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Results for “Serial Passage”

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PubMed1984

Changes in growth pattern of human squamous-cell carcinomas of the head and neck during serial passages in nude mice.

Six human squamous-cell carcinomas of the head and neck heterotransplanted to nude mice were studied with respect to changes in tumour volume doubling time (DT), cell cycle phase distribution and clonal composition during serial passages. The tumours exhibited a statistically significant decrease in DT between the first and third or fourth passages towards a constant value. The changes in DT are likely to depend on a reduction of cell loss factor. Five of the original tumours and serially passed heterotransplants were analyzed with flow cytometry. Two tumours were diploid and remained so after heterotransplantation. Three tumours had one diploid and one aneuploid cell population. At heterotransplantation there was a selection of clones and only the aneuploid cell populations could be recovered from the heterotransplants in the first as well as in later passages.

Animals↗

PubMed1994

The effect of serial passage on Sabin oral poliomyelitis vaccine virus in secondary monkey kidney versus Vero cells as measured by the monkey neurovirulence test.

The three Sabin strains of poliomyelitis seed virus were serially passaged in either secondary monkey kidney or Vero cell cultures and the tenth passage of each virus harvest compared to non-passaged Sabin reference virus of the same type using the monkey neurovirulence test. All three types were further attenuated by passage in Vero cells, whereas only type 2 became further attenuated after passage in secondary monkey kidney cells. After passage in Vero cells, type 3 poliomyelitis virus became more heat stable, as measured by its replicative capacity at 40 degrees C.

Animals↗

PubMed1998

Adaptation and serial passage of bovine coronavirus in an established diploid swine testicular cell line and subsequent development of a modified live vaccine.

A virulent bovine coronavirus isolate (newborn calf diarrheal) was adapted and serially passaged in an established diploid swine testicular cell line (ST cells). The same cells have been used to produce modified live porcine rotavirus and coronavirus vaccines that are federally licensed and sold worldwide. Growth of the bovine coronavirus resulted in cytopathic effect characterized by cellular stranding and subsequent cell lysis. Virus yields were relatively high in the ST cells and active replication was confirmed by immune electron microscopy and immunofluorescence. Adaptation of bovine coronavirus to a diploid swine cell line has not been previously reported. Different cell culture passage levels of bovine coronavirus were evaluated by oral inoculation of clean-catch, colostrum-deprived calves. A passage level of bovine coronavirus was identified that multiplied in the calf without the clinical signs of disease associated with virulent passages. The modified live bovine coronavirus vaccine remained safe and efficacious even after 5-backpassages in calves. Further efficacy studies have shown that the modified live bovine coronavirus vaccine significantly protected calves from highly virulent challenges with either winter dysentery or newborn calf diarrheal coronavirus isolates.

Adaptation, Biological↗

PubMed1987

Biochemical characterization of an aphthovirus type C3 strain Resende attenuated for cattle by serial passages in chicken embryos.

We have compared several aspects of an aphthovirus strain attenuated for cattle (C3R-O/E) with the original strain (C3Res) from which it was derived after serial passages in chicken embryos. Biochemical differences detected by protein analysis in regular polyacrylamide gels (SDS-PAGE) and on electrofocusing gels (NEPHGE) suggest the presence of mutations throughout the genome. Changes were located in coat proteins VP1 and VP3 and in the polymerase precursor P100 (P3/ABCD). No other differences were found at the protein level by means of the techniques used. Polypeptide P100 of the attenuated strain showed a faster electrophoretic mobility in SDS-PAGE with respect to that of the wild-type strain, and the change seems to be located on its amino terminus half. Several functional differences were also found between the two viruses. Both strains grew equally well in BHK cells reaching roughly similar titers in plaque assays. However, the wild-type strain maintained its titer in cells of bovine origin (BK), whereas the titer of C3R-O/E strain decreased approximately one log in this cell system; moreover, plaques elicited by the attenuated strain were much smaller than the ones produced by C3Res. A diminution in the rate of RNA synthesis induced by C3R-O/E in BK cells compared with that of the wild-type strain was also detected; this trait was not observed in BHK cells. A delay in the kinetics of RNA synthesis was also detected in this strain. The virus yield of attenuated strain in BK cells was four times lower than in BHK cells.

