Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.     

Air cleaners can reduce the risk of allergic sensitization

Exposure to house-dust mite allergens is a factor in the development of allergic symptoms in atopic patients. Several measures can be proposed to control indoor allergen levels, inducing a clinical benefit. The use of an air cleaner is one simple way of achieving this goal. We conducted a simulation trial in a proper room, to verify the usefulness of a domestic cleaner, based on mechanical air filtration, to reduce the levels of environmental allergens. We checked the presence of mite components by different methods (Aclotest and Der p 1 ELISA), in dust recovered before and after using the air cleaner. Our results indicate that this approach could be useful in significantly lowering the levels of mite allergens.     

Measurement of serum IgE antibodies against Japanese cedar pollen (Cryptomeria japonica) in Japanese monkeys (Macaca fuscata) with pollinosis.

IgE antibodies against allergens of Japanese cedar (Cryptomeria japonica, CJ) pollen in the serum of seven Japanese monkeys (Macaca fuscata) with pollinosis were measured by fluorometric indirect enzyme-linked immunosorbent assay (ELISA). All of the monkeys were found to have specific IgE to the crude pollen antigen. The specific IgE levels were well correlated with those determined by the Pharmacia CAP system. IgE antibodies were then assayed with two kinds of purified allergens (Cry j I and Cry j II) by the ELISA. We found that five monkeys had specific IgE to both allergens, although the other two had IgE only to Cry j I or Cry j II; there is different immune responsiveness to the two major allergens in the monkeys.     

Concentration of Marine Birnavirus from Seawater with a Glass Fiber Filter Precoated with Bovine Serum Albumin

In the present study an efficient method for sampling the marine birnavirus (MABV) gene from seawater was developed. MABV gene was monitored by a specific polymerase chain reaction. When Millipore filters were used, MABV was efficiently collected on a filter with 0.05-µm pore size. When both millipore and glass fiber filters were used, MABV was recovered from both filters. Use of plain glass fiber filters resulted in poor recovering efficiency. However, coating the glass fiber filters with 1% bovine serum albumin trapped MABV efficiently. Combining concentration on glass fiber filters with polymerase chain reaction is quantitative, economic and fast, suggesting that this method can be used to detect genetically identified fish disease viruses, algal viruses, and phages.     

Investigation of methods for measurement of radioactivity in tritiated DNA and applications to assays for DNA polymerase activity

Methods for collection and counting of ³H-labeled DNA on nitrocellulose and glass fiber filters have been investigated. The findings are of potential importance in determining the amount of radioactivity in DNA as well as other macromolecules. The highest counting efficiencies were observed using glass fiber filters, NCS for dissolving DNA, and a toluene seintillation mixture for counting. However, glass fiber filters, even with large amounts of co-precipitant albumin, failed to collect all of low molecular weight (approx 185,000 daltons) DNA. Thus, in many applications nitrocellulose filters proved to be more advantageous.     

High resolution X-ray structures of mouse major urinary protein nasal isoform in complex with pheromones

In mice, the major urinary proteins (MUP) play a key role in pheromonal communication by binding and transporting semiochemicals. MUP‐IV is the only isoform known to be expressed in the vomeronasal mucosa. In comparison with the MUP isoforms that are abundantly excreted in the urine, MUP‐IV is highly specific for the male mouse pheromone 2‐sec‐butyl‐4,5‐dihydrothiazole (SBT). To examine the structural basis of this ligand preference, we determined the X‐ray crystal structure of MUP‐IV bound to three mouse pheromones: SBT, 2,5‐dimethylpyrazine, and 2‐heptanone. We also obtained the structure of MUP‐IV with 2‐ethylhexanol bound in the cavity. These four structures show that relative to the major excreted MUP isoforms, three amino acid substitutions within the binding calyx impact ligand coordination. The F103 for A along with F54 for L result in a smaller cavity, potentially creating a more closely packed environment for the ligand. The E118 for G substitution introduces a charged group into a hydrophobic environment. The sidechain of E118 is observed to hydrogen bond to polar groups on all four ligands with nearly the same geometry as seen for the water‐mediated hydrogen bond network in the MUP‐I and MUP‐II crystal structures. These differences in cavity size and interactions between the protein and ligand are likely to contribute to the observed specificity of MUP‐IV.     

