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PubMed1984

Potential limitations of in vitro clonogenic drug sensitivity assays.

P388 murine leukemic cells lines which were resistant (P388R) or sensitive (P388S) to adriamycin (adr) were used to evaluate the potential utility of in vitro clonogenic assays for detecting and quantitating the number of adr-resistant cells present in a cell mixture. The progeny of P388S cells that had been exposed for 1 h or continuously to adr were as sensitive to the drug as the original P388S cells. Serial passages in the presence of adr, however, resulted in the selection of adr-resistant cells, but even these cells varied in their level of resistance. When P388S and P388R cells were mixed together and exposed to adr, the clonogenic assay consistently overestimated the proportion of P388S cells present. These studies demonstrate some of the inherent limitations of clonogenic drug sensitivity assays and may provide an explanation for the reported discrepancies between in vitro drug sensitivity and clinical responses to therapy reported in the literature.

Animals↗

PubMed1992

Infectivity of human T-lymphotropic virus type I to human nervous tissue cells in vitro.

Infectivity of human T-lymphotropic virus type I (HTLV-I) to human nervous tissue cells was explored using co-cultivation with X-irradiated, HTLV-I-producing MT2 cells. Examined cells included normal cerebellar cells, brain tumor cells (astrocytoma, medulloblastoma, meningioma, hemangioblastoma, and schwannoma), and various cell lines (astrocytoma, ependymoma, oligodendroglioma, medulloblastoma, and neuroblastoma). Successful HTLV-I infection was confirmed immunohistochemically using monoclonal antibodies to HTLV-I p19, p24, and pX product. All cell lines and primary cultures from normal cerebellar tissues and brain tumors could be infected with HTLV-I. Double immunostaining showed that glial fibrillary acidic protein-, S-100 protein- or vimentin-positive cells were susceptible to infection. Neurofilament- or neuron-specific enolase-positive cells in medulloblastoma could also be infected. Reverse-transcriptase assay revealed the productive infection in U251-MG (astrocytoma) and KG-IC (oligodendroglioma) lines. Co-cultivated U251-MG cells formed syncytial polykaryons after serial passages, and polymerase chain reaction assay detected HTLV-I genome in U251-MG syncytial polykaryons and p19+ mononuclear cells. HTLV-I viral RNA was also detected in infected U251-MG cells by in situ hybridization. These data show that HTLV-I may have a wide spectrum of infectivity in human nervous tissues.

Base Sequence↗

PubMed1991

Alteration of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa.

The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa3 relative to wild-type strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1[mi-3] allele, which suppresses both mutations.

Amino Acid Sequence↗

PubMed1989

Milk whey as a serum supplement for the growth of FMD virus in BHK-21 cells.

Two batches of experimental media containing 10% milk whey (sweet or sour) and 1% bovine serum supported the growth of BHK-21 cells in serial passages. The cell yield was greater than 2 x 10(6)/ml at 48 h. The cells were susceptible to inoculation with foot and mouth disease (FMD) virus type 'A' and the virus titres obtained were comparable with those in Eagle's MEM-G with 10% serum. The use of milk whey could reduce the consumption of serum required for the growth of FMD virus in BHK-21 cells by up to 90%.

Animals↗

PubMed1984

The potential of the nude mouse xenograft model for the study of head and neck cancer.

A total of 130 human head and neck cancers was transplanted subcutaneously in athymic nude mice in order to obtain a series of xenografts. All tumours were derived from previously untreated patients. Initial growth, which was histopathologically confirmed, was observed in 34 cases (26.1%). Serial passages were successful in 12 of 23 cases (52.1%). Of 117 squamous cell carcinomas, 30 (25.9%) showed initial take in the mice. Poorly differentiated squamous cell carcinomas tend to grow more readily than moderately differentiated and well-differentiated ones. Material from metastatic lymph nodes tends to show a higher take than material from primary tumours. In general the tumour-volume doubling time decreased to 4-6 days when the number of passages increased. Histology of the xenografted tumours showed that transplantation had caused no major changes. No macro-, or microscopic signs of metastasis were observed in any of the mice. The implications of this model for fundamental and applied research are discussed.

Animals↗

PubMed1986

Serial ultrastructural study of experimental Creutzfeldt-Jacob disease in guinea pigs.

