Effects of dog ownership in early childhood on immune development and atopic diseases.

BACKGROUND: Exposure to pets in childhood has been associated with a reduced risk of wheezing and atopy. OBJECTIVE: Our objective was to determine whether the effects of pet exposure on immune development and atopy in early childhood can be explained by alterations in exposure to innate immune stimuli in settled dust. METHODS: Two hundred and seventy-five children at increased risk of developing allergic diseases were evaluated to age 3 years for pet ownership, blood cell cytokine responses, and atopy. Can f 1, Fel d 1, endotoxin, ergosterol, and muramic acid were measured in settled dust from 101 homes. RESULTS: Dog exposure at birth was associated with decreased atopic dermatitis (AD) (12% vs. 27%; P=0.004) and wheezing (19% vs. 36%; P=0.005) in year 3. The rates of AD (23%) and wheezing (42%) in year 3 were relatively high in children who acquired dogs after birth. The prevalence of dog sensitization (10-12%) did not vary according to dog exposure. Can f 1 levels in bedroom dust were positively associated with IL-10 (r=0.26; P=0.01), IL-5 (r=0.34, P<0.001), and IL-13 (r=0.28; P=0.004) responses at age 1, and IL-5 (r=0.24; P=0.022) and IL-13 (r=0.25; P=0.015) responses at age 3. In contrast, endotoxin was associated with IFN-gamma (r=0.31; P=0.002) and IL-13 (r=0.27; P=0.01) responses at age 3 but not at age 1, and similar relationships were present for muramic acid. Adjustment for levels of innate immune stimuli in house dust did not significantly affect the relationships between Can f 1 and cytokine responses. CONCLUSIONS: Exposure to dogs in infancy, and especially around the time of birth, is associated with changes in immune development and reductions in wheezing and atopy. These findings are not explained by exposure to endotoxin, ergosterol, or muramic acid.

The malaria vector mosquito Anopheles gambiae expresses a suite of larval-specific defensin genes.

cDNAs of Anopheles gambiae Defensin 2 (AgDef2), Defensin 3 (AgDef3) and Defensin 4 (AgDef4), identified in the genome sequence, have been characterized and their expression profiles investigated. In contrast to both typical defensins and insect antimicrobial peptides generally, the newly identified defensins were not upregulated with acute-phase kinetics following immune challenge in insects or cell culture. However, mRNA abundance of AgDef2, AgDef3 and AgDef4 increased significantly during the larval stages. Promoter analysis of all three genes failed to identify putative immune response elements previously identified in other mosquito defensin genes. As previous studies failed to identify these larval-specific defensins, it seems likely that further antimicrobial peptide genes with nontypical expression profiles will be identified as more genome sequences become available.

Biological potency of German cockroach allergen extracts determined in an inner city population.

BACKGROUND: Cockroach allergy is an important cause of inner city asthma. To perform valid studies on the diagnosis and treatment of cockroach allergy, biological potencies of test extracts need to be established, and a surrogate in vitro test for biological potency should be chosen. METHODS: Sixty-two cockroach-allergic adult subjects were recruited for quantitative skin testing with three commercial German cockroach extracts. The intradermal D50 values were determined using linear interpolation, and the biologic potencies were determined from D50 data. The extracts were also analysed for relative potency, using a competition ELISA, and for specific allergen content, using a two-site ELISA. RESULTS: Estimates of each extract's D50 were analysable in 48-55 subjects, with D50s between 10.3 and 11.8. All three extracts were bioequivalent using pre-set criteria. The biological potencies of the extracts were 1738-8570 bioequivalent allergy units (BAU)/mL (geometric mean=3300), and these relative potencies were similar to those estimated by competition ELISA and specific allergen content. IgE against cockroach allergens were detected in sera from 34 subjects with analysable D50s, and 17 subjects had IgE directed against specific cockroach allergens. Although the presence of anti-Bla g 5 correlated with the subjects' skin test responses for 2/3 extracts, no single allergen was immunodominant. Antibody responses among the subjects were heterogeneous. CONCLUSIONS: Although commercial cockroach extracts are relatively low in potency, immunotherapeutic doses should be achievable. Biological potency may be estimated using D50 testing, a combination of specific allergen determinations, or by an overall potency assay such as the competition ELISA. CAPSULE SUMMARY: The biological potency of three German cockroach allergen extracts, determined in an inner city population, was 1738-8570 BAU/mL. No one allergen was immunodominant, and surrogate in vitro testing methods were examined.

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.

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.

Mouse allergen-specific immunoglobulin G4 and risk of mouse skin test sensitivity.

