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PubMed1984

Establishment and characterization of a human T-cell leukemia line (LALW-2) in nude mice.

Lymphoblasts from the peripheral blood of a 10-year-old boy who was treated for T-cell acute lymphoblastic leukemia were heterotransplanted intraocularly into nude mice. The resultant tumors and their metastases were serially passaged both intraocularly and subcutaneously in the mice. The mouse tumors closely resembled morphologically and cytochemically a human T-cell lymphoma. The primary cells, as well as cells from subsequent in vivo passages, were predominantly of a suppressor T-cell phenotype (OKT8-positive). No viral products were identified. Chromosome analysis revealed a near-diploid karyotype with a translocation between chromosomes 11 and 14. The tumorigenicity, morphology, cytochemistry, immunologic phenotype, and karyotypic pattern of the cells remained constant through six serial in vivo passages over a period of 10 months. This is the first report of direct heterotransplantation and long-term in vivo maintenance of primary human T-cells in immunologically unmanipulated nude mice.

Animals↗

PubMed1975

Interfering passages of Sindbis virus: concomitant appearance of interference, morphological variants, and trucated viral RNA.

Serial passage of Sindbis at high multiplicities of infection resulted in cyclical variations in virus titer. Decreases in virus titer were correlated with the appearance of smaller-sized virions, interference and truncated viral RNA. The smaller particles were 37 nm in diameter, exclusive of the hemagglutinin spikes as compared with a diameter of 50 nm for standard virions. Passages which contained 37-nm partilces also interfered with infectious center formation by standard, plaque-purified virus. Polyacrylamide gel analysis of RNA isolated from virions present in interfering passages demonstrated the sequential appearance of three RNA species smaller than standard RNA with approximate molecular weights of 3.3 X 106, 2.7 X 106, and 2.2 X 106. The 3.3 X 106 RNA was evident in passage 5, by passage 8 both the 3.3 X 106 and 2.7 X 106 RNAs were present, and by passage 13 all three were present with the 2.2 X 106 RNA predominating.

Cell Line↗

PubMed1998

HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus).

Cotton rats (Sigmodon hispidus and S. fulviventer) are susceptible to many viruses that infect humans (e.g., poliovirus, respiratory syncytial virus, influenza virus, adenovirus, and parainfluenza virus) and have been influential in developing therapeutic clinical intervention strategies for many viral infections of man. This study set out to determine whether cotton rats are susceptible to infection with HIV type 1 (HIV-1). Results indicate that HIV-1 does infect the cotton rat and S. fulviventer is more susceptible than S. hispidus. The virus was passaged from animal to animal for a total of three serial passages; but HIV replicated poorly in vivo, was only detectable as proviral DNA, and never exceeded one provirus per 1.8 x 10(5) cotton rat peripheral blood mononuclear cells. Infection induced a distinct and characteristic anti-HIV antibody response that, in some animals, included neutralizing antibodies, recognized all of the major HIV-1 antigens and the antibodies lasted out to 52 wk post-infection. Neonate S. fulviventer were not more susceptible to infection than adults. In vitro culture studies produced indirect evidence of viral replication by detection of viral gag gene RNA in reverse transcriptase-PCR assays on viral culture supernatants. Collectively, these results indicate that HIV-1 can replicate in a nontransgenic rodent and that this system may have potential as an animal model for HIV-1 infection if viral replication rates can be improved in vivo.

Animals↗

PubMed1976

Lysosomal enzyme levels in human amniotic fluid cells in tissue culture. IV. %A N-acetyl-beta-D-glucosaminidase.

Total and % A hexosaminidase were similar for primary cultures and later passages of amniotic fluid cells. The culture variables-through serial passage, within a passage and replicate primary cultures-resulted in some variation in total hexosaminidase, but an insignificant change in % A hexosaminidase. They are unlikely to give rise to any problems in the antenatal diagnosis of Sandhoff's and Tay-Sachs diseases.

Acetylglucosaminidase↗

PubMed1967

Growth curve and distribution of Rous sarcoma virus (Bryan) in Japanese quail.

