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PubMed1992

Markovian processes in aneuploids: I. Diploid populations.

The case of diploid selfing organisms which present monosomy and nullisomy for a certain pair of homologous chromosomes is considered. Three cases of dominance for fitness (i) no dominance, (ii) complete dominance and, (iii) overdominance are considered. Expressions for the average, coefficient of variation and coefficient of asymmetry for distribution which represent the number of generations needed to reach an absorbing state for a process which begins in a transient state are obtained. Expressions for the parameters are from by the application of Markov's theory of chains which was developed by Kemeny & Snell (1960, Finite Markov Chains, Princeton, NJ: Van Nostrand) and Bosso et al. (1969, Biometrics 22, 17-26).

Aneuploidy↗

PubMed1988

To each genotype a separate strategy--a dynamic game theory model of a general diploid system.

A dynamic game theory model is presented for a diploid system in which each genotype corresponds to a different strategy. The population mates randomly and the strategy, which is determined by a single locus, is expressed only in the male. A general analytic solution for any number of alleles does not appear to be possible, but the cases of two and three alleles are treated in detail. A stability analysis applicable to any number of alleles is presented, so that any equilibria located by numerical methods can be checked for local stability. Computer programs for doing all of these calculations are available from the author.

Alleles↗

PubMed2002

Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts.

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4h) with APC at concentration ranges of 10-40 microM. The effect of APC on cell survival after 4 h treatment was significantly higher than after 2 h treatment. The mutagenicity of APC was investigated at the HPRT locus, and the frequency of HPRT mutants was estimated by selection in medium containing 6-thioguanine (6-TG). Treatment of fibroblast cells with 20 microM of APC for 2 or 4 h resulted approximately in 5 or 10 times increase of 6-TG resistant mutant frequencies, respectively, compared to untreated control cells. The cell cycle analyses performed during the expression time (9-12 days) have shown that after 2 and 4h treatment with APC the cells were blocked in G2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G2 phase increased about two-fold (28.6-31.8% compared to 13.5% in the untreated cells). The mode of cell death during the expression time was via necrosis, rather than apoptosis, which was demonstrated by fluorescein-diacetate (FDA)-staining and terminal dUTP nick end labeling (TUNEL)-method.

Antimetabolites, Antineoplastic↗

PubMed1997

Downregulation of DNA topoisomerase I in old versus young human diploid fibroblasts.

DNA topoisomerase I (Topo I) is an enzyme that alters the superhelicity of DNA. It has been implicated in such critical cellular functions as transcription, DNA replication, and recombination. Roles for Topo I in DNA repair following DNA damage have also been studied extensively. In the present investigation, we examined the regulation of Topo I expression and activity during cellular replicative senescence. We found that the capacity of Topo I to relax supercoiled DNA molecules is significantly decreased in senescent diploid fibroblasts when compared to young (early passage) fibroblasts. We also found that the steady-state expression level of Topo I mRNA is correlated with enzyme activity, i.e., decreased in early vs. late passage cells. We also treated early and late passage cells with agents that may modulate the process of cellular senescence: UV light, retinoic acid, and interleukin-1 beta. We found that all three agents decreased the activity of Topo I in young fibroblasts and increased the activity of Topo I in senescent fibroblasts. This effect was most striking following exposure of the cells to retinoic acid, so to analyze this effect, we postulated an age-dependent kinetics of Topo I mRNA induction in response to retinoic acid. Consistent with this postulate, we found that whereas exposure of early passage cells to retinoic acid results, in a matter of hours, in a decrease in the expression of Topo I mRNA, exposure of the senescent cells to retinoic acid results in an increased expression. These observations suggest that processes that are altered in senescent fibroblasts, such as DNA replication and repair, may be due, in part, to alteration in the expression and activity of DNA Topo I.

Cells, Cultured↗

PubMed1998

DNA signals for G2 checkpoint response in diploid human fibroblasts.

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included gamma-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4-12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.

Calibration↗

PubMed2000

Cytoplasmic retention of p-Erk1/2 and nuclear accumulation of actin proteins during cellular senescence in human diploid fibroblasts.