BACKGROUND: High serum levels of cat-specific IgG and IgG4 are associated with protection against allergic sensitization to cat, but whether this association applies to other animal allergens remains unclear. OBJECTIVE: To determine if high levels of mouse-specific IgG and IgG4 are associated with a decreased risk of mouse skin test sensitivity. METHODS: Two hundred and sixty workers of a mouse facility underwent skin prick testing and completed a questionnaire. Serum levels of mouse-specific IgG and IgG4 were quantified by solid-phase antigen binding assays. Room air samples were collected and airborne Mus m 1 was quantified by ELISA. RESULTS: Forty-nine participants had a positive skin prick test to mouse. Mouse-specific IgG was detected in 219 (84%) participants and IgG4 was detected in 72 (28%) participants. A detectable mouse-specific IgG4 level was associated with an increased risk of mouse skin test sensitivity (odds ratios (OR) 6.4, 95% confidence intervals (CI) 3.3-12.4). Mouse-specific IgG and IgG4 were both positively correlated with mouse allergen exposure (r(s)=0.31, P=0.0001, and r(s)=0.27, P=0.0006, respectively). The odds of skin test sensitivity peaked at moderate levels of IgG4, but decreased at the highest levels of mouse-specific IgG4. In contrast, the odds of skin test sensitivity increased monotonically with IgG levels. CONCLUSIONS: A detectable level of mouse-specific IgG4 is associated with an increased risk of skin test sensitivity to mouse. However, the highest IgG4 levels appear to be associated with an attenuated risk of mouse skin test sensitivity, suggesting that induction of high levels of IgG4 through natural exposure may protect against the development of allergic sensitization.

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.

Mouse allergen-specific immunoglobulin G and immunoglobulin G4 and allergic symptoms in immunoglobulin E-sensitized laboratory animal workers.

BACKGROUND: High levels of allergen-specific IgG have been associated with clinical efficacy in immunotherapy studies, but whether this antibody isotype is associated with clinical tolerance in the setting of environmental exposure remains unclear. OBJECTIVE: To determine if mouse allergen-specific IgG (mIgG) and IgG4 (mIgG4) levels are associated with mouse-related symptoms among IgE-sensitized laboratory workers. METHODS: Fifty-eight workers with either skin test or serologic evidence of IgE-mediated mouse sensitization were studied. Symptom data were obtained by a questionnaire. Serum levels of mouse-specific IgG, IgG4, and IgE were quantified by a solid-phase antigen-binding assay (IgG) and RAST (IgG4 and IgE), and the relationships between mouse-specific serologic responses and mouse-related symptoms were analysed. RESULTS: Twenty-three (39.7%) participants reported mouse-related symptoms. Mouse-specific IgG and IgG4 levels were not associated with mouse-related symptoms among the study population as a whole. Among the 29 (50%) participants with detectable mouse-specific IgE (mIgE), higher mouse-specific IgG and IgG4 levels were associated with a decreased risk of symptoms, after adjusting for mIgE level (odds ratio (OR) 0.3, 95% confidence interval (CI): 0.1-1.4, and OR 0.3, 95% CI: 0.04-2.6, respectively). Higher levels of mIgG and mIgG4 remained associated with a decreased risk of symptoms after additional adjustment for sex and handling of mice (OR 0.1, 95% CI: 0.02-0.7, and OR 0.2, 95% CI: 0.02-2.1, respectively). Higher mIgG : IgE and mIgG4 : IgE ratios were also associated with a decreased risk of symptoms after adjusting for these confounders (OR 0.1, 95% CI: 0.02-0.7, and OR 0.2, 95% CI: 0.02-0.92, respectively). CONCLUSION: Among workers with detectable mIgE, higher mIgG and mIgG4 levels are associated with a decreased risk of mouse-related symptoms. High serum levels of mIgG or mIgG4 may be markers for clinical tolerance among laboratory mouse workers with detectable mIgE, but these findings need to be confirmed in larger, prospective studies.

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.

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).

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.

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.

Cockroach allergen abatement with extermination and sodium hypochlorite cleaning in inner-city homes.

BACKGROUND: Although the importance of cockroach allergen in chronic asthma has now been well defined, little progress has been made in the control of cockroach allergen in infested homes. OBJECTIVE: The objective of this study was to examine the ability of a combination of professional pest extermination and household cleaning using a solution of sodium hypochlorite to reduce cockroach infestation and allergen levels in cockroach infested homes. METHODS: Seventeen cockroach-infested homes were studied with three homes serving as controls. In the intervention homes, a professional exterminator applied 0.05% abamectin twice at 2-week intervals at study entry and a professional cleaner cleaned the homes before and after the extermination. All washable surfaces were cleaned throughout the study with a solution of 0.5% hypochlorite. Monthly home visits were conducted to inspect the home, interview the homeowner, place passive cockroach traps, and to collect settled dust samples from the kitchen, bedroom, and TV/living room. RESULTS: The number of cockroaches in the passive traps decreased rapidly after the initial intervention in most homes. Median Blatella germanica allergen 1 levels in the settled dust samples fell by 91% in the kitchen, 78% in the bedroom, and 77% in the living room over the course of the study in the intervention homes but gradually rose in the control homes. The overall reductions were very similar to those seen in a previous study with a similar protocol except for the use of the sodium hypochlorite. CONCLUSIONS: Successful extermination is possible in most inner-city homes and cockroach allergen levels can be reduced by 80% to 90%. However, 0.5% sodium hypochlorite did not seem to improve allergen reduction, and in many homes, allergen levels remained above the proposed threshold of 8 U/g of dust throughout the study.

