data exist to estimate their capacity to potentiate disease. However, since a cotton rat model is available to detect potentiated disease resulting from infection of cotton rats previously immunized with formalin-inactivated RSV vaccine, it is now possible to systematically evaluate new vaccines in experimental animals for disease potentiation before studies are initiated in humans. It is likely within the next several years that one or more of these PIV or RSV vaccines will be tested in humans for safety and immunogenicity.

Animals↗

PubMed1989

Characterization of vaccinia virus deletion mutants isolated from persistently infected Friend erythroleukemia cells.

Persistent viral infections in vitro are useful to study the evolution of virus populations in the absence of immunological pressure. Several deletion mutants have been isolated in this laboratory from Friend erythroleukemia cells persistently infected with vaccinia virus strain IHD-W, designated SQAvac. Two of the mutants, which remain stable after serial passage in L cells, have been characterized. The deletion which range between 20 to 22 kb, has been localized at the left terminus comprising HindIII fragments C and N. In addition, HindIII B fragment lost the sequences that hybridize to pAG5, a plasmid containing the 3.5 kb terminal sequences and acquired different restriction sites. Phenotypic characterization of these mutants revealed that they were not replication defective since they grew in all cell lines tested and produced plaques of the same size as the wild-type. However, they were less effective in suppressing host protein synthesis. The LD50 for the mutants titered in NIH Swiss female mice was greater than 10(9) PFU as compared to 10(6) PFU for the wild-type, indicating reduced virulence in vivo. These mutants, which display different properties to previously described mutants with deletions at the left terminus, provide another valuable system to study the molecular basis of virulence of vaccinia.

Animals↗

PubMed1989

Infectivity of vesicles prepared from chilo iridescent virus inner membrane: evidence for recombination between associated DNA fragments.

Treatment of CIV particles with octylglucoside at high ionic strength leads to the solubilization of the inner viral membrane. Incubation of permissive cells (Cf124 cells) with vesicles obtained after dialysis of the detergent shows that this fraction is infectious. This infectivity, which is very low, could only be detected after two serial passages on permissive cells. This phenomenon is, however, reproducible. Isopycnic centrifugation analysis shows that some DNA cosediments with the vesicles. Extraction and purification of this DNA confirm the presence of a large DNA fragment of about 50.10(6) Da. Digestion with restriction endonucleases demonstrated that this DNA did not correspond to a particular fragment but to a population of DNA fragments of homogeneous size arising from various regions of the viral genome. Purified viral DNA was not infectious, the presence of DNA in the vesicles could not account therefore for their infectivity. Experiments of non-genetic reactivation of purified CIV DNA by UV-irradiated virus suggest that one (or several) structural component(s) of CIV particles must be involved in the first stages of the viral replication cycle. In addition, transfection of cells with large overlapping DNA fragments could generate infectious particles when the cells were superinfected with UV-irradiated virus. It can be supposed that the vesicle suspensions, which probably contain the reactivating factor, are composed of a population of vesicles which are all different in their DNA content. Infectivity of such suspensions would be the consequence of a recombination between large overlapping DNA fragments.

Aedes↗

PubMed1990

Foot-and-mouth disease virus strains isolated from persistently infected cell cultures are attenuated for mice and cattle.

It was previously reported that during serial passage of a BHK-derived cell line persistently infected with foot-and-mouth disease virus, (termed C1-BHK-Rc1) the virus became increasingly more virulent for BHK-21 cells (de la Torre et al 1988). Virus strains isolated from different cell passage levels were tested for virulence in mice and cattle. The results showed that in the course of persistence in BHK cells, FMDV became progressively less virulent for mice and cattle.

Animals↗

PubMed1991

Changes in cell gene expression in human leukemic cells persistently infected with vaccinia virus.

Persistent viral infections in vitro are useful systems to study the coevolution of virus and cell populations. Persistent infection of mouse Friend erythroleukemic cells (FEL) with vaccinia virus results in profound changes of the virus as well as of the cells. To investigate phenotypic changes of other cell types, we have established a persistent infection with vaccinia virus in a human leukemic cell line (K562). This cell line can be induced to differentiate along the erythroid pathway synthesizing embryonic and fetal globins, thus providing a system in which specific genes can be stimulated. After serial passage, the persistently infected cells (K562vac) became spontaneously differentiated, as shown by the increase in the number of cells producing hemoglobin (benzidine positive cells), and resistant to superinfection. These phenotypic changes of the cells were not accompanied by changes in the viral population. Hybridization of cellular RNA with cloned embryonic and fetal globin genes indicated that uninduced K562 cells do not express these genes, whereas cells induced by hemin or butyrate express G gamma (fetal globin) epsilon and zeta (embryonic globins) genes. By contrast vaccinia infected cells spontaneously express the G gamma gene. These results demonstrate that persistent infection with vaccinia virus elicited phenotypic changes in the infected cell population; in this case the constitutive expression of fetal hemoglobin.