Animals↗

PubMedDate not supplied

Serial passage of hepatitis delta virus in chronic hepatitis B virus carrier chimpanzees.

Five consecutive passages of hepatitis delta virus in hepatitis B virus carrier chimpanzees were performed in order to further characterize the infectious and pathogenic nature of this naturally occurring defective virus. Three animals received identical inocula at fourth passage in order to assess individual animal variation as a factor in the course of infection and disease. Acute hepatitis delta virus infection occurred in all hepatitis B virus carrier chimpanzees as demonstrated by coincident intrahepatic hepatitis delta antigen, serum hepatitis delta antigen and serum hepatitis delta virus RNA followed by seroconversion to antibody to hepatitis delta antigen. In all animals, acute hepatitis was temporally associated with hepatitis delta virus infection and was self-limited. The incubation period to hepatitis shortened with passage, whereas biochemical and histologic evidence of liver diseases increased. The marked increase in liver disease with passage was not associated with increasing markers of hepatitis delta virus replication or expression, thus indicating that adaptation to the chimpanzee by serial passage resulted in increased hepatitis delta virus virulence. The duration of hepatitis due to hepatitis delta virus infection in three chimpanzees which received the same inoculum varied from 1 to 8 months. The observations of passage adaptation and individual host variation in this experimental model of hepatitis delta virus disease parallel known pathogenic variations in human hepatitis delta virus infection.

Animals↗

PubMed1987

Measles virus persistence in a hamster brain cell line: cyclical fluctuation of viral expression during serial passages.

Measles virus persistence in a hamster brain cell line was examined for the appearance of viral antigens and for the synthesis of infectious virus, viral RNA, and virus-specific proteins during 19 serial passages. Cyclical fluctuation was detected at all levels of measles virus replication. After reaching the maximal activity at the passages 9 to 11 viral synthesis diminished rapidly, being lowest after 2 to 3 subsequent passages. No release of infectious virus was detected at the passages 14 and 15 and only 20% of the cells contained measles virus antigens when tested by immunofluorescence. After the nonproductive phase, the cells released virus again and the number of antigen-positive cells increased. The alternation in the amount of the measles virus-specific proteins and viral RNA correlated directly with the parameters mentioned above. No apparent defects in the synthesis of individual viral proteins were observed.

Animals↗

PubMed1999

Extended serial passaging of mammalian neural stem cells in suspension bioreactors.

Neural stem cells (NSCs) are primitive cells that are the "parent" cells of all the cells in the central nervous system (CNS). Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Huntington's disease, which affect millions of people worldwide and cost billions of dollars in health care each year. This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture. We examined stationary culture passaging protocols and developed our own optimal procedure. Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time. The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10(7) with no decrease in growth rate, maximum cell density, or viability. The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS-neurons, astrocytes, and oligodendrocytes. For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics. A semicontinuous scheme for large-scale production is also presented.

Animals↗

PubMed1977

Role of endogenous murine leukemia virus in immunologically triggered lymphoreticular tumors. I. Development and use of oncogenic cellfree preparations serially passaged in vivo.