Guinea pigs inoculated with brain homogenate from serially passaged Creutzfeldt-Jakob disease (CJD) were killed biweekly starting at week 2 until terminal illness (about 200 days following inoculation). A mild swelling of postsynaptic dendrites and an increase in the number of glial filaments in astrocytic processes was seen at week 4, followed by increased swelling and lucency of axons and dendrites by week 6 post inoculation (p.i.). Severe undulation and focal interruptions of synaptic membranes were also observed both at weeks 4 and 6. By week 8, one could see cystically dilated cellular processes. These sometimes showed continuity with adjacent swollen processes through focally disrupted plasma membranes, and most likely represent a progressive enlargement of vacuoles through fusion and subsequent addition of adjoining processes. The spongiform changes increased mildly between week 8 and week 10 and remained essentially the same in subsequent weeks. After week 24 there was a sharp increase in both the number and size of vacuoles. At week 24 severe structural alterations were present both in the neurons and astrocytes, and numerous intranuclear inclusions were demonstrated in many neuronal nuclei. This study shows that morphological changes in the brain occur considerably earlier than the clinical manifestations of the disease. In the early phase of the disease, there were significant alterations on the dendrites and synapses.

Animals↗

PubMed1995

Partial characterization of a unique 84-kDa polypeptide stimulated by ascorbic acid in skin fibroblasts.

Ascorbic acid stimulated the synthesis of a noncollagenous 84 kDa polypeptide by up to 3.5-fold in primary cultures of chick skin fibroblasts. Stimulation was observed with concentrations ranging 0.4 to 50 micrograms/ml and was dose-dependent within the concentrations up to 10 micrograms/ml during 24 h treatment under conditions in which procollagen synthesis was increased. This protein was found to be translated with a molecular size of 84 kDa in an in vitro cell-free translation experiment, indicating that the 84 kDa polypeptide was a primary translate. The stimulation was accompanied by a 1.7-fold increase in translational activity of the mRNA for this polypeptide. Its synthesis rapidly reduced when the cells were serially passaged until the third subcultivation, whereas procollagen synthesis remained essentially constant. These results indicate that the stimulation of this unique 84 kDa polypeptide by ascorbic acid was modulated at least in part at a translational level and its synthesis appears to be related to in vitro cellular aging.

Animals↗

PubMed1993

The EnDA endometrial adenocarcinoma: an oestrogen-sensitive, metastasizing, in vivo tumour model of the rat.

A high percentage of endometrial carcinomas contain oestrogen and progesterone receptors. For endocrine therapy of recurrent endometrial carcinoma, only high-dose progestins are in clinical use. As, therefore, the development of new endocrine treatment strategies is of great interest, suitable animal models for this tumour are essential. Up to now, only human tumour xenografts transplanted in immune-deficient nude mice, but no syngeneic in vivo tumour models, have been available. In the present article we describe the hormone sensitivity of the EnDA endometrial adenocarcinoma of the DA/Han rat growing as s.c. implants in DA/Han rats and athymic nude mice in serial passage. In both species, the tumour expresses oestrogen, but no progesterone receptors. Transplanted in DA/Han rats or nude mice, ovariectomy reduced tumour weight by 64% and 46% respectively. In both species substitution of ovariectomized animals with oestradiol restored tumour weights to intact control levels. Oestradiol substitution of intact animals did not further enhance tumour growth. The growth of the primary tumour was inhibited by medroxyprogesterone acetate (MPA) at a dose of 100 mg/kg by 67% and by tamoxifen at a dose of 20 mg/kg by 38%. Lung metastases were regularly seen in both species, although to a lesser extent in nude mice than in DA/Han rats. Tamoxifen treatment did not alter the number of lung metastases, whereas MPA or ovariectomy produced a significant reduction in the number of lung metastases. The EnDA endometrial carcinoma of the DA/Han rat with respect to its oestrogen sensitivity, oestrogen receptor expression, morphology and metastatic growth, grossly resembles a typical endometrial adenocarcinoma and can therefore be regarded as a useful in vivo experimental model for the evaluation of new endocrine treatment strategies.

Adenocarcinoma↗

PubMed1990

Accelerated age-related decline in replicative life-span of Duchenne muscular dystrophy myoblasts: implications for cell and gene therapy.

