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Biomedical subjects

D Ewing

Publications and source records attributed to D Ewing.

At least 19 recordsLinked to original sources
PubMed2001

Synergistic neutralizing antibody response to a dengue virus type 2 DNA vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing GM-CSF.

We have previously shown that a dengue virus type 1 DNA vaccine expressing premembrane (prM) and envelope (E) genes was immunogenic in mice and monkeys and that rhesus monkeys vaccinated with this construct were completely to partially protected from virus challenge. In order to improve the immunogenicity of dengue DNA vaccines, we have evaluated the effect of lysosome targeting of antigens and coimmunization with a plasmid expressing GM-CSF on antibody responses. A dengue virus type 2 candidate vaccine containing prM and E genes was constructed in which the transmembrane and cytoplasmic regions of E were replaced by those of the lysosome-associated membrane protein (LAMP). The modified vaccine construct expressed antigen that was colocalized with endogenous LAMP in lysosomal vesicles of transfected cells, whereas the antigen expressed from the unmodified construct was not. It was hypothesized that targeting of antigen to the lysosomal compartment will increase antigen presentation by MHC class II, leading to stronger CD4-mediated immune responses. Mice immunized with the modified construct responded with significantly higher levels of virus neutralizing antibodies compared to those immunized with the unmodified construct. Coimmunization of mice with a plasmid expressing murine GM-CSF enhanced the antibody response obtained with either the unmodified or the modified construct alone. The highest antibody responses were noted when the modified construct was coinjected with plasmid expressing the GM-CSF gene. These results could form the basis for an effective tetravalent dengue virus DNA vaccine.

3T3 Cells↗
PubMed2001

International Child Care Practices Study: infant sleeping environment.

BACKGROUND: The International Child Care Practices Study (ICCPS) has collected descriptive data from 21 centres in 17 countries. In this report, data are presented on the infant sleeping environment with the main focus being sudden infant death syndrome (SIDS) risk factors (bedsharing and infant using a pillow) and protective factors (infant sharing a room with adult) that are not yet well established in the literature. METHODS: Using a standardised protocol, parents of infants were surveyed at birth by interview and at 3 months of age mainly by postal questionnaire. Centres were grouped according to geographic location. Also indicated was the level of SIDS awareness in the community, i.e. whether any campaigns or messages to "reduce the risks of SIDS" were available at the time of the survey. RESULTS: Birth interview data were available for 5488 individual families and 4656 (85%) returned questionnaires at 3 months. Rates of bedsharing varied considerably (2-88%) and it appeared to be more common in the samples with a lower awareness of SIDS, but not necessarily a high SIDS rate. Countries with higher rates of bedsharing appeared to have a greater proportion of infants bedsharing for a longer duration (>5 h). Rates of room sharing varied (58-100%) with some of the lowest rates noted in centres with a higher awareness of SIDS. Rates of pillow use ranged from 4% to 95%. CONCLUSIONS: It is likely that methods of bedsharing differ cross-culturally, and although further details were sought on different bedsharing practices, it was not possible to build up a composite picture of "typical" bedsharing practices in these different communities. These data highlight interesting patterns in child care in these diverse populations. Although these results should not be used to imply that any particular child care practice either increases or decreases the risk of SIDS, these findings should help to inject caution into the process of developing SIDS prevention campaigns for non-Western cultures.

Beds↗
PubMed2000

Immunogenicity of dengue virus type 1 DNA vaccines expressing truncated and full length envelope protein.

Recombinant plasmid DNA constructs expressing truncated or full-length dengue-1 envelope (E) with or without the pre-membrane (prM) were tested for immunogenicity in mice, as candidate dengue DNA vaccines. Two plasmids, one expressing the N-terminal 80% E and the other expressing prM and full length E were immunogenic in intradermally inoculated mice. The vaccinated mice produced dengue-1 specific antibodies that were both neutralizing and long lasting. Data suggested that the plasmid expressing prM and full length E produced virus like particles in transfected cells, and is probably a better immunogen compared to that expressing 80% E.

Animals↗
PubMed2000

Dengue virus type 1 DNA vaccine induces protective immune responses in rhesus macaques.

A candidate DNA vaccine expressing dengue virus type 1 pre-membrane and envelope proteins was used to immunize rhesus macaques. Monkeys were immunized intramuscularly (i.m.) or intradermally (i.d.) by three or four 1 mg doses of vaccine, respectively. Monkeys that were inoculated i.m. seroconverted more quickly and had higher antibody levels than those that were inoculated i.d. The sera exhibited virus-neutralizing activity, which declined over time. Four of the eight i.m.-inoculated monkeys were protected completely from developing viraemia when challenged 4 months after the last dose with homologous dengue virus. The other four monkeys had reduced viraemia compared with the control immunized monkeys. The i.d. -inoculated monkeys showed no reduction in viraemia when challenged with the virus. All vaccinated monkeys showed an anamnestic antibody response, indicating that they had established immunological memory. Vaccine-induced antibody had an avidity index similar to that of antibody induced by virus infection; however, no clear correlation was apparent between antibody avidity and virus neutralization titres.