Cell Line↗

PubMed1994

Differential regulation of cation transport of vascular smooth muscle cells in a high glucose concentration milieu.

To gain new insights into the pathogenesis of diabetic angiopathy, the influence of high glucose concentration on cation transport of vascular smooth muscle cell (VSMC) membrane was investigated by measuring Na, K and Ca transport in serially passaged cultured VSMC. (1) Na-K pump activity, described as ouabain sensitive 86Rb uptake, and Na-K cotransport, described as bumetanide sensitive 86Rb washout of VSMC, grown in high glucose concentration medium (460 mg/dl), was lower than that grown in normal glucose concentration medium (100 mg/dl). A smaller 5-N,N-hexamethylene amiloride (HMA) sensitive 22Na uptake (Na-H antiport) in high glucose concentration medium. accounted for this difference. (2) 45Ca uptake was also smaller in VSMC cultured in high glucose concentration medium. However, the washout rate constant for 45Ca was comparable between high and normal glucose cultured VSMC. (3) Both intracellular concentration of Na and cytosolic free Ca concentration concentration ([Ca]i) of high glucose cultured VSMC were greater than normal glucose cultured VSMC. (4) Intracellular water volume based on the equilibrium distribution of 3-O-methyl-[14C]glucose was not different between normal and high glucose cultured VSMC. It is concluded that VSMC grown in high glucose concentration milieu manifests a decreased Na-K, and Ca transport in conjunction with an increase in intracellular concentration of Na and [Ca]i. These results suggest that high glucose, per se, may alter membrane permeability to cations, possibly leading to changes in VSMC contractility and/or proliferation. This abnormality seen in the diabetic state may closely link to the pathogenesis of diabetic angiopathy, thus as a result risking hypertension and vascular disease.

Amiloride↗

PubMed1992

Phenotypical modulation of liver fat-storing cells by retinoids. Influence on unstimulated and growth factor-induced cell proliferation.

In conditions of chronic liver inflammation, liver fat-storing cells (FSC) differentiate into 'myofibroblast-like cells'. This transition is characterized by a gradual loss of vitamin A stores, and previous studies suggest a possible relationship between the intracellular retinoid content and the proliferative potential of this cell type. In the present study, we further characterized this aspect of FSC biology by monitoring ultrastructural changes and growth characteristics during several serial passages in culture. Our observations suggest that the complete transition to the 'myofibroblast-like phenotype' is paralleled by a sudden and remarkable increase in the growth rate. At this stage, cell growth appears rather independent from the presence of mitogens in the culture medium, suggesting cell transformation. Accordingly, the mitogenic effects of platelet-derived growth factor and epidermal growth factor appears reduced when compared to those observed in FSC retaining the original 'storing' phenotype. Incubation of vitamin A-depleted FSC with retinol and retinoic acid resulted in the partial recovery of intracellular retinoid stores and in a significant reduction of basal growth rate and basal and growth factor-induced DNA synthesis. In summary, these in vitro observations suggest that intracellular retinoids play a central role in the control of unstimulated and growth factor-induced FSC proliferation and may help understand in vivo mechanisms leading to liver fibrosis.

Analysis of Variance↗

PubMed1986

Disinfectant induced changes in the antibiotic sensitivity and phage typing pattern in Staphylococcus aureus.

Serial passage through disinfectants at sub-minimal inhibitory concentrations induced antibiotic resistances in 30.5% of the derived phenotypic variants of five strains of Staphylococcus aureus. A proportion of these phenotypic variants showed changes in their phage typing pattern by becoming susceptible to an increased number of phages, and a change from phage non-typable to typable was also observed. A loss of temperate phages was noted in some variants when subjected to reverse phage typing.

Disinfectants↗

PubMedDate not supplied

Establishment of human mucosal microvascular endothelial cells from inferior turbinate in culture.

Human microvascular endothelial cells were isolated and cultured from the mucosa of inferior turbinates. Using dish-coated collagen and a medium composed of a 1:1 mixture of sarcoma 180-conditioned medium and Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum and 75 micrograms/mL endothelial cell growth factors prepared from bovine pituitary glands), these cells grew rapidly to confluence and survived serial passages until the 16th population doubling level. The cells were identified as endothelial cells by their morphology, immunostaining of factor VIII antigen, and cytochemical staining with Ulex europeus agglutinin. Furthermore, Weibel-Palade bodies and numerous pinocytotic vesicles were confirmed by electron microscopy. Proliferation experiments demonstrated the need for either endothelial cell growth factor or tumor-conditioned medium. An exogenous matrix was also required for these cells in tissue culture. A tubule-like morphology appeared in the original monolayer of human microvascular endothelial cells after 1 month in the same plate, indicating that these cells have the ability to form tubules in the presence of sarcoma 180-conditioned medium.