In order to investigate the role of signal transduction and the related changes of actin cytoskeleton organization in the process of cellular senescence, H-ras double mutants--V12S35, V12G37 and V12C40--proteins were expressed constitutively in human diploid fibroblast (HDF) cells by retrovirus infection at PD26. Constitutive expression of V12S35, V12G37 and V12C40 proteins induced premature senescence at PD38, PD47 and PD50, respectively, in contrast to the control cells at PD59. Premature senescence was evidenced by the slow cellular growth rate and SA-beta-galactosidase expression accompanied by morphological changes such as flat and large cell shape. Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells as well as V12S35 and V12G37 expressers were unable to export actin fibers from nucleus to cytoplasm, form stress fibers through the MAPK and Ral.GDS pathways. Perinuclear expression of Racl was prominent in the HDF cells and V12C40 expresser, while translocation of Racl from perinucleus to nucleus and strong expression of RhoA were observed in the V12S35 expresser. In summary, the induced premature senescence by H-ras double mutants were accompanied by nuclear accumulation of actin and Racl proteins, cytoplasmic retention of p-Erk1/2 and marked induction of RhoA expression mainly through dysregulation of the MEK pathway.

Actins↗

PubMed2000

mRNA levels of the linker histone variant, H1o, in mitotically active human diploid fibroblasts as a function of the phases of the cell cycle and cumulative population doublings.

Senescence and differentiation have many similarities with respect to certain aspects of gene expression and cell cycle related events. One linker histone variant tightly associated with differentiation is the H1 variant, H1o. The work of this investigation has focused on the expression of H1o during the phases of the cell cycle and as a function of increasing cumulative population doublings (CPD) in an in vitro model ageing cell system, namely, human diploid fibroblasts. Increased H1o mRNA levels were found during the S phase of the cell cycle contrary to H1o protein relative synthesis rates, which were found to be increased during the Go phase of the cell cycle. These results were obtained in actively proliferating cell populations. However when the proliferative rate of the overall population begins to drop (CPD 50), H1o mRNA levels tend to remain stable throughout the Go, G1 and S phases. On the other hand, no changes in the H1o relative synthesis rates were found as a function of increasing CPD. Uncoupling of H1o protein and mRNA levels has been observed in numerous differentiating systems. The analogous mode in which H1o gene expression is regulated in both these two systems reinforces the opinion that senescence and differentiation may have similarities at the level of chromatin remodelling.

Cell Cycle↗

PubMed1998

Expressional changes in alternative splicing affecting genes during cell passage of human diploid fibroblasts.

Normal human diploid cells have a limited proliferative lifespan in in vitro cultures. Changes in gene expression have been examined for understanding control mechanisms of limited proliferative lifespan. and enhanced expression of growth suppressing genes such as p21 was reported in late-passaged cells. We screened genes which were expressed preferentially in mid-passaged cells by the differential plaque screening of the subtracted cDNA libraries prepared from young, life-extended, and immortalized SV40-transformed human fibroblasts. Among isolated clones, ASF/SF2, which was known to affect alternative splicing, was expressed in normal fibroblasts with a peak at mid-passage. Relative expression levels of SC35 and hnRNPA1, which are also known to affect alternative splicing, was also highest at mid-passage. Changes in alternative splicing at mid-passage, if it occurred, may play a crucial role in the process of cellular senescence.

Alternative Splicing↗

PubMed1987

The role of specific IgE and beta-propiolactone in reactions resulting from booster doses of human diploid cell rabies vaccine.

Reactions after booster injections of human diploid cell rabies vaccine (HDCV) were investigated to determine the possibility of IgE-type antibody involvement. Although normal manufacture of HDCV involves the inactivation of the virus with beta-propiolactone (BPL), the effect of BPL on nonviral vaccine components, such as host cell components or stabilizing proteins, may be typical of the haptenic action of small molecular weight chemicals. Specific IgE to commercial HDCV preparations, BPL-treated preparations of noninfected host MRC5 cell sonicate (BPL-MRC5), and a human albumin (HA) (BPL-HA) used as a stabilizing agent were detected in sera from five individuals who reported reactions after booster doses of HDCV. However, these patients had no detectable IgE to normal HA. Sera from nonvaccinated individuals, vaccinated individuals who reported no reaction after HDCV booster, and pollen-allergic individuals had no detectable IgE to HDCV, BPL-MRC5, or BPL-HA. Changes in the ratios of pre- to postbooster serum levels of specific IgE to HDCV and BPL-HA were significantly different in a group of 19 individuals who reported reactions to HDCV boosters; these changes in pre- to postbooster IgE levels in nonreactive vaccinees were not significant. Prebooster serum IgE RAST ratios to HDCV or BPL-HA were not predictive of potential reactions to HDCV. A number of experimental BPL-HA reaction mixtures were assayed to examine the effect of variable concentrations of BPL to HA. Increasing relative molar concentrations of BPL to HA resulted in increased electrophoretic mobility, whereas the highest relative specific IgE binding was detected in BPL-HA molar reaction mixtures of approximately 12.5:1.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody Specificity↗

PubMed1983

A single-blind, placebo-controlled comparison of the reactivity and antigenicity of trivalent oral poliomyelitis vaccine prepared in monkey kidney or human diploid cell substrates.