Animals↗
PubMed1998

The oxygen fixation hypothesis: a reevaluation.

The oxygen fixation hypothesis (OFH), developed in the late 1950s, is widely regarded as the most satisfactory explanation of why oxygen is a radiation sensitizer. Central to this hypothesis is the explicit belief that DNA lesions (originally called "target lesions") that are produced by x-rays with the chemical participation of oxygen pose a special threat to cell survival because these lesions cannot be chemically restored to an undamaged state. According to this hypothesis, oxygen sensitizes because these "nonrestorable" lesions ultimately increase the amount of stable DNA damage--and thus the extent of lethality--from a give dose. Using three wild-type strains of Escherichia coli, the maximum insignificant dose (MID, the maximum dose that does not reduce the survival from 1.0) was measured. The MID values are clearly strain dependent. This result is inconsistent with the OFH since the production of "nonrestorable" DNA-O2 radicals should not be affected by the genetic identity of the irradiated cell. By focusing solely on rapid events in radiation chemistry, this hypothesis does not address any role that might be played by enzymatic DNA repair. In fact, if enzymatic repair is successful, then the issue of chemical restorability is not relevant to lethality or to radiation sensitization by oxygen. Overall, these results emphasize that the OFH does not satisfactorily explain why oxygen is a sensitizer. Although the basic chemistry proposed in the OFH is undoubtedly correct, the hypothesis does not explain why the reaction products pose a special risk to the cell. Attention must also be given to enzymatic DNA repair and to its success.

Cell Survival↗
PubMed1997

Genetic, antigenic and serologic characterization of human immunodeficiency virus type 1 from Indonesia.

To examine the genetic and antigenic characteristics of HIV-1 in Indonesia, samples from 19 HIV-positive volunteers were studied. By a combination of PCR typing and DNA sequence analysis, 12 of the 19 volunteers were determined to be infected with HIV-1 clade B and seven with clade E. Six of the seven Indonesian clade E isolates were from volunteers associated with the Indonesian Military during a peacekeeping mission in Cambodia. Infectivity reduction neutralization assays showed that the Indonesian E viruses were effectively neutralized by Thailand clade E HIV-1 antisera but not by U.S. clade B antisera. The Indonesian clade B virus tested was neutralized by U.S. clade B antisera and not by the Thailand E antisera. Using a previously described serologic typing ELISA based on clade B and E V3 peptides, genetic clade was accurately determined in eight of eight sera tested. This is the first report of the genetic and antigenic analysis of HIV-1 isolates from Indonesia. The data indicate that at least two genetic and antigenic HIV-1 clades (clade E and B) circulate in Indonesia.

Amino Acid Sequence↗
PubMed1997

Production of radiation-resistant E. coli strains by daily X-irradiation.

Exposure of E. coli AB1157 (a K-12 wild-type strain) to very large, daily X-ray doses has produced mutant strains resistant to both X-rays and UV photons. Results reported here are with KS0(160), the most resistant strain isolated thus far. Relative to its parent, KS0(160) is about 2.3 x more resistant to X-rays and about 2.2 x more resistant to ultraviolet photons (ratios of sensitivities in air). Two other characteristics of KS0(160) make its responses to X-rays different from those of AB1157: KS0(160) has an oxygen enhancement ratio of only 1.8 compared with 2.7 for its parent; glycerol reduces the sensitivity of AB1157 by about 75% (in air), but the maximum effect in KS0(160) is only a 49% reduction in response. P1 transduction experiments showed that all the acquired resistance in KS0(160) is lost when SOS repair activity is genetically blocked by an inserted lexAl, indicating that the mutation(s) associated with the acquired resistance in KS0(160) are in wild-type genes involved with induced DNA repair (i.e. SOS repair activity).

Dose-Response Relationship, Radiation↗
PubMed1996

The X-ray induction of SOS repair activity in E. coli: euoxic vs. anoxic DNA damage.

The induction of DNA repair (the "SOS Response") by X rays has been studied in E. coli GE94, irradiated under euoxic versus anoxic conditions. GE94 contains a recA-lacZ gene fusion that allows the radiation-induced transcription of recA to be followed by measuring the changes in beta-galactosidase levels. At doses up to about 450 Gray, more recA transcription (indicating more DNA damage capable of inducing SOS repair activity) occurs in cells irradiated in air instead of 100% nitrogen. Although the presence of dissolved oxygen cannot change the initial number of DNA radicals (initial "energy-deposition events"), apparently more of these radicals become stable lesions when oxygen is present.

DNA Damage↗
PubMed1995

The directed evolution of radiation resistance in E. coli.

E. coli AB1157 (a wild-type, K-12 strain having no known defects in DNA repair capability) was irradiated daily with a very large X-ray dose to develop a series of strains unusually resistant to both X rays and ultraviolet (UV) photons. An understanding of how wild-type strains mutate and become more resistant should lead to a better understanding of DNA repair processes and their effects on radiation sensitivity.

Biological Evolution↗
PubMed1995

Hydroxyl radicals and DNA double-strand breaks in X-irradiated E. coli.

