The comparative characteristics of different forms of allergens from mites of the genus Dermatophagoides]

The comparative study of allergenic preparations of Dermatophagoides mites [correction of ticks] from different manufacturers and the international standard preparations of such allergens was made. D. pteronyssinus and D. farinae allergens from the USSR and the Netherlands were studied. The samples under study were evaluated in a complex of in vivo and in vitro experiments. The preparations produced in the USSR and in the Netherlands exhibited pronounced specificity, but the allergens of both species from the Netherlands were more active. Some differences in the immunochemical characteristics of the preparations were noted.     

Sampling dust from human dwellings to estimate the prevalence ofDermatophagoides mite and cat allergens

Summary Quantification of specific allergens in household dust samples may provide important information for selecting appropriate allergen control methods, and monitoring efficacy and compliance. The purpose of this study was to investigate the source of variation in mite and cat allergen measurements associated with dust sample collection. Discrete and composite dust samples were collected on a filter using a special vacuum sampling device. Aqueous extracts of the dust samples were prepared thenDer p I,Der f I, andFel d I were quantitated by enzyme immunoassays (EIA). Mite and cat allergens were frequently detected in dust samples from human dwellings, and the amounts of these allergens varied significantly (p<0.01) among dwellings. The differences of allergen measurements among duplicate samples taken immediately and up to three weeks later appear to be much smaller than differences among houses and between rooms. Variation among dust samples taken from living rooms and bedrooms of the same dwelling suggest differences in allergen reservoirs. Composite samples formed by sampling specific objects within a room may provide a reliable estimate of allergen exposure in that room. Dust samples from discrete objects are useful to find and monitor specific reservoirs of mite and cat allergens.     

Recombinant allergens for analysing T-cell responses

T-cell responses constitute a central element of allergic disease and a model for studying Th1 and Th2 cytokine pathways. Most studies to date have used extracts of allergens which contain variable quantities of different allergens and non-allergenic antigens. Recombinant allergens provide the tools for studying the responses to allergens in a reproducible and dose-dependent manner and the different T-cell responses of allergic and non-allergic subjects provide a method for verifying the responses and their relationship to allergic sensitisation. Most allergies show dominant responses to one or a few major allergens. These allergens have been described for the common allergies and have been produced as recombinant allergens. A particular problem for allergens is that many are mixtures of proteins from multi-gene families or are highly polymorphic. Information now exists so the sequence variation can be represented. Purified recombinant allergens produced by standard expression systems stimulate the expected T-cell responses from the peripheral blood of allergic and non-allergics to allergen extracts. Although stimulation with recombinant allergens which are not produced with a natural IgE binding activity can provide a measure of allergenicity, the altered tertiary structure can reduce Th2 responses. The sequence information now available provides the means to use PCR to produce cDNA for the production of recombinant allergens from readily available sources. The production of the highly reactive recombinant Der p 2 allergen of house dust mite from natural sources is described.     

Immunological identification and characterization of individual food allergens

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

Evaluation of the biological activity of allergens by different in vivo and in vitro immunological methods

The specific activity of different allergens has been studied in vivo and in vitro by certain immunological methods (complement inactivation, mastocyte degranulation, neutrophil damage index, lymphocyte blast transformation). The results obtained by the skin allergic tests have been found to correlate well with those obtained by the in vitro methods. This allows one to use these methods for a more complete characterization of allergens and their standardization.     

Early Exposure to Respiratory Allergens by Placental Transfer and Breastfeeding

The relationship between allergen exposure and the onset of or protection from allergic diseases remains unclear. Many factors could be related to immunological responses, such as the age when the exposure occurs, type of allergen, timing, dose, and allergen route. In this study, we investigated whether exposure to respiratory allergens could occur in pregnancy or early life. In particular, we assessed whether Der p 1 and Blo t 5, as well as specific antibodies against these allergens, could be detected in 90 paired cord blood and colostrum samples. Der p 1 was detected in 58.6% of colostrum and 29% of cord blood samples, whereas Blot 5 was positive in 41.3% and 9.6% of the samples, respectively. Similar to specific IgA, which could be detected in all samples for both mites, specific IgG was found in a high number of colostrum samples, 93.5% and 94.8% for Dp and Bt, respectively. Although allergens were not detected in all cord blood samples, a high percentage of them (≥95%) were positive for specific IgM to both mites in cord blood samples, suggesting that neonates can be exposed and sensitized to airborne allergens during pregnancy. Many studies have attempted to correlate allergen exposure or its prevention in early infancy with the onset of or protection from allergic diseases. However, conflicting and inconsistent data do not show a clear correlation with or suggest a way to prevent allergen sensitization. Nevertheless, these unconvincing results could be better understood if the relationship with many aspects of allergen exposure after pregnancy could be clarified. Thus, it is necessary to address basic issues related to allergen exposure, including the development of reproducible, standardized and reliable methods, and to determine how and where the exposure occurs.     

