Computational tools for the study of allergens

Allergy is a major cause of morbidity worldwide. The number of characterized allergens and related information is increasing rapidly creating demands for advanced information storage, retrieval and analysis. Bioinformatics provides useful tools for analysing allergens and these are complementary to traditional laboratory techniques for the study of allergens. Specific applications include structural analysis of allergens, identification of B- and T-cell epitopes, assessment of allergenicity and cross-reactivity, and genome analysis. In this paper, the most important bioinformatic tools and methods with relevance to the study of allergy have been reviewed.

Allergen databases

Allergies represent a significant medical and industrial problem. Molecular and clinical data on allergens are growing exponentially and in this article we have reviewed nine specialized allergen databases and identified data sources related to protein allergens contained in general purpose molecular databases. An analysis of allergens contained in public databases indicates a high level of redundancy of entries and a relatively low coverage of allergens by individual databases. From this analysis we identify current database needs for allergy research and, in particular, highlight the need for a centralized reference allergen database.

Impact of voluntary folate fortification on plasma homocysteine and serum folate in Australia from 1995 to 2001: a population based cohort study

Study objective: To investigate the effect of the voluntary folate fortification policy in Australia on serum folate and total plasma homocysteine (tHcy) concentrations. Design: Population based cohort study. Setting: Perth, Western Australia. Participants: Men and women aged 27 to 77 years (n = 468), who were originally randomly selected from the Perth electoral roll. The cohort was surveyed in 1995/96 before widespread introduction of folate fortification of a variety of foods, and followed up on two occasions, firstly in 1998/99 and again in 2001, when a moderate number of folate fortified foods were available. Subjects with abnormal serum creatinine concentrations at baseline were excluded from this analysis. Main results: Repeated measures analysis of variance was used to determine changes in serum folate and tHcy over the three surveys and to assess whether time trends were related to age, sex, MTHFR C677T genotype, or consumption of folate fortified foods. An increase (38%) in mean serum folate (p < 0.0005) and a decrease (21%) in mean tHcy (p < 0.0005) were seen after introduction of the voluntary folate fortification policy in Australia. Serum folate was consistently higher (p = 0.032) and tHcy was consistently lower (p = 0.001) in subjects who consumed at least one folate fortified food compared with subjects who did not consume any folate fortified foods in the previous week. The time related changes in serum folate and tHcy were affected only by intake of folate fortified foods (p < 0.0005) and not by any other measured variables including age, sex, or MTHFR genotype. Conclusion: Voluntary fortification of foods with folate in Australia has been followed by a substantial increase in serum folate and decrease in tHcy in the general population.

Weight loss during prolonged lactation in rural Bangladeshi mothers

To determine the duration of lactation which is associated with weight loss in rural Bangladeshi mothers and also to determine the relationship with consumption patterns of principal food items, a cross-sectional study was carried out among 791 lactating rural Bangladeshi mothers aged 18-40 years. Results were compared with 333 non-pregnant and non-lactating mothers of a similar age group. The duration of lactation was up to 60 months. The mean difference in body-weight and body mass index (BMI) of lactating mothers who breastfed their children up to 24 months was significantly lower compared to non-lactating mothers of the same age group, but no differences were observed for those who breastfed beyond 24 months. The frequency of consumption of principal food items was comparable between the non-lactating and the lactating mothers who breastfed beyond 24 months. Results of multiple linear regression analysis showed that body-weight of mothers was negatively correlated with 1-12 month(s) and 13-24 months of lactation after controlling for height, education, and food consumption (slope -1.04, p < 0.05 and slope -1.23, p < 0.05 respectively). Height and consumption of meat and milk were significantly positively correlated with body-weight (slope 0.53, p < 0.001; slope 1.44, p < 0.001; and slope 0.75, p < 0.05 respectively). The study concluded that Bangladeshi women who breastfed up to 24 months were of lower weight than non-lactating mothers, most likely due to the effect of lactation. These mothers were not taking any additional foods during their lactating period. Based on the findings of the study, it is recommended that mothers consume additional energy-rich foods during the first 24 months of lactation to prevent weight loss.