Cellfree extracts (CFEs) prepared from (BALB/cJ X A/J)F1 (CAF1) and (BALB/cJ X C57BL/6J)F1 (CB6F1) mice in which a graft-versus-host reaction (GVHR) has been induced are known to be oncogenic, but only after a protracted latent period (mean, 16 mo). Serial passage of such CFEs in successive generations of syngeneic mice inoculated at birth led to the development of two separate oncogenic preparations, the CA serioes in CAF, mice and the CB series in CB6F, mice, in which the mean latent period was reduced to 6 and 12 months, respectively. Both oncogenic preparations contained infectious B-tropic murine leukemia virus (MuLV) and particles with the ultrastructural characteristics of MuLV. No other kind of virus particle was seen. When these preparations were injected into infant syngeneic mice, B-tropic MuLV could be detected in the reticular tissues as early as 2 weeks thereafter. The virus persisted in the reticular tissues and was present in the lymphoreticular tumors that subsequently developed. However, if the same preparation was injected into young adult recipients, there may have been transient MuLV replication, but the virus subsequently disappeared from the reticular tissues and no lymphoreticular tumors developed. Previous experiments showed that MuLV was present in CFEs prepared from CAF, animals with the GVHR but absent in those of normal control mice. Since the lymphoreticular tumors arising in mice with the GVHR were the same as those induced by the CA and CB MuLV preparations, it was concluded that tumorigenesis in mice with the GVHR was caused by endogenous B-tropic MuLV activated by the immunologic disturbance.

Animals↗

PubMed2002

Presence of Intact vpu and nef genes in nonpathogenic SHIV is essential for acquisition of pathogenicity of this virus by serial passage in macaques.

Use of the macaque model of human immunodeficiency virus (HIV) pathogenesis has shown that the accessory genes nef and vpu are important in the pathogenicity of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV). We examined the ability of two nonpathogenic SHIVs, SHIV(PPC) and DeltavpuDeltanefSHIV(PPC), to gain pathogenicity by rapid serial passage in macaques. In this study, each virus was passaged by blood intravenously four times at 4-week intervals in macaques. Animals were monitored for 40 weeks for levels of CD4 T cells and quantitative measures of virus infection. DeltavpuDeltanefSHIV(PPC) maintained a limited phase of productive replication in the four animals, with no loss of CD4(+) T cells, whereas SHIV(PPC) became more pathogenic in later passages, judging by plasma viral load and viral mRNA in lymph nodes, infectious peripheral blood mononuclear cells and CD4(+) T cell loss. The nef, LTR, and env of the SHIV(PPC) viruses underwent numerous mutations, compared to DeltavpuDeltanefSHIV(PPC). This study confirms the seminal role that nef, LTR, and vpu could play in regulation of pathogenesis of HIV infection.

Acquired Immunodeficiency Syndrome↗

PubMed1985

Biochemical characterization of an aphthovirus type 0(1) strain campos attenuated for cattle by serial passages in chicken embryos.

The biochemical properties of a virulent and an attenuated strain of foot-and-mouth disease virus (FMDV) Type 0(1) Campos (0(1)C) were compared in order to establish differences that could account for their altered biological functions. The avirulent strain (0(1)C-O/E) was derived from the virulent strain 0(1)C by serial passages in chicken embryos. Analysis of the RNase T1-generated oligonucleotides of the viral RNA through one- and two-dimensional (2D) gel electrophoresis (fingerprints) revealed a few changes in the genome structure of the 0(1)C-O/E strain compared to the wild type strain. In addition there was a significant decrease in the length of the poly(C) rich tract of the 0(1)C-O/E RNA. All virion structural proteins, except VP4, their precursors, and the viral RNA polymerase (p56a) show charge differences. In addition a significant decrease in the apparent molecular weight of polypeptide p100 (primary translational product from the 3' end region of the genome) of the attenuated strain was observed.

Animals↗

PubMed1990

Extensive heterogeneity of the protein composition of Chlamydia trachomatis following serial passage in two different cell lines.