On primary infection with the Bryan strain of Rous sarcoma virus (RSV), the growth curve of the virus in the brain of Japanese quail was similar to that observed in chicks and turkey poults. Infectious virus disappeared from the brain after inoculation. After an eclipse period during which no virus was detectable, infectious virus began to appear at 2 days and reached maximal titers in the brain samples at 7 days after inoculation. When Japanese quail were infected intracerebrally with RSV, relatively high titers of virus were recovered from brain tissue but not from liver, lung, kidney, or blood of moribund birds. Only tumors produced in the wing web of quail infected subcutaneously yielded high titers of virus. Other tissues yielded no virus, even though wing web tumors appeared as early as in chicks similarly infected. RSV could be propagated in the wing web of quail for at least 14 passages without any loss of infectivity. On the other hand, serial passage in quail brain resulted in a progressive loss of infectivity until virus was completely lost.

Animals↗

PubMed1990

A human glioma cell line retaining expression of GFAP and gangliosides, recognized by A2B5 and LB1 antibodies, after prolonged passage.

A malignant astrocytoma was cultured from a tissue biopsy taken at surgical resection, and serially passaged 65 times over a period of 4 years. Early culture cells showed a variety of morphological types, but were all positively labelled with a polyclonal antibody directed against glial fibrillary acidic protein (GFAP). As the number of passages increased small, GFAP-positive cells became the predominant cell type. Flow cytometry demonstrated changes in GFAP expression between early and late passages; early passages showing greater fluorescence intensity than those at later passages. All cells, however, remained positive for GFAP. In addition, this line (IPSB-18) was both vimentin and glutamine synthetase positive and no change in the expression of these two proteins was detectable on continued sequential passaging. Fibronectin was not detectable at any stage. As the number of passages increased, the population doubling time was shortened from 72 h at passage 5, to 28 h at passage 30. This line is unusual in its retention of GFAP expression through 65 sequential passages; the majority of astrocytoma lines lose this capacity after a few passages in vitro. The monoclonal antibody, A2B5, which recognizes surface gangliosides, has been previously shown to identify sub-set type 2 astrocytes in normal neural tissue in vitro. The expression of A2B5 gangliosides by 5-20% of GFAP-positive IPSB-18 cells, and their co-expression with the ganglioside GD3 identified by the LB1 monoclonal antibody, shows that cells with a similar phenotype to the type 1 and type 2 astrocytes of optic nerve are present in cultured gliomas, even after long term sequential passaging.

Antibodies, Monoclonal↗

PubMed1975

[Studies on the adaptation of various strains of orthomyxovirus type A, subtype A/equi 2, passaged either on bovine kidney cells or on chicken fibroblasts or on RK-13 cells or by those passages alternating with "in ovo" passages].

The Authors have researched to adapt Orthomyxovirus, subtype A/equi-2 (Naples/213/67, Andria/60/68 and Miami/63), by serial passages on kidney calf cells, chick embryo cells and RK-13 cell line or by one or two passages on these colture media alternated with one passage "in ovo". The results obtained show that only kidney calf cells are most suitable because the viruses replicate for eleven passages. On the chick embryo cells and RK-13 cell line the viruses replicate only for two passages and these results are not modified when one or two passages are alternated with one passage "in ovo". The Authors have observed the production of infectious but not haemadsorbing and haemagglutining virus. The phenomenon is discussed and integrated with collateral tests.

Adaptation, Biological↗

PubMed1992

Malignant transformation of human fibroblasts previously immortalized with 60Co gamma rays.

Normal human fibroblasts obtained from a whole embryo were malignantly transformed with only 60Co gamma rays, but after extensive passage. The fibroblasts were exposed intermittently to 60Co gamma rays, 13 times, 2,800 rads in total, from the primary culture to the 40th passage level. At the 50th passage level the cells became morphologically changed, showing chromosomal abnormalities, and serial passages showed them to be immortalized. They were not tumorigenic on transplantation into nude mice, but became malignant after extensive passage; i.e., at the 547th passage level and 2,800 days after initiation of the culture. Our results indicate that several different genotypic changes are necessary for malignant transformation of human cells over long periods. No mutation at codons 12 and 61 of H-, K- and N-ras was detected in the malignant cells. Thus, this system may be useful to detect other cellular genes that may contribute to the malignant phenotypes of human fibroblasts.

