Biomedical subjects

P Eggleston

Publications and source records attributed to P Eggleston.

At least 19 recordsLinked to original sources

PubMed2006

High efficiency site-specific genetic engineering of the mosquito genome.

Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.

Aedes↗

PubMed2006

A novel association between clustered NF-kappaB and C/EBP binding sites is required for immune regulation of mosquito Defensin genes.

A comparative analysis identified key cis-acting regulatory elements responsible for the temporal control of mosquito Defensin gene expression. The promoters of Anopheles gambiae Defensin 1 and two isoforms of Aedes aegypti Defensin A are up-regulated by immune challenge. This stimulated activity depends upon a cluster of three NF-kappaB binding sites and closely associated C/EBP-like motifs, which function as a unit for optimal promoter activity. Binding of NF-kappaB and C/EBP like transcription factors is confirmed by electrophoretic mobility shift assay, including supershifts with antibodies to C/EBP. KappaB-like motifs are abundant within antimicrobial peptide gene promoters and most are very closely associated with putative C/EBP binding sites. This novel association between NF-kappaB and C/EBP binding sites may, therefore, be of widespread significance.

Aedes↗

PubMed2006

A tapeworm molecule manipulates vitellogenin expression in the beetle Tenebrio molitor.

Metacestodes of Hymenolepis diminuta secrete a molecule that decreases vitellogenin (Vg) synthesis in the beetle host, Tenebrio molitor. The 5608 bp T. molitor Vg cDNA represents a single-copy gene encoding a single open reading frame of 1821 amino acids with a predicted molecular mass of 206 kDa. Northern blot analysis revealed detectable levels of transcripts only in adult females. In vivo, Vg mRNA abundance was significantly higher in fat bodies from infected females compared with control females at all but the earliest time point. In vitro, Vg mRNA abundance was significantly increased in fat bodies incubated with live stage I-II parasites. The apparent conflict between increased Vg mRNA abundance and decreased Vg protein in fat bodies from infected females is discussed.

Amino Acid Sequence↗

PubMed2005

Evaluating the costs of mosquito resistance to malaria parasites.

Costly resistance mechanisms have been cited as an explanation for the widespread occurrence of parasitic infections, yet few studies have examined these costs in detail. A malaria-mosquito model has been used to test this concept by making a comparison of the fitness of highly susceptible lines of mosquitoes with lines that are resistant to infection. Malaria infection is known to cause a decrease in fecundity and fertility of mosquitoes; resistant mosquitoes were thus predicted to be fitter than susceptible ones. Anopheles gambiae were selected for refractoriness/resistance or for increased susceptibility to infection by Plasmodium yoelii nigeriensis. Additional lines that acted as controls for inbreeding depression were raised in parallel but not exposed to selection pressure. Selections were made in triplicate so that founder effects could be detected. Resistance mechanisms that were selected included melanotic encapsulation of parasites within 24 h postinfection and the complete disappearance of parasites from the gut. Costs of immune surveillance were assessed after an uninfected feed, and costs of immune deployment were assessed after exposure to infection and to infection and additional stresses. Mosquito survivorship was unaffected by either resistance to infection or by an increased burden of infection when compared with low levels of infection. In most cases reproductive fitness was equally affected by refractoriness or by infection. Resistant mosquitoes did not gain a fitness advantage by eliminating the parasites. Costs were consistently associated with larval production and egg hatch rate but rarely attributed to changes in blood feeding and never to changes in mosquito size. No advantages appeared to be gained by the offspring of resistant mosquitoes. Furthermore, we were unable to select for refractoriness in groups of mosquitoes in which 100% or 50% of the population were exposed to infection every generation for 22 generations. Under these selection pressures, no population had become completely refractory and only one became more resistant. Variations in fitness relative to control lines in different groups were attributed to founder effects. Our conclusion from these findings is that refractoriness to malaria is as costly as tolerance of infection.

Animals↗

PubMed2003

Juvenile hormone titre and egg production in Tenebrio molitor infected by Hymenolepis diminuta: effect of male and/or female infection, male age and mating.