To determine if the host-modulated adherence characteristics of the intracellular bacterial pathogen Chlamydia trachomatis were due to the acquisition of altered surface-exposed proteins, highly purified chlamydiae grown in two different host cells were analysed. Two serovars, L1 and E, were grown for multiple passages in both HeLa and McCoy host cells. Numerous protein differences in the chlamydial elementary bodies (EB) of each serovar grown in the two different hosts were detected by two-dimensional (2-D) gel electrophoresis and fluorography of radioactively labelled proteins. At least four to six serial passages in the alternative host were necessary before the changes were apparent. Iodination of suspensions of purified chlamydiae and 2-D electrophoresis revealed several surface proteins that were determined by the host cells in which the bacteria had replicated. These iodinated chlamydial proteins were removed by treatment of the iodinated EB with trypsin, indicating their location at the bacterial surface. Two of the major constituents of the outer-membrane complex, the cysteine- and methionine-rich 60 kDa and 40 kDa proteins, remained unchanged in both molecular mass and charge during the host adaptation. Several chlamydial proteins capable of binding iodinated host membrane preparations also exhibited host-dependent alterations. Immunoblotting experiments with a rabbit and a human polyclonal sera indicated that distinct host-specified chlamydial proteins were reactive with the two sera.

Animals↗

PubMed1989

Modification of foot-and-mouth disease virus after serial passages in the presence of antiviral polyclonal sera.

Foot-and-mouth disease virus (FMDV) shows a remarkable antigenic variability. Like other RNA viruses, this virus has a high rate of mutation. It has been proposed that selection exerted by the host's antibodies could play a major role in the rapid evolution of FMDV. The present work reports the selection of FMDV antibody-resistant populations (Nr), after serial passages of cloned FMDV A24 Cruzeiro strain on secondary monolayers of bovine fetal kidney cells in the presence of subneutralizing antiviral polyclonal sera (APS). After a limited number of passages under selective pressure, the virus population showed the following characteristics: (1) increased resistance to neutralization by APS; (2) altered electrophoretic mobility of structural viral proteins (VP1); (3) remarkable plaque size reduction, (4) a pronounced thermosensitivity (ts); and (5) decreased pathogenicity for mice, in both uncloned and cloned small plaque size populations. This indicates that FMDV populations under antibody pressure in vitro, have acquired, in addition to expected characteristics of natural FMDV variants (resistance to neutralization and altered viral structural proteins), phenotypic markers which correspond to attenuated, less virulent variants.

Antibodies, Viral↗

PubMed1987

Podospora anserina does not senesce when serially passaged in liquid culture.

A procedure was developed for the prolonged growth of the ascomycete fungus Podospora anserina in liquid culture to determine the effects of such growth on the senescence phenotype. Senescence in P. anserina, which is maternally inherited and associated with the excision and amplification of specific mitochondrial plasmids, occurs when this species is grown on solid medium. In two independent experiments no evidence of senescence was observed as mycelia were serially passaged in liquid culture. Further, when separable mycelial masses, termed puff balls, from the liquid cultures were plated on solid medium, a significant increase in their average longevity was observed. The apparent immortality of P. anserina in liquid culture was not dependent upon mitochondrial DNA rearrangements, nor was it affected by the presence of a previously described senescence plasmid, alpha senDNA. Evidence was obtained indicating that growth in liquid culture exerts selective pressure to maintain the wild-type mitochondrial genome.

Ascomycota↗

PubMed1990

Serial passage of infectious transcripts of a cucumber mosaic virus satellite RNA clone results in sequence heterogeneity.

Populations of the D-satellite RNA (D-sat) of cucumber mosaic virus (CMV) have a major site of heterogeneity at which a significant proportion of the molecules vary from each other. The generation of this heterogeneity was investigated by using infectious transcript RNA of a prototype D-sat clone as an inoculum of uniform sequence to initiate serial passages in five different host plant species. RNase protection assays indicated that heterogeneity at the same site seen in natural D-sat RNA populations developed within two to four passages in most of the hosts tested, and the proportion of the population exhibiting this heterogeneity accumulated to a much higher level in tobacco than in the other hosts. Characterization of the heterogeneity which developed in tobacco showed it to be qualitatively different from that in the original D-sat population. This analysis of a CMV satellite RNA is the first example in which infectious transcript RNA from a clone has been used to investigate the rapid generation and selection of heterogeneity in a genomic RNA population replicating in plants.

Base Sequence↗

PubMed2000

Tissue-engineered cartilage using serially passaged articular chondrocytes. Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM).