Targeting Antigen-loaded Liposomes to Myeloid Antigen Presenting Cells

Objective: To target antigen-loaded liposomes to myeloid APC in vivo for immunotherapy and to manipulate immune function through liposome composition. Method: Liposomes were loaded with ovalbumin, the lipophilic red fluorescent marker, DiI, with or without QuilA adjuvant then injected either i.v. or s.c. to naı¨ ve C57Bl/6 mice. Spleen, liver and draining LN were stained with MHC class II and various myeloid markers to determine the uptake of liposomes. Frozen sections of spleen and draining LN were stained with FITC-labeled mAb to determine which cells take up the liposomes. To determine the effect on OVA-specific T cell responses, liposomes were administered to Balb/c mice which received DO11.10 OVAspecific TCR transgenic T cells labelled with CFSE. Results: The DiI fluorescence was visualized in MHC class II+ macrophages and DC in draining lymph nodes after s.c. injection and in spleen and liver after i.v injection. Immunofluorescence microscopy shows liposome uptake in marginal zone macrophages and some DC in the T cell areas of the spleen after i.v. injection. Administration of ova-liposomes with or without QuilA stimulated a specific T cell response as measured by CFSE dilution. Conclusion: APC of liver, spleen and LN, and subsequent antigen presentation to T cells can be targeted for immunotherapy by the administration of liposomes encapsulating antigen and adjuvant. Varying the composition and routes of liposome administration is expected to alter the function of the targeted APC and the T cell response.

Modelling of species in hood fires by conditional moment closure

Carbon monoxide, the chief killer in fires, and other species are modelled for a series of enclosure fires. The conditions emulate building fires where CO is formed in the rich, turbulent, nonpremixed flame and is transported frozen to lean mixtures by the ceiling jet which is cooled by radiation and dilution. Conditional moment closure modelling is used and computational domain minimisation criteria are developed which reduce the computational cost of this method. The predictions give good agreement for CO and other species in the lean, quenched-gas stream, holding promise that this method may provide a practical means of modelling real, three-dimensional fire situations. (c) 2005 The Combustion Institute. Published by Elsevier Inc. All rights reserved.

Biotechnology and reduced spoilage

This book provides information on new and existing developments and the ways they can be combined to preserve particular foods.

Evaluation of short dietary questions from the 1995 National Nutrition Survey

Six of the short dietary questions used in the 1995 National Nutrition Survey (see box below) were evaluated for relative validity both directly and indirectly and for consistency, by documenting the differences in mean intakes of foods and nutrients as measured on the 24-hour recall, between groups with different responses to the short questions. 1. Including snacks, how many times do you usually have something to eat in a day including evenings? 2. How many days per week do you usually have something to eat for breakfast? 3. In the last 12 months, were there any times that you ran out of food and couldn’t afford to buy more? 4. What type of milk do you usually consume? 5. How many serves of vegetables do you usually eat each day? (a serve = 1/2 cup cooked vegetables or 1 cup of salad vegetables) 6. How many serves of fruit do you usually eat each day? (a serve = 1 medium piece or 2 small pieces of fruit or 1 cup of diced pieces) These comparisons were made for males and females overall and for population sub-groups of interest including: age, socio-economic disadvantage, region of residence, country of birth, and BMI category. Several limitations to this evaluation of the short questions, as discussed in the report, need to be kept in mind including: · The method for comparison available (24-hour recall) was not ideal (gold standard); as it measures yesterday’s intake. This limitation was overcome by examining only mean differences between groups of respondents, since mean intake for a group can provide a reasonable approximation for ‘usual’ intake. · The need to define and identify, post-hoc, from the 24-hour recall the number of eating occasions, and occasions identified by the respondents as breakfast. · Predetermined response categories for some of the questions effectively limited the number of categories available for evaluation. · Other foods and nutrients, not selected for this evaluation, may have an indirect relationship with the question, and might have shown stronger and more consistent responses. · The number of responses in some categories of the short questions eg for food security may have been too small to detect significant differences between population sub-groups. · No information was available to examine the validity of these questions for detecting differences over time (establishing trends) in food habits and indicators of selected nutrient intakes. By contrast, the strength of this evaluation was its very large sample size, (atypical of most validation studies of dietary assessment) and thus, the opportunity to investigate question performance in a range of broad population sub-groups compared with a well-conducted, quantified survey of intakes. The results of the evaluation are summarised below for each of the questions and specific recommendations for future testing, modifications and use provided for each question. The report concludes with some general recommendations for the further development and evaluation of short dietary questions.