Allergenic proteins are fragmented in low concentrations of sodium hypochlorite.

BACKGROUND: To facilitate allergen removal from indoor environments, it would be helpful to have household cleaning products that modified allergenic activity. Because NaOCl dissolves proteins in high concentrations and is both capable of killing bacteria and viruses and inactivating viral antigens at somewhat lower concentrations, we explored its effects on Mus m 1 and other indoor allergens. OBJECTIVE: To examine the ability of NaOCl to reduce the allergenicity of Mus m 1 and other indoor allergens. METHODS: Using purified mouse urinary allergen, we examined the effect on protein measured by Coomassie protein assay and on Mus m 1 measured by ELISA. We also examined the effects using SDS/PAGE and Western blots probed with sheep anti-Mus m 1 and with allergic human serum. RESULTS: When NaOCl and Mus m 1 were combined in a molar ratio of 100 : 1, IgE binding to Mus m 1 on Western blot was significantly reduced. At higher NaOCl concentrations the protein appeared to fragment and eventually became undetectable. Fragmentation appeared to be random in that peptides of a wide range of apparent molecular weight were produced. The reaction was complete within 1-2 min at OCl : pr ratios of greater than 200 : 1 and was optimal at pH 7.4. Immunological activity of other allergens (Fel d 1, Bla g 1, Der p 1) was decreased in vitro and dried allergen extracts were removed from surfaces. Adding an extraneous protein, BSA, to NaOCl:Mus m 1 solutions decreased the effect of NaOCl on the allergen. CONCLUSIONS: We concluded that NaOCl at concentrations commonly used in household products is capable of dramatically affecting allergenic protein.

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.

Ecology and elimination of cockroaches and allergens in the home.

Cockroach infestations have been indicated as a major contributor to asthma throughout the world. Several studies have shown that large numbers of asthmatic patients are sensitized to cockroach allergens. Eliminating this pest from homes, schools, and public buildings involves a long-term commitment to a rational extermination process. This article covers the characteristics of the major cockroach species that invade homes, assesses the role of environmental exposure to cockroaches in asthma, and provides an intervention program for their extermination.

House dust mite and cockroach exposure are strong risk factors for positive allergy skin test responses in the Childhood Asthma Management Program.

BACKGROUND: Children with asthma have a high prevalence of environmental allergies, especially to indoor allergens. The relationships of exposure to indoor allergens (dust mites, cat, dog, cockroach, and molds) and other host factors to allergy sensitization have not been evaluated simultaneously in a large cohort. OBJECTIVES: We studied 1041 children aged 5 to 12 years with mild-to-moderate asthma to determine risk factors associated with having positive allergy skin test responses to indoor allergens. Also, we described, compared, and contrasted 6 allergens in the home environments of these children from 8 North American cities. METHODS: Data were used from baseline visits of the Childhood Asthma Management Program. Patients' sensitivities to house dust mites (Dermatophagoides farinae and Dermatophagoides pteronyssinus), cats, dogs, cockroaches, and molds were examined for relationships to demographic variables, home dust allergen exposures, number of other positive allergy skin test responses, total serum IgE levels, and smoking in the home. RESULTS: San Diego (78.5%) and Toronto (59.3%) had the topmost percentages of homes with moderate-to-high house dust mite levels. Boston (21.5%), St Louis (16.3%), and Baltimore (13.4%) had the highest percentages of homes with detectable levels of cockroach allergen. For house dust mites, the higher the level of allergen exposure, the more likely patients were to have positive allergy skin test responses, with relative odds of 9.0 (95% confidence interval, 5.4-15.1) for those exposed to high mite levels (>10.0 microg/g dust) relative to those unexposed. Even exposure to low levels of mite allergen (0.020-2.0 microg/g) was found to be a significant risk factor for sensitization. For cockroach allergen, those with detectable home exposure were more likely to have positive skin test responses (relative odds, 2.2; 95% confidence interval, 1.3-3.8) than those with undetectable exposure. In contrast, levels of exposure to cat, dog, and mold allergens were not related to sensitization rates. For cat allergen, this may reflect lower rates of cat ownership among highly sensitized subjects. Furthermore, the number of allergy skin test responses that were positive, excluding the test for the outcome of interest for each model, and total serum IgE levels were strong independent predictors of sensitization. CONCLUSIONS: Levels of exposure determined by house dust analysis are important determinants of sensitization for dust mite and cockroach allergen. This relationship was not demonstrable for cat, dog, or mold allergens, possibly because of confounding factors. For all allergens studied, the degree of atopy, determined by the total number of positive skin test responses or by total serum IgE levels, is an important contributing risk factor for sensitization.

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.