Infection of Tenebrio molitor with Hymenolepis diminuta induces curtailment of female fertility. We examined ovulation and oviposition, and associated titres of juvenile hormone (JH), in relation to parasitism and mating. Oviposition was significantly increased in infected mated and virgin beetles by days 6 and 9 post-emergence. Ovulation was not changed by infection; by the end of the 18-day experiment, the total number of laid eggs was not significantly altered. On day 6, JH levels were significantly higher in virgin infected insects, compared to non-infected controls (236+/-37.7 and 107+/-9.62 pg/g wet weight). Oviposition increased after mating, but total eggs ovulated remained the same. JH levels were higher in mated females on days 12 and 18 post-emergence, for infected and control insects. Previous studies suggested that male reproductive potential might rise following infection, because uninfected females lay more eggs when mated to infected males. We tested whether this caused an increase in female JH. Males were mated on days 5 or 12, when significant changes in their reproductive physiology begin to be observed, and are maximal, respectively. However, male age was of greater significance in promoting JH levels in females (p=0.001), than infection status of either partner (p=0.33).

Age Factors↗

PubMed2001

A sensitive and rapid assay for homologous recombination in mosquito cells: impact of vector topology and implications for gene targeting.

BACKGROUND: Recent progress in insect transgenesis has been dramatic but existing transposon-based approaches are constrained by position effects and potential instability. Gene targeting would bring a number of benefits, however progress requires a better understanding of the mechanisms involved. Much can be learned in vitro since extrachromosomal recombination occurs at high frequency, facilitating the study of multiple events and the impact of structural changes among the recombining molecules. We have investigated homologous recombination in mosquito cells through restoration of luciferase activity from deleted substrates. The implications of this work for the construction of insect gene targeting vectors are discussed. RESULTS: We show that linear targeting vectors are significantly more efficient than circular ones and that recombination is stimulated by introducing double-strand breaks into, or near, the region of homology. Single-strand annealing represents a very efficient pathway but may not be feasible for targeting unbroken chromosomes. Using circular plasmids to mimic chromosomal targets, one-sided invasion appears to be the predominant pathway for homologous recombination. Non-homologous end joining reactions also occur and may be utilised in gene targeting if double-strand breaks are first introduced into the target site. CONCLUSIONS: We describe a rapid, sensitive assay for extrachromosomal homologous recombination in mosquito cells. Variations in substrate topology suggest that single-strand annealing and one-sided invasion represent the predominant pathways, although non-homologous end joining reactions also occur. One-sided invasion of circular chromosomal mimics by linear vectors might therefore be used in vitro to investigate the design and efficiency of gene targeting strategies.

Animals↗

PubMed2001

Gene targeting in mosquito cells: a demonstration of 'knockout' technology in extrachromosomal gene arrays.

BACKGROUND: Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. RESULTS: We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an extrachromosomal array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. CONCLUSIONS: These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species.

Aedes↗

PubMed2001

Maternal depressive symptoms and emergency department use among inner-city children with asthma.

CONTEXT: Inner-city minority children with asthma use emergency departments (ED) frequently. OBJECTIVE: To examine whether maternal depressive symptoms are associated with ED use. DESIGN, SETTING, AND PATIENTS: Baseline and 6-month surveys were administered to mothers of children with asthma in inner-city Baltimore, Md, and Washington, DC. MAIN OUTCOME MEASURES: Use of the ED at 6-month follow-up was examined. Independent variables included asthma morbidity, age, depressive symptoms, and other psychosocial data. RESULTS: Among mothers, nearly half reported significant levels of depressive symptoms. There were no demographic or asthma-related differences between the children of mothers with high and low depressive symptoms. However, in bivariate analyses, mothers with high depressive symptoms were 40% (prevalence ratio [PR], 1.4; 95% confidence interval [CI], 1.0-3.6; P =.04) more likely to report taking their child to the ED. Mothers aged 30 to 35 years were more than twice as likely (PR, 2.2; 95% CI, 1.9-9.3; P =.001) to report ED use, as were children with high morbidity (PR, 1.9; 95% CI, 1.4-7.1; P =.006). Child age and family income were not predictive of ED use. After controlling for asthma symptoms and mother's age, mothers with depressive symptoms were still 30% more likely to report ED use. CONCLUSIONS: Depression is common among inner-city mothers of children with asthma. Beyond asthma morbidity, maternal age and depressive symptoms are strong predictors of reports of ED visits. Identifying and addressing poor psychological adjustment in mothers may reduce unnecessary ED visits and optimize asthma management among inner-city children.