Cell Division↗

PubMed1986

Ectopic G-29 and G-37 glucagon secretion by hypercalcemic infantile renal tumors.

Four hypercalcemic infantile renal tumors were shown to secrete glucagon-like peptides. These unusual tumors were histologically classified as rhabdoid tumors of the kidney (3 cases) and a cellular mesoblastic nephroma (1 case). Elevated G-29 and G-37 glucagon levels were detected in the plasma and tumor extracts as well as in the supernatants of cultured tumor explants. Three of these tumors were heterotransplanted into the nude mice and serially passaged from a mouse to another. The glucagon level decreased in the transplanted tumor extracts with the number of passage.

Animals↗

PubMed1988

Non-neurovirulent, genetically stable clone of poliovirus (Sabin type III vaccine strain).

A clone of poliovirus type III (TRU-3) was derived from the Sabin-3 vaccine strain by the cDNA:RNA hybrid molecular cloning method. The virus recovered was found to be considerably less neurovirulent for primates than its progenitor Sabin III vaccine strain. Upon serial passages in humans, TRU-3 did not increase its neurovirulence (unlike the Sabin strain) and maintained the base sequence of the attenuated strain in a critical part of the 5' region of the genome (unlike the Sabin strain). The substitution of TRU-3 clone for Sabin type III in oral polio vaccine could eliminate the vast majority of cases of vaccine-associated poliomyelitis.

Animals↗

PubMed1983

Comparison between macroscopic and microscopic evaluation of tumour responsiveness using the subrenal capsule assay.

Using the subrenal capsule assay in normal mice, a histologic evaluation was made of 8 human primary ovarian tumours and 3 human colon, 2 lung and 5 ovarian carcinomas growing in serial passage in nude mice. The results of the evaluation indicated that there is a tumour- and drug-dependent correlation between the macroscopically and microscopically evaluated effects, with cyclophosphamide demonstrating excellent concordance but adriamycin and cisplatin both demonstrating consistently more tumour cell killing on histologic analysis than could be appreciated macroscopically. Leukocyte infiltration and fibrosis were greatly increased by the latter 2 drugs, leading to unrepresentative macroscopic measurements. Variable amounts of host cell infiltration can also be demonstrated in the untreated control when normal mice are used. The use of nude mice decreases the discrepancy between macroscopic and microscopic evaluation.

Animals↗

PubMed1985

Effects of long-term treatment of human carcinoma cells with interferon alpha.

Three tumor cell lines derived from human colon, urinary bladder and ovarian carcinoma were serially passaged in the continuous presence of human interferon alpha for extended periods of time. Phenotypic changes induced by interferon differed among these three cell lines. Thus interferon enhanced colon tumor cell aggregation but inhibited the aggregation of bladder tumor cells. The antiproliferative activity of interferon was more pronounced in bladder and ovarian cells than in colonic cells. However, the tumorigenicity of parental and cloned colon tumor cells injected i.p. or s.c. was markedly reduced by passage of the cells with interferon. Interferon treatment reduced the tumorigenicity of ovarian tumor cells when these cells were injected i.p. but not when injected s.c. The tumorigenicity of bladder tumor cells was not affected by interferon.

Animals↗

PubMed1986

Growth characteristics of head and neck squamous cell carcinoma in nude mice.

Growth behaviour of human squamous cell carcinoma from the head and neck region was studied in nude mice. Tumour growth was observed in 10 out of 13 tumours transplanted with a primary take rate of 77 and 100% for serial passaging. The take rate percentage related to the number of tumour inocula used, varied from 10 to 90% in the first passage and from 70 to 100% in the subsequent passages. No significant difference could be established in the growth character, except from the first to the second passage. Histological studies demonstrated preservation of the original histopathological features, micro- and macroinvasion of two tumour lines into the fibrous capsule and cyst formation. Comparison of growth curves with histological features demonstrated that growth curves are not a reliable measure for the number of vital tumour cells present.

Animals↗

PubMed1984

A method for directly establishing short-term cultures of human gestational choriocarcinoma in vitro.

A procedure is described which offers a method for establishing, without recourse to a serial passage within animal hosts, short-term cultures of human gestational choriocarcinoma in vitro. The method involves the use of either post mortem or surgical biopsy tissue from patients with known choriocarcinomas. The tissues are subsequently sectioned on a freezing microtome and viable tumour selected for explantation in vitro. Radioimmuno assays of culture media supernatant confirm the trophoblastic nature of the cells growing in vitro, cytogenetic analyses of these cultures reveal several aneuploid modes.

Cells, Cultured↗