Phthalocyanine-dyed fibers adsorb allergenic proteins

Summary. Phthalocyanine (Pc)-dyed fiber is reported to reduce atopic symptoms in some patients when they use underwear made of the fiber. We investigated the adsorption of allergens on Pc-fiber. Pc-fiber trapped house dust/pollen/food allergens with varied molecular weight and pI. The adsorbed allergens were released in the presence of mild detergent. Pc-fiber did not change the molecular weight or disulfide bonding of the allergens. These observations imply that Pc-fiber is applicable as an “allergen trap” for a wide variety of products.     

Effectiveness of benzyl benzoate in elimination of house dust mites

The effect of elimination treatment with benzyl benzoate was examined in 30 adults with asthma caused by sensitivity to house dust mites. The patients were randomised into a control and an active group, who treated their mattresses with benzyl benzoate (Acarosan^®). The study lasted 12 months and the effect of the treatment was monitored by monthly dust sampling, analyzed for major allergens fromDermatophagoides pteronyssinus andfarinae by the ELISA method and for guanine by the Acarex^® method. The clinical effect was assessed by measuring lung function, daily peak flows, symptoms and medicine consumption as well as skin prick tests, and specific IgE to mite allergens. For both groups, a significant decrease was observed in house dust mite allergens, but there was no significant difference between the groups. No considerable differences were observed in clinical parameters within or between the groups. A good correlation was observed between ELISA and Acarex^®. but the latter showed a major variation in the different classes. In conclusion. treatment of mattresses with benzyl benzoate had no effect in a group of patients with asthma due to house dust mite allergy. Regular vacuum cleaning of the bed may reduce house dust mite allergen exposure.     

Major house dust mite allergens Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1 degrade and inactivate lung surfactant proteins A and D

Lung surfactant proteins (SP) A and D are calcium-dependent carbohydrate-binding proteins. In addition to playing multiple roles in innate immune defense such as bacterial aggregation and modulation of leukocyte function, SP-A and SP-D have also been implicated in the allergic response. They interact with a wide range of inhaled allergens, competing with their binding to cell-sequestered IgE resulting in inhibition of mast cell degranulation, and exogenous administration of SP-A and SP-D diminishes allergic hypersensitivity in vivo. House dust mite allergens are a major cause of allergic asthma in the western world, and here we confirm the interaction of SP-A and SP-D with two major mite allergens, Dermatophagoides pteronyssinus 1 and Dermatophagoides farinae 1, and show that the cysteine protease activity of these allergens results in the degradation of SP-A and SP-D under physiological conditions, with multiple sites of cleavage. A recombinant fragment of SP-D that is effective in diminishing allergic hypersensitivity in mouse models of dust mite allergy was more susceptible to degradation than the native full-length protein. Degradation was enhanced in the absence of calcium, with different sites of cleavage, indicating that the calcium associated with SP-A and SP-D influences accessibility to the allergens. Degradation of SP-A and SP-D was associated with diminished binding to carbohydrates and to D. pteronyssinus 1 itself and diminished capacity to agglutinate bacteria. Thus, the degradation and consequent inactivation of SP-A and SP-D may be a novel mechanism to account for the potent allergenicity of these common dust mite allergens.     

Evaluation of countermeasures for reduction of mouse airborne allergens

Three kinds of countermeasures for reduction of mouse airborne allergens were evaluated with use of an air sampler and immunochemical methods. Mouse cages and the sampler were placed inside a flexible-film isolator, and concentrations of mouse major allergens in the air were measured. The levels of the airborne allergens, prealbumin and albumin, generated by 10 males, were 3,050 and 492 pg/m3, respectively. Those by 10 females were lower, 317 and 270 pg/m3, respectively. When mouse cages were covered with a filter cap, the airborne allergens inside the isolator were decreased by 90%. When corncob was used as bedding in place of wood shavings, the airborne allergens were decreased by 57 and 77%, respectively. Therefore, for reduction of mouse airborne allergens, we recommend using female mice, covering the cages with filter caps, and using corncob bedding.     