Base Sequence↗

PubMed2006

Progressive truncation of the Non-Structural 1 gene of H7N1 avian influenza viruses following extensive circulation in poultry.

In order to support eradication efforts of avian influenza (AI) infections in poultry, the implementation of "DIVA" vaccination strategies, enabling the Differentiation of Infected from Vaccinated Animals have been recommended by international organisations. A system, based on the detection of antibodies to the Non-Structural 1 (NS1) protein of AI has been proposed but the success of such a system lies in the conservation of the NS1 protein among different AI isolates. With this in mind, the ns1 gene of 40 influenza A viruses isolated from a spectrum of avian species was sequenced and compared phylogenetically. The isolates included both low pathogenicity (LPAI) (n=22) and highly pathogenic (HPAI) (n=18) viruses of the H7 subtype and were representative of the avian influenza viruses that circulated in Northern Italy from 1999 to 2003. Size variation in the predicted amino acid sequence of each NS1 was revealed with two different levels of carboxy-terminal truncation being observed. Of the 40 isolates analysed, 16 had a full-length NS1 protein of 230 aa, 6 had a truncated protein of 220 aa and 18 had an intermediate truncation resulting in a protein of 224 aa. All of the H7N1 HPAI isolates possessed the intermediate carboxy-terminal truncation. In addition, all of the H7N1 LPAI viruses circulating at the beginning of the epidemic had a full length NS1 while those circulating towards the end of the period had a truncated protein. To determine whether modifications to NS1 could be a result of laboratory manipulation, two strains (A/ty/Italy/977/99 and A/ck/Italy/1082/99) with a full length NS1 were inoculated into 10-day-old embryonated chicken and 12-day-old embryonated turkey eggs via the allantoic route for 20 blind passages and sequenced at passages 3, 10, and 20. No truncation was observed following these serial passages. To determine whether the truncation involved an immunogenic region of the NS1 protein a peptide spanning residues 219 aa to 230 aa was synthesized and tested in an indirect ELISA against sera obtained from turkeys experimentally infected with a virus strain known to have a full length NS1 protein. The peptide proved to be immunogenic highlighting the fact that the variations of the NS1 protein presented in this work must to be taken into consideration when developing a diagnostic test based on the identification of antibodies to the NS1 protein.

Amino Acid Sequence↗

PubMed1993

Propagation of hepatitis A virus in a renal cell line JTC-12.P3 of cynomolgus monkey origin.

Human hepatitis A virus (HAV) derived from 10% HAV infected marmoset liver homogenate and faeces from acute hepatitis A was successfully propagated in vitro in a new cell line, JTC-12.P3. The cell line originated from the renal cortex of cynomolgus monkey which was adapted to growth in a serum free, protein free, chemically defined synthetic medium. Replication of the virus was followed by solid phase RIA, immunofluorescent staining, and immunoelectron microscopy. The propagation of HAV occurred over several passages, with the 1st and 2nd passages requiring at least 8 weeks each. However, with the increasing serial passage of virus, the period needed to detect it was shortened, suggesting the adaptation of HAV to the cells. The identity of the newly synthetized virus particles with HAV was established by immunoelectron microscopy and immunofluorescent blocking effect with human convalescent serum. The HAV propagated in JTC-12.P3 cells banded predominantly at a density of 1.32 g/cm3 in CsC1 gradient. The infected cells showed no specific signs of CPE. Ultrastructurally, clusters of virus particles 27 nm in diameter were observed mainly in the lysosomal vesicles and freely in crystalline array in the cytoplasm, too. Addition of 0.1% of various anti-HAV negative sera or of prostaglandin E1 to the culture medium caused accelerated propagation of HAV.

Animals↗

PubMed2000

Mutations in 23S rRNA and ribosomal protein L4 account for resistance in pneumococcal strains selected in vitro by macrolide passage.

The mechanisms responsible for macrolide resistance in Streptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A), erm(A), erm(B), erm(C), erm(TR), msr(A), mph(A), mph(B), mph(C), ere(A), ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia coli numbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids ((63)KPWRQKGTGRAR(74)) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.

Amino Acid Sequence↗

PubMed1984

Endothelial cell cycle kinetics. Changes in culture and correlation with endothelial properties.