Adult↗

PubMed2001

Children with asthma and nebulizer use: parental asthma self-care practices and beliefs.

We examined demographic characteristics, patterns of medication use, asthma morbidity, and asthma self-management practices and beliefs among inner-city children currently using a nebulizer. We also describe the relationship between asthma self-management practices and beliefs and anti-inflammatory (AI) therapy. We observed a high rate of morbidity, including frequent emergency room visits, hospitalizations, symptom days and nights, and school absences in this group of school-aged children with asthma. More than three-quarters (81%) reported asthma symptoms consistent with mild persistent or greater severity of asthma, and therefore these subjects should be taking AI medications. Another 16% (36 of 231) of these children reported symptoms consistent with mild intermittent asthma. Only 1 out of 7 children in this study reported taking AI medications. We found that parents of children taking daily AI medications were more likely to agree with the belief that children should use asthma medications daily even when the child is not reporting any symptoms.

Administration, Inhalation↗

PubMed2000

Nebulizer use in inner-city children with asthma: morbidity, medication use, and asthma management practices.

OBJECTIVE: To assess the frequency of nebulizer use, describe morbidity and patterns of medication administration, and examine the potential relationships between inhaled anti-inflammatory medication administration, asthma morbidity, and asthma management practices in children with asthma using a nebulizer compared with children with asthma not using a nebulizer. RESEARCH DESIGN: A cross-sectional, descriptive survey of previous events. SETTING: Elementary schools and participants' homes in Baltimore, Md, and Washington, DC. PARTICIPANTS: Six hundred eighty-six families of children aged 5 to 12 years with a diagnosis of at least mild, persistent asthma. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Asthma morbidity, health care utilization, pattern of asthma medication administration, nebulizer use, and asthma management data were collected by telephone survey administered to caregivers. Nebulizer use was defined as use at least 1 or more days per month during the last 6 months. Of 686 children identified, 231 (33%) reported current nebulizer use. Nebulizer users had significantly increased lifetime hospital admissions, hospitalizations, and emergency department visits in the last 6 months compared with nonnebulizer users. Inhaled corticosteroid administration was low for both groups (nonnebulizer users, 8%; nebulizer users, 15%). In the nebulizer users group, administration of inhaled anti-inflammatory medications was associated with increased asthma morbidity (increased hospitalizations, days and nights with symptoms, and oral steroid use). CONCLUSIONS: Nebulizer use by inner-city children with asthma is higher than anticipated but is not associated with reduced asthma morbidity. This group of high-risk children was undertreated with inhaled corticosteroids for long-term control of asthma despite reports of adequate monitoring by a primary care physician.

Administration, Inhalation↗

PubMed2000

Emergency department visits by urban African American children with asthma.

BACKGROUND: Asthma morbidity among African American children has been identified as a significant national health concern. High emergency department use is one index of this morbidity and may reflect disease severity, disease management, and social factors. OBJECTIVE: This study examined the prevalence and correlates of emergency department use and other indices of asthma morbidity among a sample of urban, low-income, African American children. METHODS: Parents of 392 elementary school children with asthma who had consented to participate in an asthma education program were interviewed by phone according to a standardized protocol. RESULTS: Children had a mean of 6.2 days of restricted activity (SD 8.1) and 7.9 symptomatic nights (SD 8.1). The mean number of school days missed because of asthma was 9.7 (SD 13.5). Among children with asthma symptoms in the past 12 months, 73.2% could identify a specific physician or nurse who provided asthma care. For those families without an identified asthma primary care provider, 39.3% received their usual asthma care from the emergency department. A total of 43.6% of the children had been to the emergency department for asthma care without hospitalization in the previous 6 months. Close to 80% of children reported using one or more prescribed asthma medication, and of these only 12% reported using inhaled anti-inflammatory medications. Families of children who had used the emergency department in the prior 6 months reported more asthma symptoms, lower social support, problems paying for health care, and the absence of a hypoallergenic mattress cover and that they had seen a physician for regular asthma care in the past 6 months. CONCLUSIONS: We conclude that asthma management for children in the inner city relies on episodic care and emergency care, that asthma medication management does not conform to current guidelines, and that asthma symptoms resulting in school absences and workdays lost are prevalent.