A comparative study of the expediency of using different chemical methods for determining protein in allergen preparations]

Parallel use of some known chemical procedures for measurements of protein in commercial forms of allergens revealed considerable variations in the results, which was due to the nature of the methods used and to admixtures of different substances in the preparations under study. The conclusion on the necessity of using at least two methods of protein determination for the standardization of allergens was made. Preference was given to Bradford's method. As the standardization of allergens manufactured in the USSR is carried out on the basis of the quantitative determination of protein (in PNU), this problem is of not only theoretical, but also practical importance.     

Comparative characteristics of the diagnostic properties of staphylococcal allergens in an experiment

The diagnostic value of five staphylococcal allergens prepared from a single S. aureus strain by different methods and in different institutions has been tested on the experimental models of delayed, immediate and mixed (immediate and delayed) hypersensitivity in guinea pigs. The advantages of the allergens prepared in Kazan (USSR) for the detection of delayed hypersensitivity and the ultrasonicated allergen, as well as the allergen made in Czechoslovakia, for the detection of immediate hypersensitivity have been noted.     

Contact allergy and sociodemographic characteristics

The aim of the study was to determine the frequency of positive patch test reaction to different contact allergens according to patients age, sex, occupation and clinical features. Between 1999 and 2003, patch testing was performed in 3,293 patients with respective clinical diagnoses. Patch testing was done by the standard technique proposed by the International Contact Dermatitis Research Group (ICDRG). Study results showed statistically significant differences in patch test response according to sex and age for three allergens (cobalt chloride, nickel sulphate and thiomersal); according to occupation for nine allergens (cobalt chloride, nickel sulphate, balsam of Peru, fragrance mix, thiuram mix, wood tars, neomycin sulphate, thiomersal and detergents), and clinical diagnosis for two allergens (nickel sulphate, and wood tars). The most common and relevant allergens were: nickel sulphate, cobalt chloride and carba mix. They were found in all examinees regardless of age, sex, occupation and diagnoses. The increased awareness of allergens and their potential sources may help to limit the usage of these chemicals in manufacture of consumer products.     

Analytical methodology for assessment of food allergens: opportunities and challenges

This review summarizes the available in vitro, in vivo, and informatic methods designed to evaluate different aspects of the capacity of proteins to act as true food allergens. By now, there is no single method to fully assess the potential allergenicity of proteins. The characterization of many food allergens will help to uncover the sequential and structural motifs that determine the behaviour of proteins as food allergens.     

Isolation of food allergens

The identification of food allergens is a priority in the management of food allergy, because of the need to obtain standardized extracts and pure allergens for diagnosis and therapy. It is thus important to develop methods for purification of allergenic molecules in order to study their biological and immunological characteristics. Protocols for protein extraction from foods and for allergen purification are reviewed in this paper. We report published methods for extraction of allergens from either animal and vegetable foods and detailed purification methodologies including ion-exchange, gel filtration and reversed-phase chromatography of well known allergens.     

Computational analysis of the relationship between allergenicity and digestibility of allergenic proteins in simulated gastric fluid

Background  

Safety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types.     

Quantitation of latex allergens

Minimizing allergen concentration in latex goods to prevent sensitization to natural rubber latex (NRL) and thereby the development of clinical allergy is acknowledged as of mutual interest for rubber manufacturers and regulatory health authorities. However, measuring total protein, the principal currently available method, cannot be deemed a satisfactory regulatory measure to control allergen content. Specific methods based on human IgE-containing reagents, such as radioallergosorbent test (RAST) inhibition, have been available in certain laboratories for demonstrating NRL allergens in rubber products but the methods lack standardization. Currently, one commercial test has become available for measuring individual NRL allergens by capture ELISA-based assays using monoclonal antibodies and purified or recombinant allergens. Such methods are specific, they can be properly standardized, and they are of sufficient sensitivity and reproducibility. Results from medical gloves collected in two national market surveys in Finland in 1995 and 1999, respectively, show that Hev b 6.02 and Hev b 5, the two major allergens for NRL-allergic adults, are the most abundant allergens regularly detectable in high- and moderate-allergen gloves. In addition, Hev b 3 and Hev b 1, the two major allergens for children with spina bifida, are also commonly found. In general, when the sum of the four allergens exceeded 1 microg/g, most NRL-allergic patients showed positive skin prick test reactions against them. Using these new methods assessment of threshold levels that could in due course become guidelines for the rubber industry and regulatory health authorities is becoming possible. Eventually, this progress is expected to lead to a declining incidence of latex allergy.     