In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.

Alcian Blue↗

PubMed1997

Mineralization in serially passaged human alveolar bone cells.

Well-characterized human bone cell cultures have been regarded as a useful tool to study bone control mechanisms and also to analyse bone/biomaterials interactions. In the present study, human alveolar bone cells were cultured in alpha-minimal essential medium (alpha-MEM) containing 10% foetal bovine serum (FBS), 50 microg/ml ascorbic acid, 10 mM sodium beta-glycerophosphate and either in the presence or in the absence of 10 nM dexamethasone (Dexa). Cultures were characterized concerning cell viability/proliferation, alkaline phosphatase (ALP), acid phosphatase (ACP) and tartraric acid resistant phosphatase (TRAP) activities, and formation of mineralized areas. Cell proliferation increased gradually for approximately 20 days. In the presence of Dexa, cells formed isolated or interconnected multilayered clusters that increased with culture time. Histochemical assays revealed strong positive reactions for ALP and calcium and phosphates deposits, mainly in relation t! o ce lls associated with the clusters. High levels of ALP activity (biochemical determination) were observed. Cells cultured in the absence of Dexa showed significantly lower ALP activity and no calcium and phosphates deposits were present. Serially passaged cells kept the proliferation rate constant but a decrease in ALP activity was observed either in the presence or in the absence of Dexa. The ability to form mineralized areas (cultures fed with Dexa) also decreased on serial subculture.

Journal Article↗

PubMed2006

Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

West Nile Virus (WNV) is a mosquito-borne flavivirus that was introduced into the U.S. in the New York City area in 1999. Despite its successful establishment and rapid spread in a naive environment, WNV has undergone limited evolution since its introduction. This evolutionary stability has been attributed to compromises made to permit alternating cycles of viral replication in vertebrate hosts and arthropod vectors. Outbreaks of a close relative of WNV, St. Louis encephalitis virus (SLEV), occur in the U.S. periodically and are also characterized by limited genetic change overtime. We measured both phenotypic and genotypic changes in WNV and SLEV serially passaged in mosquito cell culture in order to clarify the role of an individual host cell type in flavivirus adaptation and evolution. Genetic changes in passaged WNV and SLEV were minimal but led to increased relative fitness and replicative ability of the virus in the homologous cell line C6/36 mosquito cells. Similar increases were not measured in the heterologous cell line DF-1 avian cells. These phenotypic changes are consistent with the concept of cell-specific adaptation in flaviviruses.

Adaptation, Physiological↗

PubMed1997

Restoration of the differentiated functions of serially passaged chondrocytes using staurosporine.

Among the various directions explored in order to have a large number of differentiated articular chondrocytes easily available, the restoration of the differentiated properties after cell multiplication in monolayer has been proposed. It has been clearly shown that the synthesis of cartilage proteoglycans and type II collagen synthesis is coincident with the presence of a faint microfibrillar architecture but is absent in chondrocytes showing well-defined actin cables. Staurosporin, mainly described as a protein kinase C inhibitor, has also been shown to rapidly induce the disruption of the actin microfilaments. The purpose of this paper was to investigate whether properties of differentiated chondrocytes were reinitiated upon staurosporin treatment of serially passaged chondrocytes. Results showed, after staurosporine treatment of cells at Passage two for 5 d, complete suppression of type I and type III collagen synthesis and induction of type II collagen synthesis and of Alcian blue stainable matrix. Additionally, we showed that staurosporin restored metabolic responses that chondrocytes in primary culture exhibit upon interleukin-1 beta treatment (decrease of Alcian blue- positive cells, induction of expression of the 92 kDa gelatinase, nitric oxide production). We conclude that staurosporin is a potent redifferentiating agent of articular chondrocytes that have been subcultured up to Passage two for multiplication. Taking into account that the cellularity of cartilage is very low, staurosporine-treated chondrocytes could be useful as an alternative cellular model to evaluate pharmacotoxicological effects of drugs.

Actins↗