Analytical validation for a series of marker compounds used to assess renal drug elimination processes

Renal drug elimination is determined by glomerular filtration, tubular secretion, and tubular reabsorption. Changes in the integrity of these processes influence renal drug clearance, and these changes may not be detected by conventional measures of renal function such as creatinine clearance. The aim of the current study was to examine the analytic issues needed to develop a cocktail of marker drugs (fluconazole, rac-pindolol, para-aminohippuric acid, sinistrin) to measure simultaneously the mechanisms contributing to renal clearance. High-performance liquid chromatographic methods of analysis for fluconazole, pindolol, para-aminohippuric acid, and creatinine and an enzymatic assay for sinistrin were developed or modified and then validated to allow determination of each of the compounds in both plasma and urine in the presence of all other marker drugs. A pilot clinical study in one volunteer was conducted to ensure that the assays were suitable for quantitating all the marker drugs to the sensitivity and specificity needed to allow accurate determination of individual renal clearances. The performance of all assays (plasma and urine) complied with published validation criteria. All standard curves displayed linearity over the concentration ranges required, with coefficients of correlation greater than 0.99. The precision of the interday and intraday variabilities of quality controls for each marker in plasma and urine were all less than 11.9% for each marker. Recoveries of markers (and internal standards) in plasma and urine were all at least 90%. All markers investigated were shown to be stable when plasma or urine was frozen and thawed. For all the assays developed, there were no interferences from other markers or endogenous substances. In a pilot clinical study, concentrations of all markers could be accurately and reproducibly determined for a sufficient duration of time after administration to calculate accurate renal clearance for each marker. This article presents details of the analytic techniques developed for measuring concentrations of marker drugs for different renal elimination processes administered as a single dose to define the processes contributing to renal drug elimination.

Recent dioxin contamination from Agent Orange in residents of a southern Vietnam city

Marked elevation of dioxin associated with the herbicide Agent Orange was recently found in 19 of 20 blood samples from persons living in Bien Hoa, a large city in southern Vietnam. This city is located near an air base that was used for Agent Orange spray missions between 1962 and 1970. A spill of Agent Orange occurred at this air base more than 30 years before blood samples were collected in 1999. Samples were collected, frozen, and sent to a World Health Organization-certified dioxin laboratory fm congener-specific analysis as part of a Vietnam Red Cross project. Previous analyses of more than 2200 pooled blood samples collected in the 1990s identified Bien Hoa as one of several southern Vietnam areas with persons having elevated blood dioxin levels from exposure to Agent Orange. In sharp contrast to this study, our previous research showed decreasing tissue dioxin levels over time since 1970. Only the dioxin that contaminated Agent Orange, 2,3, 7, 8-tetrachlmodibenzo-p-dioxin (TCDD), was elevated in the blood of 19 of 20 persons sampled from Bien Hoa. A comparison pooled sample from 100 residents of Hanoi, where Agent Orange was not used, measured blood TCDD levels of 2 parts per trillion (ppt). TCDD levels of up to 271 ppt, a 135-fold increase, were found in Bien Hoa residents. TCDD contamination was also found in some nearby soil and sediment samples. Persons new to this region and children born after Agent Orange spraying ended also had elevated TCDD levels. This TCDD uptake was recent and occurred decades after spraying ended. We hypothesize that a major route of current and past exposures is from the movement of dioxin from soil into river sediment, then into fish, and from fish consumption into people.