An assessment of the replicative life-span of myoblasts is of fundamental importance in designing treatment strategies for Duchenne muscular dystrophy (DMD) based on cell or gene therapy. To ascertain myoblast life-span, or the total number of cell divisions of which a myoblast was capable, we serially passaged and counted the progeny of individual myoblasts until they senesced. We compared the life-span of myoblasts from eight DMD patients with controls: three individuals with no known neuromuscular disease, three DMD carriers, and three patients with other muscle degenerative diseases. A decline in replicative capacity was observed with increasing donor age, which was markedly accelerated for DMD relative to control myoblasts. The average myoblast from a 5-year-old control was capable of 56 doublings, or a potential yield of approximately 10(17) cells per cell. By contrast, at 2 years of age, the typical age at clinical onset, only 6% of DMD myoblasts had a life-span of 50 doublings in tissue culture, and by age 7 DMD myoblasts capable of 10 doublings were rare. Our results suggest that the myoblasts (satellite cells) of even the youngest DMD patients have undergone extensive division in an attempt to regenerate degenerating myofibers. These findings have implications for therapeutic intervention in DMD involving genetic engineering and myoblast implantation.

Age Factors↗

PubMed1994

Role of H-ras in the malignant progression of rat tracheal epithelial cells.

The effect of an activated H-ras oncogene on the progression of neoplasia was studied in transformed rat tracheal epithelial cells. Nude mouse tumours produced by injection of these cells exhibited a higher fraction of cells containing the mutant ras gene that did the injected cells, while a subclone that lacked the ras mutation was much less tumorigenic than parental cells. Serial passage of one cell line containing a ras mutation resulted in an increase in the fraction of ras-mutated cells, which suggests that, in this line, ras activation may confer a selective advantage in vitro as well. However, this was not seen in another ras-containing line, suggesting the importance of alternative pathways in malignant progression of rat tracheal epithelial cells.

Animals↗

PubMed1982

Autofluorescence as an index of ageing in human fibroblasts in culture.

An exponential increase in autofluorescence (AF) during serial passaging of human diploid fibroblasts, strain MRC-5, was observed using a fluorescence-activated cell sorter. Skin fibroblasts from patients with premature-ageing syndromes had high levels of AF, whereas virus-transformed cell lines had negligible amounts of AF.

Adult↗

PubMed1981

Propagation of the Kakegawa strain of bovine coronavirus in suckling mice, rats and hamsters.

The Kakegawa strain of bovine coronavirus was easily propagated in suckling mice. Infected animals died with nervous symptoms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. The 3rd passage viral material from infected mice evoked the same disease in suckling mice, rats and hamsters inoculated by the intracerebral or by the subcutaneous route. Viruses recovered from mice, rats and hamsters could be clearly differentiated from mouse hepatitis virus strain 2 by the neutralization test.

Animal Population Groups↗

PubMed1978

Some biological and serological properties of large and small plaque variants of Getah virus.

Two plaque types of Getah virus were isolated on BHK-21 cells from an emulsion of suckling mouse brain infected with strain AMM-2021 and were designated as large plaque (LP) and small plaque (SP) strains. Some of the properties of LP and SP strains were studied with the following results. The difference in plaque size of the LP and SP strains was quite distinct on BHK-21 cells and there was little difference in size at the 3rd--8th serial passage level in suckling mouse brain. The LP strain showed high hemagglutinating activity but the SP strain produced no detectable HA activity. The LP strain was shown to be more virulent than the SP strain to suckling mice by intracerebral or intraperitoneal inoculation. The LP strain was not neutralized as effectively as the SP strain with antisera to original, LP and SP strains. An enhancement of infectivity was observed when the LP strain was senstized with either anti-LP or anti-SP hyerimmune mouse sera. This phenomenon was not pronounced withe the SP strain.

Animals↗

PubMed1981

Isolation of cytopathic porcine rotavirus in cell roller culture in the presence of trypsin.

Cytopathic porcine rotavirus was isolated in roller tube cultures of MA-104 cells. Faeces of a piglet suffering from diarrhoea, were treated with trypsin which was also added to the maintenance medium. Using stationary cultures, virus was not isolated from the same materials. The cytopathic effect was clearly observed after 8 serial passages and the virus titer at the 14th passage level was 10(7) median tissue culture infective doses per ml, or higher. At the 27th passage, rotavirus particles were seen by negative contrast electron microscopy at a density of 1.36 to 1.38 g/cm3 in CsCl centrifugation gradients. There are partial cross-neutralization between the Lincoln strain of bovine rotavirus and porcine rotavirus from pigs or tissue cultures. Gnotobiotic piglets were inoculated with pig-passaged virus and viral antigen was detected in epithelial cells of small intestinal villi by immunofluorescence. The highest titer of virus was detected in faeces 72 hours after inoculation. The cell culture adapted virus which produce a cytopathic effect was designated the strain S 80 of porcine rotavirus.