Vaccination with a single dose of trivalent oral poliomyelitis vaccine elicited fourfold or greater antibody responses to one or more poliomyelitis virus types in 59% of volunteers (16/27) receiving vaccine prepared from virus grown in monkey kidney cells and in 69% of volunteers (16/23) receiving vaccine prepared from virus grown in MRC5 human diploid cells. Type for type the antibody titres and percentages of volunteers responding to the two vaccines were broadly equivalent. The clinical reactivities of both vaccines were similar to that of a placebo in terms of the overall incidence, duration and severity of reactions. The nature of the reactions observed did, however, vary, in that headaches were more frequently reported by recipients of vaccine prepared from virus grown in monkey kidney cells. This difference was not, however, statistically significant.

Adult↗

PubMed1975

Immunogenicity and acceptability of a human diploid-cell culture rabies vaccine in volunteers.

A rabies vaccine, prepared in human diploid-cell culture, was given intradermally (i.d.) or intramuscularly (i.m.) to 35 volumteers who had not previously received prophylactic immunisation. Rabies neutralising antibody titres rose sharply after the first dose, and 4 weeks after the second dose all titres were above 1/80 (1-7 i.u. per ml.). Immunisation via the i.d. and i.m. routes was equally successful in terms of likely immune status, but the i.d. rate was associated with many more local sideeffects.

Adolescent↗

PubMed1976

Human diploid cell strain rabies vaccine. Rapid prophylactic immunisation of volunteers with small doses.

The clinical and antibody responses of volunteers to three intradermal schedules of human diploid cell strain rabies vaccine (0.4 ml on day 0; 0-1 ml on days 0, 1, 2, 3; and 0-1 ml on days 0, 3, 7, and 14) are described. Vaccine was administered to 114 contacts of two rabid patients in order to evoke a rapid antibody response and the antibody titres of 30 of those who were vaccinated and bled were measured. High antibody titres were obtained in all subjects irrespective of their immunisation schedule; there were only minimal local reactions. All volunteers had titres greater than 1/78 (1.7 I.U./ml) by day 14, and 7 of 10 receiving 0.1 ml into each limb on day 0 had detectable antibody by day 7.

Antibodies, Viral↗

PubMed1981

Multisite intradermal antirabies vaccination. Immune responses in man and protection of rabbits against death from street virus by postexposure administration of human diploid-cell-strain rabies vaccine.

Lymphocyte transformation, production of neutralising antibody, and the development of antirabies IgG antibody were studied in ten healthy volunteers in response to 0.8 ml of human diploid-cell strain (HDCS) rabies vaccine administered on one occasion in divided doses in 8 intradermal (i.d.) sites. All ten volunteers rapidly developed substantial titres of rabies antibody, and eight of the ten had T lymphocytes that were immunologically stimulated by HDCS rabies-virus antigen. Postexposure treatment with 0.8 ml of HDCS vaccine given at 4 i.d. sites completely protected fourteen rabbits from death by street virus. The results suggest that in developing countries patients could be protected with small volumes of potent tissue-culture vaccine administered intradermally shortly after exposure.

Animals↗

PubMed1983

An economical regimen of human diploid cell strain anti-rabies vaccine for post-exposure prophylaxis.

Vaccine regimens using 0.1 ml human diploid cell strain vaccine (HDCSV) given intradermally (id) in single and multiple sites, or with aluminum hydroxide adjuvant given subcutaneously (sc), were compared with the regimens of HDCSV and Semple vaccine currently suggested by WHO. Some groups were also given human rabies-immune globulin (HRIG). Neutralising antibody titres were monitored for 3 months. Antibody was detected earliest in subjects given 0.1 ml HDCSV id at each of eight sites. The highest antibody titres from day 14 onwards were found after intramuscular (im) administration of HDCSV, but the multiple-site id regimen, which requires only one quarter of the volume of vaccine required for the im regimen, gave similar results, provided that a booster was given on day 91. This finding suggests that a treatment schedule based on this regimen would be suitable for post-exposure prophylaxis. Adjuvanted vaccine gave similar results to the same amount of antigen given id. Semple vaccine produced the lowest titres. HRIG, given at the high dose of 40 IU per kg, suppressed the antibody response to some of the regimens.