Glass transition in Australian honeys

Differential scanning calorimetry (DSC) was used to study the glass transition in 10 Australian honeys by scanning at 10 degrees Cmin(-1) from -130 to 50 degreesC after annealing at -50 degreesC. The honeys had moisture contents 14.9 to 18.0%, seven were from Eucalyptus species. The glass transition temperatures (T-g) ranged from -46 degrees to -38 degreesC and were significantly (p

In situ zymography: topographical considerations

In situ gelatin zymography is a simple technique providing valuable information about the cellular and tissue localization of gelatinases. Until recently, the use of this technique has been confined to soft, relatively homogeneous tissue. In this report in situ zymography has been utilized to assess the sub-lamellar location of gelatinases in the hard, semi-keratinized epidermal layer and the adjacent soft connective tissue matrix of the dermis of the equine hoof. We show that alterations in the orientation at which the tissue is dipped and withdrawn from the emulsion cause profound alterations in emulsion thickness. Microscopic Variations in the surface topography of frozen tissue sections also influence emulsion thickness making interpretation of the results difficult. Given these results, researchers must be aware of potential variations in zymographic analysis may be influenced by physical tissue parameters in addition to suspected gelatinase activity. (C) 2001 Elsevier Science B.V. All rights reserved.

Detection of dengue viral RNA in Aedes aegypti (Diptera : Culicidae) exposed to sticky lures using reverse-transcriptase polymerase chain reaction

Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 mul of a 10(4) pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23-30 degreesC. Following 7,10,14, and 28 d application, 10 mosquitoes each were removed from the lure pooled and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7, 10, 14, 21 and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after application to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation.

Relationship of high energy expenditure and variation in dietary intake with reporting accuracy on 7 day food records and diet histories in a group of healthy adult volunteers

Objectives: To assess the accuracy of reporting from both a diet history and food record and identify some of the characteristics of more accurate reporters in a group of healthy adult volunteers for an energy balance study. Design: Prospective measurements in free-living people. Setting: Wollongong, Australia. Subjects: Fifteen healthy volunteers (seven male, eight female; aged 22 -59 y; body mass index (BMI) 19 - 33 kg/m(2)) from the local community in the city of Wollongong, Australia. Interventions: Measurement of energy intake via diet history interview and 7 day food records, total energy expenditure by the doubly labelled water technique over 14 days, physical activity by questionnaire, and body fat by dual-energy X-ray absorptiometry. Results: Increased misreporting of energy intake was associated with increased energy expenditure (r = 0.90, P < 0.0001, diet history; r(s)=0.79, P=0.0005, food records) but was not associated with age, sex, BMI or body fat. Range in number of recorded dinner foods correlated positively with energy expenditure (r(s)=0.63, P=0.01) and degree of misreporting (r(s)=0.71, P=0.003, diet history; r(s)=0.63, P=0.01, food records). Variation in energy intake at dinner and over the whole day identified by the food records correlated positively with energy expenditure (r=0.58, P = 0.02) and misreporting on the diet history (r=0.62, P=0.01). Conclusions: Subjects who are highly active or who have variable dietary and exercise behaviour may be less accurate in reporting dietary intake. Our findings indicate that it may be necessary to screen for these characteristics in studies where accuracy of reporting at an individual level is critical. Sponsorship: The study was supported in part by Australian Research Council funds made available through the University of Wollongong.

Improved detection of lacZ reporter gene expression in transgenic epithelia by immunofluorescence microscopy

The bacterial lacZ gene is commonly used as a reporter for the in vivo analysis of gene regulation in transgenic mice. However, several laboratories have reported poor detection of beta-galactosidase (the lacZ gene product) using histochemical techniques, particularly in skin. Here we report the difficulties we encountered in assessing lacZ expression in transgenic keratinocytes using classic X-gal histochemical protocols in tissues shown to express the transgene by mRNA in situ hybridization. We found that lacZ reporter gene expression could be reliably detected in frozen tissue sections by immunofluorescence analysis using a beta-galactosidase-specific antibody. Moreover, we were able to localize both transgene and endogenous gene products simultaneously using double-label immunofluorescence. Our results suggest that antibody detection of beta-galactosidase should be used to verify other assays of lacZ expression, particularly where low expression levels are suspected or patchy expression is observed.