Endothelial cells in culture have emerged as a model system for evaluation of endothelial properties. Culture conditions may affect expression of these endothelial properties or growth characteristics. Therefore, we evaluated cell cycle kinetics, prostacyclin release, and angiotensin-converting enzyme activity in primary and subcultured cells from bovine pulmonary and systemic vessels. Prostacyclin release was highly variable during passage in culture. However, release following stimulation with ionophore A23187 declined with serial passage. Angiotensin-converting enzyme activity also declined with repeated subculturing. Greater than 10% of primary confluent bovine endothelial cells were cycling compared to 2 to 8% of human umbilical vein endothelial cells. With passage in culture, 25% of bovine endothelial cells were cycling at confluence. Occasional strains of subcultured bovine endothelium arrested in G2 but would reenter G1 upon subculturing. Exposure to trypsin did not appear to contribute to this phenomenon. These studies have demonstrated variability in endothelial cell properties and growth characteristics during in vitro propagation. The existence of this variability must be considered when studying properties or responses of subcultured endothelium.

Animals↗

PubMed1995

[Zinc sulfate and heparin for local therapy of herpes. Antiherpetic drugs, not leading to selection of HSV variants].

The massive widespread use of a substance to treat HSV could lead to the spread of resistant HSV strains and hence to a failure of treatment in a vital indication. In Germany, three defined substances suitable for treating HSV are available over the counter: Zinc sulfate, heparin and acyclovir. Experiments show that heparin retains its activity even after 20 passages whereas acyclovir produces a completely resistant virus after only a single passage. The inactivation of free HSV by zinc sulfate is reduced after serial passages. However, this partial resistance disappears again spontaneously and may be considered clinically irrelevant. With respect to drug efficacy and resistance, therefore, a combination of zinc sulfate and heparin would appear superior to zinc sulfate alone. The use of the genotoxic DNA polymerase inhibitor acyclovir should be restricted to severe cases of HSV.

Acyclovir↗

PubMed1993

Secretion of alpha-amylase in human parotid gland epithelial cell culture.

The secretions of the salivary gland system are essential for the maintenance of oral health. The nature of cell-specific secretions of the various glands and their regulation is not completely understood. The objective of this study was to establish epithelial cell cultures from the human parotid gland that exhibit the tissue-specific function of alpha-amylase secretion. A specimen of normal human parotid gland was obtained at surgery and used to obtain primary cultures by the explant/outgrowth procedure. The cultures were maintained in keratinocyte basal medium, supplemented with insulin (5 micrograms/ml), EGF (10 ng/ml), hydrocortisone (0.5 micrograms/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. The cultures were passaged using 0.125% trypsin to dissociate the cells. Phase contrast and ultrastructural observations showed that the cells were polygonal and exhibited desmosomes. Their cytoplasm contained tonofilament bundles and abundant rough endoplasmic reticulum and Golgi complexes. Immunofluorescence studies showed that all cells were positive for cytokeratins. Immunoblot analysis revealed keratins with molecular weights of 58, 56, 52, 50, 48, 46, and 40 KD, which are characteristic of secretory epithelia. The cells have been passaged 35 times so far, undergoing a cumulative 120-140 population doublings. The serially passaged epithelial cell cultures produced and secreted alpha-amylase, a major component of parotid gland acinar cell secretion. The beta-adrenergic agonist, isoproterenol (ISP), stimulated alpha-amylase secretion, which was accompanied by increased intracellular concentrations of cAMP. ISP-induced stimulation of amylase and cAMP was blocked by the beta-adrenergic antagonist, propranolol. Further, dibutyryl cAMP also enhanced the secretion of amylase. Thus we have established a long-term epithelial cell culture model of human parotid gland epithelial cells that exhibits differentiated function and retains the intact beta-adrenergic receptor system.

Adrenergic beta-Agonists↗

PubMed2000

Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion.

Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.

Animals↗

PubMed1987

The in vitro growth, heterotransplantation, and immunohistochemical characterization of the blastemal component of Wilms' tumor.