Adolescent↗

PubMed1983

Stability of human diploid-cell-strain rabies vaccine at high ambient temperatures.

A batch of lyophilised human diploid-cell-strain rabies vaccine was divided into three batches, which were exposed to different temperatures during their distribution to and storage at three centres in Pakistan. Vaccine potency after exposure to these temperatures was tested by measuring antibody response in those given the vaccine, and by three different laboratory tests. The results indicate that the vaccine retains its antigenicity for man and for laboratory tests despite continuous exposure to high ambient temperatures for up to 11 weeks. These findings, together with current developments in vaccine manufacture and treatment schedules, offer considerable hope to many countries with low budgets for health care and where rabies is poorly controlled.

Adolescent↗

PubMed1984

Failure to achieve predicted antibody responses with intradermal and intramuscular human diploid cell rabies vaccine.

In a study to compare the immunogenicity of human diploid cell rabies vaccine (HDCV) given by intramuscular or automatic intradermal jet injection, neither method of administration resulted in antibody levels predicted by previous studies. 49 days after starting a series of three 0.1 ml doses of HDCV given intradermally, 85 volunteers had a geometric mean titre (GMT) of neutralising antibody to rabies of 1:170. 9 concurrent control subjects who received 1.0 ml doses of vaccine intramuscularly had a GMT of only 1:269. Although standard potency testing did not demonstrate that the vaccine used was subpotent , these results strongly suggest that the immunogenicity of HDCV is substantially less than previously reported.

Antibodies, Viral↗

PubMed1985

Economical multiple-site intradermal immunisation with human diploid-cell-strain vaccine is effective for post-exposure rabies prophylaxis.

An economical post-exposure regimen of Mérieux human diploid-cell-strain vaccine (HDCSV) was compared with Semple vaccine (SV), the most widely used vaccine in Asia. 155 patients bitten by animals proved to be rabid received either conventional courses of SV (34 severe and 43 mild cases) or HDCSV, 0.1 ml intradermally, at eight sites on day 0, at four sites on day 7, and at one site on days 28 and 91 (36 severe and 42 mild cases). All severely bitten patients were given equine anti-rabies serum (EARS), 80 IU/kg on day 0. There were no deaths from rabies in either group. Follow-up was 97.5% at 1 year and 93% at 2 years. 88% of patients given HDCSV alone had detectable neutralising antibody on day 7 in contrast to 2% given SV alone. Antibody persisted until 1 year in all sera tested from HDCSV patients in contrast to only 48% of SV sera. The high dose of EARS resulted in pronounced suppression of response to HDCSV. There were no serious systemic side-effects but local side-effects were significantly more common in the SV group. The multiple-site intradermal HDCSV regimen was at least as effective as SV. The amount of HDCSV used was 30% of the conventional dose.

Adolescent↗

PubMed1989

Rapid detection of influenza virus infections in human fetal lung diploid cell cultures.

Haemadsorbing foci were found in human fetal lung (HFL) diploid cell cultures 12 h after inoculation with influenza viruses A and B. The size and number of the foci were maximal after 48 h of incubation, being limited by production of an unidentified inhibitor. By contrast, inoculation with parainfluenza virus type 3 led to haemadsorption which increased during 10 days of incubation. For the detection of influenza viruses A and B maximum sensitivity was achieved by changing the medium, the day before use to one that was serum free. The number of foci at 15.5 h post-infection and infectivity for primary African green monkey kidney (AGMK) cultures were similar. Virus infectivity and production of haemagglutinin in HFL cells were entirely cell-associated; they were not affected by treatment with trypsin. Nevertheless, influenza viruses A and B antigens were identified in the infected cells by means of immunofluorescence at 15.5 h and virus was recovered by passage of frozen and thawed cells in AGMK cultures. For rapid routine diagnosis of viral infections, the early haemadsorption test was shown to have the same sensitivity as immunofluorescence tests on specimens and virus detection by the shell-vial technique but was cheaper and simpler to perform.

Cells, Cultured↗