Absenteeism↗

PubMed2000

Genomic organization and immune regulation of the defensin gene from the mosquito, Anopheles gambiae.

The defensin gene from the mosquito, Anopheles gambiae, is present as a single copy per haploid genome. Two exons, encoding a 102 residue preprodefensin, are separated by a 105 bp intron bounded by consensus splice sites. The upstream regulatory sequence includes a TATA box, arthropod initiator and numerous motifs homologous to insect and mammalian immune response elements. This promoter is capable of upregulation by immune challenge in cultured cells and activity is further stimulated by Gambif1, a mosquito Rel protein known to translocate to the nucleus and bind NF-kappa B sites in target promoters. Activity is inhibited by p50, a mammalian Rel protein that competitively binds NF-kappa B sites, and virtually abolished by p40, an avian I kappa B protein that inhibits nuclear translocation.

Amino Acid Motifs↗

PubMed2000

Factors associated with medication self-administration in children with asthma.

This study investigated factors associated with early self-administration of inhaled asthma medications by minority children. Specifically, the study evaluated: (1) the reasons parents allow early administration of inhaled medications, (2) childhood activities associated with early medication administration, (3) parent's perception of the child's ability to use a metered-dose inhaler (MDI), (4) the child's actual ability to use an MDI, and (5) concordance/discordance between physician-parent reports and parent-child reports of asthma medications. Study results indicated that 93% of the children were taking inhaled asthma medications without adult supervision. Early self-administration of asthma medications was related to the parent's employment status and the performance of other childhood behaviors such as completion of homework independently and crossing the street alone. Only 7% of the children had effective MDI skills, but 60% of the parents rated their child's MDI skills as excellent. Twenty percent, 67%, and 50%, respectively, of the parents' reports of beta-agonists, daily inhaled steroids, and cromolyn were discordant with the physician's actual prescriptions. Sixty-two percent, 57%, and 79%, respectively, of the children's reports for inhaled beta-agonists, daily inhaled steroids, and cromolyn were discordant with their parents' reports. Implications for anticipatory guidance, future educational strategies, and supervision of MDI technique are provided.

Adolescent↗

PubMed2000

Assessing pediatric clinical asthma practices and perceptions: a new instrument.

Over the past 20 years, the most substantial increases in prevalence, morbidity, and mortality of asthma have been observed among children aged 5-14 years. A survey instrument designed to measure clinical asthma management practices of primary care physicians was developed and evaluated. Study participants included 127 practitioners providing pediatric asthma care in inner-city communities in Baltimore, MD and Washington, DC. Study results found that the instrument assessed four separate dimensions of clinical assessments and five dimensions of physician perceptions. These dimensions should be considered in future research protocols and may be used to design tailored interventions to improve asthma care.

Adult↗

PubMed2000

Effectiveness of a clinical pathway for inpatient asthma management.

BACKGROUND: Clinical pathways for asthma are tools that have the potential to improve compliance with nationally recognized management guidelines, but their effect on patient outcomes has not been documented. OBJECTIVES: To determine the effect of an asthma clinical pathway on patients' length of stay, use of nebulized beta-agonist therapy while hospitalized, and use of acute care clinics for 2 weeks after discharge. DESIGN/METHODS: The study was a randomized, controlled trial. Patients between the ages of 2 and 18 years admitted with an asthma exacerbation and not under the care of an asthma specialist were eligible for the study. Patients were randomized either to a conventional ward (control group) or to a ward using the clinical pathway (intervention group). For 2 weeks after discharge, we collected data to determine whether patients visited a health care provider for worsening asthma. RESULTS: One hundred ten patients (26%) were enrolled. Control and intervention groups had similar demographic and asthma severity profiles. The intervention group had an average length of stay 13 hours shorter than did the control group. In addition, at every dosing interval, the intervention group received less nebulized beta-agonist therapy. There were no deaths in either group. CONCLUSION: A clinical pathway for inpatient asthma decreased the length of stay and beta-agonist medication use with no adverse outcomes or increased acute-care encounters through 2 weeks after discharge.