We have used glycerol to study the relationship between hydroxyl radicals, one of the primary radiolytic products, and the production of DNA double-strand breaks in selected E. coli strains. Our results suggest that when bacteria are irradiated at doses up to about 120 Gray, hydroxyl radicals produce DNA lesions, but not double-strand breaks.

DNA Damage↗
PubMed1995

The use of glycerol to link DNA damage from hydroxyl radicals with the activities of DNA repair enzymes.

We show that glycerol can be used to study the relationship between DNA damage caused by hydroxyl radicals and the activities of different DNA repair enzymes. We suggest that such tests with glycerol will lead to a better understanding of the mechanistic basis for radiation-induced cell death, as well as provide valuable information on the specific roles of different DNA repair enzymes.

DNA Damage↗
PubMed1995

Can an X-ray dose threshold be measured for the induction of SOS repair activity in E. coli.

Survival ("clonogenic ability") and induced DNA repair (SOS repair) have been studied at low X-ray doses in two Escherichia coli strains (GE94 and KY943), both of which contain a recA-lacZ protein fusion. X-ray doses greater than about 0.5 Gray clearly induce SOS repair activity. However, the threshold dose that just activates SOS repair is probably much lower than 0.5 Gray, perhaps as low as 0.001 Gray. It seems unlikely that this dose threshold can be more accurately measured with the recA-lacZ fusion technique.

Dose-Response Relationship, Radiation↗
PubMed1993

N E. coli, inducible DNA repair is error-prone with regard to lethality.

The umuC gene is a member of the inducible SOS repair network in E. coli. The experiments reported here indicate that the product of the umuC locus can make lethal mistakes during its normal repair function. Such "lethal repair" may be a major factor in establishing a cell's sensitivity to radiation.

Dose-Response Relationship, Radiation↗
PubMed1991

Is N2O-dependent damage 'fixed' by action of the recA locus?

Nitrous oxide (N2O) is often a radiation sensitizer of procaryotic cells, although it has little or no effect in recA- strains of E. coli and Saccharomyces. Here we test the hypothesis that N2O-dependent damage is in itself not lethal, but that lethality occurs when this damage is incorrectly repaired by the recA+ strains. Because sensitization by N2O requires the radiolytic production of H2O2, the hypothesis can be tested with reagent H2O2. If the 'inaction' of the recA locus prevents N2O sensitization in the recA- strains, then those strains should not be sensitized by reagent H2O2. Our data show that all these strains, including the recA+ parent, are efficiently sensitized by reagent H2O2 in N2 and also in N2O; thus or data do not support that hypothesis. We propose instead that the correlation between N2O sensitization and the recA- genotype occurs because of the inherent anoxic sensitivity of the recA- strains; the doses used to assay survival, and thus to test for sensitization by N2O, are simply too low to produce sufficient H2O2 for sensitization to occur.

DNA Damage↗
PubMed1991

Do intracellular thiol or peroxidase levels block radiation sensitization by nitrous oxide in some E. coli strains?

Although nitrous oxide (N2O) is often a radiation sensitizer in procaryotic cells, it fails to sensitize some strains of bacteria, some yeast strains, and most eucaryotic cell lines. At present this inconsistency cannot be satisfactorily explained. The experiments here use eight strains of E. coli, some of which are not sensitized by N2O, to test the hypothesis that N2O's failure to sensitize might be based on high thiol content or on low peroxidase activity. Our data contradict those hypotheses. In addition, further data show that the strains not sensitized by N2O contain no unique cellular component or compound which blocks damage from N2O.

Cell Survival↗
PubMed1991

The relationship between the anoxic sensitivity and the extent of sensitization by nitrous oxide.

Nitrous oxide reacts during irradiation to increase the yield of .OH, a radical many believe to be a major cause of lethality. Logically, one would expect N2O to be a radiation sensitizer. In some instances it is, while in others it is not. In some cases we can explain why N2O fails to sensitize; factors such as dose rate, cell concentration, buffer composition and ionic strength all influence when N2O will sensitize and, if it sensitizes, by what magnitude. Based on the results presented here with multiple strains of procaryotic and eucaryotic cells, we believe the anoxic sensitivity is another critical factor that governs whether N2O will sensitize. Our data, with data from the literature, show a relationship between the anoxic sensitivity and the N2O enhancement ratio. N2O does not sensitize in vitro unless the anoxic sensitivity (inactivation constant, k) is less than approximately 0.2 daGy-1.

Animals↗
PubMed1991

Can .OH scavengers protect against direct UV-C damage in vivo?

It has previously been shown that ethanol and tert-butanol protect poly(U) against strand breaks from pulsed laser ultraviolet (UV) photons (lambda = 248 nm). This protection is believed to be based on a modification of direct DNA damage, not on scavenging hydroxyl radicals. In this paper, several .OH scavengers were tested in vivo with Escherichia coli DNA repair-deficient mutants to see if protection could also be demonstrated with non-laser UV photons (lambda approximately 254 nm). No protection was observed.

Butanols↗