Wilms' tumor has been proposed to originate from a developmental abnormality of the metanephric blastema. This undifferentiated component of Wilms' tumors has previously eluded efforts for in vitro growth. Blastema from a "classical" Wilms' tumor was transplanted into nude mice and passaged through 12 generations of heterotransplantation. Tumors from heterotransplants were grown for 12 serial passages in a serum-free growth medium supplemented with hormones and conditioned media from human kidney proximal tubule cells. The blastema initially grew on a collagen-fetal calf serum matrix as multicellular spheroids, and the cells proliferating from the rim of the spheroids had a flattened shape. Pulse-labeling with bromodeoxyuridine (BrdU) identified the proliferating cell population as blastemal in origin. Except for a loss of extracellular matrix, ultrastructural studies demonstrated morphologic similarities in the cultured cells, compared with the primary tumor and heterotransplants. Lectin histochemical stains for the peanut lectin (PNA) and immunohistochemical stains for cytokeratin (CYTO), vimentin (VIM), and epithelial membrane antigen (EMA) were performed on the original tumor, successive heterotransplants, and cells grown in vitro. The PNA stained the surface of the blastemal cells after sialidase digestion in the original tumor, heterotransplants, and cultured cells. The blastema of the original tumors was negative for CYTO and EMA but reactive for vimentin. This lack of differentiation was maintained in heterotransplants through 12 passages. However, blastemal cells demonstrated coexpression of CYTO and VIM intermediate filaments when grown in a serum-free medium on a matrix material. These studies demonstrate that the blastemal component of Wilms' tumor can be successfully grown in culture, passaged in nude mouse heterotransplants, and shown to undergo early stages of blastemal differentiation in vitro by growth in serum-free medium. This in vitro system provides a model for testing the factors that influence the growth and differentiation of the blastemal component of Wilms' tumors.

Animals↗

PubMed1982

Low density lipoprotein receptor binding in aging human diploid fibroblasts in culture.

High affinity cell surface receptors for low density lipoproteins (LDL) are inducible in cultured human lung fibroblasts by the removal of lipoproteins from the cell culture medium. The binding, uptake, and degradation of 125I-LDL by fibroblasts decrease with increasing number of population doublings. The affinity of LDL receptor binding, however, remained unchanged at different population doublings levels. Hence, the difference in LDL binding activity in the aging fibroblasts can be attributed to a reduction in the number of receptor sites on the cell membrane. Cellular uptake of [4-14C]cholesterol and 2-deoxy-D-[1-14C]glucose mediated through mechanisms independent of the LDL receptor pathway revealed no significant difference in early and late passage fibroblasts. This suggests that the alteration in the LDL receptor binding in serially passaged fibroblasts is an "age-related" phenomenon. The late population doubling fibroblasts require more LDL in the culture medium for feedback inhibition of LDL receptor synthesis. Thus, aging fibroblasts are both progressively less inducible and less suppressible in the regulation of their cell membrane LDL receptors. Similar results were also obtained with respect to the regulation of DL-3-hydroxy-3-methyl-glutaryl coenzyme A reductase in the aging fibroblasts in culture; the enzyme has become less inducible and less supressible as the fibroblasts approach the limit of their in vitro lifespan. These age-related alterations in the cellular metabolism of LDL and cholesterol might contribute to our understanding of the increased risk of athlerosclerosis in our aging population.

Cell Survival↗

PubMedDate not supplied

Establishment of a cell line from a neuroendocrine carcinoma of the endometrium and an invasion model.

A new cell line (YKK) was established from a primary tumor of neuroendocrine carcinoma of the endometrium. Cultured YKK cells are polygonal in shape. The modal chromosome number is 46 XX. The doubling time of the cells, after the 19th passage, is 38 hours and this cell line has been propagated continuously by serial passage over 50 passages for the past 2 years. YKK displays characteristics resembling the original tumor cell from the donor patient: it contains the neuron-specific enolase and chromogranin antibody, produces neuron-specific enolase, and is sensitive to cisplatin, carboplatin, and etoposide. When YKK cells are transplanted subcutaneously together with Matrigel into nude mice they form a very large tumor invading the subcutaneous muscular tissue. This experimental model is useful for studying the mechanism(s) regulating the invasion of this tumor into muscular tissue, and for the establishment of new anti-cancer treatments for neuroendocrine carcinoma.

Animals↗