Administration, Inhalation↗

PubMed1999

A randomized clinical trial to reduce asthma morbidity among inner-city children: results of the National Cooperative Inner-City Asthma Study.

OBJECTIVE: To evaluate a family-focused asthma intervention designed for inner-city children 5 to 11 years old with moderate to severe asthma. STUDY DESIGN: Randomized, multisite, controlled trial to minimize symptom days (wheeze, loss of sleep, reduction in play activity) measured by a 2-week recall assessed at 2-month intervals over a 2-year follow-up period. The intervention was tailored to each family's individual asthma risk profile assessed at baseline. RESULTS: Averaged over the first 12 months, participants in the intervention group (n = 515) reported 3.51 symptom days in the 2 weeks before each follow-up interview compared with 4.06 symptom days for the control group (n = 518), a difference of 0.55 (95% CI, 0.18 to 0.92, P =.004). The reduction among children with severe asthma was approximately 3 times greater (1.54 d/2 wk). More children in the control group (18.9%) were hospitalized during the intervention compared with children in the intervention group (14. 8%), a decrease of 4.19% (CI, -8.75 to 0.36, P =.071). These improvements were maintained in the intervention group during the second year of follow-up, during which they did not have access to the asthma counselor. CONCLUSIONS: We demonstrated that an individually tailored, multifaceted intervention carried out by Masters-level social workers trained in asthma management can reduce asthma symptoms among children in the inner city.

Asthma↗

PubMed1999

Comparative analysis of promoters for transient gene expression in cultured mosquito cells.

Three heterologous promoters (hsp70 and actin 5C from Drosophila melanogaster and IE1 from the immediate early gene of the Bombyx mori baculovirus) were assessed for their ability to drive transient luciferase expression in mosquito cells. Overall, the actin 5C promoter was considerably more effective at driving luciferase expression than either hsp70 or IE1 in cell lines derived from Anopheles, Aedes and Culex species. hsp70 functioned well when induced by heat shock and was also induced to a lesser extent by chemicals such as sodium arsenite. IE1 was also an effective initiator of transcription, particularly in two Anopheles cell lines, but generally it performed less well than the actin 5C promoter and was also outperformed by hsp70 in Anopheles gambiae cells.

Actins↗

PubMed1998

Factors affecting the retention of dust mite allergen on carpet.

Carpeting is one of the most common and perhaps the most important reservoir of aeroallergens and other components of dust found in residential or commercial settings. This research evaluated factors that affect the retention of dust mite allergens on different types of tufted carpet commonly found in homes and offices. Twenty-six types of carpet were custom manufactured as part of a two-stage factorial experiment using 182 carpet samples. Carpets differed with respect to fiber denier, cross-sectional shape, presence of fluorocarbon treatment, carpet style, pile height, and pile density. The major hypothesis tested was that the amount of allergen-containing dust recovered from vacuum samples of tufted carpet sources was dependent on micro (fiber) or macro (construction) retention characteristics of the carpets. Carpet samples were doped in a uniform and reproducible manner using an allergen-containing reference dust. A standardized vacuum surface sampler was used to recover dust from samples. Allergen was assayed using a standard, monoclonal antibody ELISA. Carpet-surface area and presence of fluorocarbon were found to have the largest effects on retention and recovery of dust and allergen. Specifically, fluorocarbon treatment of fibers, square-hollow fiber shape, high-denier fiber, low-pile height in cut-pile carpets, and low-pile density in loop carpets were significantly associated with increased release and recovery of allergen. Results from this study suggest that carpet type be considered for both allergen avoidance and allergy and asthma risk assessment.

Allergens↗