Two-dimensional electrophoresis replica blotting: a valuable technique for the immunological and biochemical characterization of single components of complex extracts

The combination of the high resolution electrophoresis (2-DE) with subsequent transfer onto a protein-binding membrane (blotting), immunological detection, and/or N-terminal sequencing is a powerful tool to identify and characterize single components of complex protein mixtures. Direct comparison of protein staining, immunological detection, and biochemical characterization of single protein spots was achieved by the replica blotting technique. The proteins were transferred from one two-dimensional gel onto several blotting membranes one after another. A canon of methods has been employed to identify and characterize allergens from different allergen sources. We have studied single major allergens as well as related major allergens from different grass species ("allergen groups") using patients' sera and allergen-specific monoclonal antibodies. The biochemical structure of the allergenic components has been analyzed by N-terminal and internal protein sequencing, precise mass determinations by matrix-assisted laser desorption/ionization mass spectrometry and investigations on post-translational modifications such as glycosylation. Here, we give a general survey of methods, and we describe an array of techniques suitable for characterization and identification of components of complex extracts, even if there is little or no previous information available.     

On The History of Soviet Military Psychology

Military psychology is a branch of psychology, which is in addition included among the military sciences. In it the psychological features of different kinds of military and, above all, of combat activity are studied in relation to social and historical conditions, military technology, personal qualities of the fighting men, and methods of military and political preparation. The general purpose of Soviet military psychology is to discover principles of human mental activity under the conditions of combat and of training for combat, and to use these principles for raising the fighting capacity of the Soviet armed forces.     

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.     

Use of a coagglutination reaction based on a Soviet staphylococcal reagent for serogrouping Streptococcus group B and meningococci

The coagglutination (COA) test is used for the serogrouping of streptococci, group B, and meningococci. The trial of a Soviet commercial preparation (staphylococcal reagent) has shown good prospects for its use in the COA test and high specificity of this test method. The availability of highly specific immune sera permits making standard kits for the identification of different infective agents in the COA test.     

SU-8 based microprobes with integrated planar electrodes for enhanced neural depth recording

A gold nanoparticle, localised plasmon array biosensor using light scattering has been employed in the detection of allergen-specific antibodies in whole blood and sera. The array sensor was functionalized with four different allergens, cat dander (Fel d1), dust mite (Der p1), peanut allergen (Ara h1) and dog dander (Can f1) and immuno-kinetic assay was performed to detect their respective anti-allergen IgG antibodies. Specific positive responses to antibodies at a concentration of 25 nM were observed for Fel d1, Der p1, and Ara h1 allergens, while the Can f1 channel served as a reference control. The sensitivity was further enhanced using a secondary anti-IgG detection antibodies to give a limit of detection of 2 nM. The results indicate the potential for nanoparticle scattering multiplexed arrays to screen unprepared blood samples at point-of-care for assays of complex samples such as the whole blood.     

Animal models to test respiratory allergy of low molecular weight chemicals: a guidance

At present, there are no widely applied or fully validated test methods to identify respiratory LMW allergens, i.e. compounds that are considered capable of inducing allergic asthma. Most tests have been investigated using strong respiratory allergens. Moreover, they are meant to detect the potential of a chemical to induce respiratory sensitisation at relatively high doses. Consequently, the sensitivity of the tests is not well-known, and they do not provide information on low doses such as generally found in occupational situations, and on threshold levels to be used in risk assessment. In addition, the various test methods use different application routes, i.e. intradermal, topical or inhalation exposure, and different parameters. Therefore standardised and validated dose-response test methods are urgently required in order to be able to identify respiratory allergens and to recommend safe exposure levels for consumers and workers. In the present paper, methods or testing strategies are described to detect respiratory sensitisation and/or allergy. Overall, assays that utilize only an induction phase may serve as indicators of respiratory sensitisation potential whereas assays that use both an induction and an elicitation or challenge phase may provide information on potency and presence of thresholds. The dermal route as sensitisation route has the advantage of the respiratory tract not being exposed to the allergen prior to challenge which facilitates the distinction between irritant and allergic effects.     

Bayesian analysis of serial dilution assays

In a serial dilution assay, the concentration of a compound is estimated by combining measurements of several different dilutions of an unknown sample. The relation between concentration and measurement is nonlinear and heteroscedastic, and so it is not appropriate to weight these measurements equally. In the standard existing approach for analysis of these data, a large proportion of the measurements are discarded as being above or below detection limits. We present a Bayesian method for jointly estimating the calibration curve and the unknown concentrations using all the data. Compared to the existing method, our estimates have much lower standard errors and give estimates even when all the measurements are outside the "detection limits." We evaluate our method empirically using laboratory data on cockroach allergens measured in house dust samples. Our estimates are much more accurate than those obtained using the usual approach. In addition, we develop a method for determining the "effective weight" attached to each measurement, based on a local linearization of the estimated model. The effective weight can give insight into the information conveyed by each data point and suggests potential improvements in design of serial dilution experiments.