Animals↗

PubMed1975

Effect of alkaline maintenance medium upon the growth of rabies virus in chick embryo cells.

HEP Flury strain of rabies virus was propagated in chick embryo cells under maintenance media of different pH. It was found that viral growth was better and reached a markedly higher maximum titer when the initial pH of maintenance medium was 8.2 to 9.0 than when it was 7.4. The enhancement of viral growth was not ascribable to mere neutralization of acids produced from infected cells, because the different media became almost equally neutral within an early phase of growth curve. Serial passage of the virus in chick embryo cells using pH 8.2 maintenance medium resulted in altered growth characteristics of the progeny virus; first, the virus so passaged could now grow equally well under alkaline and neutral maintenance media, and, secondly, autointerference observable with the parent virus eventually lowered virus yield when neutral maintenance medium was used, but this effect of undiluted passage was eliminated by the use of pH 8.2 maintenance medium.

Animals↗

PubMed1987

Pathogenic and structural properties of wild type infectious bursal disease virus (IBDV) and virus grown in vitro.

Large plaque (LP) and small plaque (SP) variants of Infectious Bursal Disease Virus (IBDV) which are formed in vitro after serial passages in CE-cells at low or high multiplicities of infection were tested for their pathogenic properties in susceptible chickens. LP virus caused clinical manifestations and destruction of the Bursa of Fabricius (BF) without killing the animals. No signs of a disease appeared after infection with SP virus, and only limited necrotic foci developed in the BF. Coinfection with the SP variant and the highly pathogenic wild type (wt) virus reduced mortality and the severity of the disease. In contrast to the SP variant, which forms incomplete particles of low density, with one or the other of the two genomic RNA segments missing, the two RNA segments characteristic for IBDV were present in approximately equal amounts in wt particles with a buoyant density of 1.29 g/ml isolated from the BF. In spite of an almost total replacement of one of the two major structural polypeptides with a molecular weight of 32 kd by a 28 kd protein, infectivity in this low density fraction was only slightly reduced. This finding underlines the importance of the type of post-translational modification in lymphoid cells or fibroblasts.

Animals↗

PubMed1975

A mouse hepatotropic variant of influenza virus.

A hepatotropic variant of avian influenza virus A/Turkey/England 63 (Hav 1, Nav 3) was selected by serial passages in mouse liver. Adaptation to this organ was established after 13 in vivo passages and was found to improve during further passages as shown by increasing rates of replication in livers of ICR mice. The mutant virus finally selected was stable and differed from the original virus mainly in lethality upon intraperitoneal injection in mice, in its ability to grow to high titers in livers of susceptible animals and in plaque morphology in chick embryo fibroblasts. No differences were detected in hemagglutination inhibition and neutralization by standard mouse antisera. Pathogenicity for the liver was independent of the route of inoculation, included other laboratory animals sensitive to influenza virus and could be inhibited by amantadine. Fatal hepatitis in 50 per cent of susceptible mice by the intraperitoneal route required from 10 to 20 EID50-. Pathological changes consisted of severe necrosis of liver parenchyma accompanied by release of F antigen into the serum and were apparently due to virus replication in hepatic cells as evidenced by immunofluorescence. The main implications of this animal model for studies on experimental hepatitis and on myxovirus-host interactions in an organ not usually associated with influenza are discussed.

Adaptation, Physiological↗

PubMed1978

Growth characteristics of influenza virus type C in avian hosts. Brief report.

Influenza virus type C could be propagated to high yield in primary chick embryo kidney cell culture (PCEK) provided that trypsin (2 microgram/ml) was used as a medium supplement. The virus could also be titrated by plaque assay using PCEK host cells and influenza C virus that had been plaque-purified in PCEK cells could then be serially passaged to high titer using the allantoic route of 10--11-day-old embryonated eggs.

Cells, Cultured↗