Improving the suitability of glass fiber filters for use as culture supports

Summary Commercially available glass fiber filters are useful as physical supports for cultures; however, as received from the manufacturers, the filters frequently contain substances that render them unsuitable for some types of experimental studies. These substances contribute to the formation of precipitates in the culture media, alter the media pH, and repress synchronous development among embryo cultures of eastern white pine (Pinus strobus L.). A simplified technique was developed to remove the contaminants and to saturate the cation exchange sites on the glass fibers with specific ions. The use of trade, product, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture or the Forest Service of any product or service to the exclusion of others that also may be suitable.     

Influence of various dust sampling and extraction methods on the measurement of airborne endotoxin.

The influence of various filter types and extraction conditions on the quantitation of airborne endotoxin with the Limulus amebocyte lysate test was studied by using airborne dusts sampled in a potato processing plant. Samples were collected with an apparatus designed to provide parallel samples. Data from the parallel-sampling experiment were statistically evaluated by using analysis of variance. In addition, the influence of storage conditions on the detectable endotoxin concentration was investigated by using commercially available lipopolysaccharides (LPS) and endotoxin-containing house dust extracts. The endotoxin extraction efficiency of 0.05% Tween 20 in pyrogen-free water was seven times higher than that of pyrogen-free water only. Two-times-greater amounts of endotoxin were extracted from glass fiber, Teflon, and polycarbonate filters than from cellulose ester filters. The temperature and shaking intensity during extraction were not related to the extraction efficiency. Repeated freeze (-20 degrees C)-and-thaw cycles with commercial LPS reconstituted in pyrogen-free water had a dramatic effect on the detectable endotoxin level. A 25% loss in endotoxin activity per freeze-thaw cycle was observed. Storage of LPS samples for a period of 1 year at 7 degrees C had no effect on the endotoxin level. House dust extracts showed a decrease of about 20% in the endotoxin level after they had been frozen and thawed for a second time. The use of different container materials (borosilicate glass, "soft" glass, and polypropylene) did not result in different endotoxin levels. This study indicates that the assessment of endotoxin exposure may differ considerably between groups when different sampling, extraction, and storage procedures are employed.     

Efficacy of Selected Respiratory Protective Equipment Challenged with Bacillus subtilis subsp. niger

The efficacy of powered air-purifying respirators, surgical masks, dust/mist respirators, and high-efficiency respirators were tested with a biological aerosol under simulated breathing conditions. Protective ability ranged from 67 to 99.95%. The majority of penetration in negative-pressure respirators occurred at the face-mask interface rather than through the filter material.     

A comparison of airborne and dust-borne allergens and toxins collected from home,office and outdoor environments both in New Haven,United States and Nanjing,China

In this study, the airborne and dust-borne concentrations of endotoxin, (1,3)-β-d-glucan and five house dust allergens were measured in office, home, and outdoor environments both in New Haven, United States and Nanjing, China. Air samples were collected using a BioSampler at a flow rate of 12.5 l/min for 30 min. Dust samples were simultaneously collected using a surface sampler. Dust samples went through extraction and dilution before analysis, while air samples were analyzed directly. Limulus Amoebocyte Lysate (LAL) Pyrochrome and Glucatell assays were used to quantify endotoxin and (1,3)-β-d-glucan concentration levels, respectively. Enzyme-linked sorbent assay was used to measure the dust mites, cat, dog, and cockroach allergens. The experimental results indicated that endotoxin, (1,3)-β-d-glucan and allergen concentrations vary greatly both with samples and environments. In all tested environments, endotoxin concentration ranged from 0.8 to 83.7 ng/m³ for air, and 7.8 to 14.3 ng/mg for dust. (1,3)-β-d-glucan concentration ranged from 0.1 to 9.8 ng/m³ for air, and 6.6 to 110 ng/mg for dust. Cockroach allergens were detected only in New Haven office and outdoor environments, and other allergens ranged from 0.1 to 90 ng/mg for dust samples, and from 1.5 to 1,282 ng/m³ for air samples. In general, similar profiles of allergens and toxins were observed in New Haven and Nanjing environments. Linear regression analysis showed that there were better endotoxin and (1,3)-β-d-glucan linear correlations (R ² = 0.78, 0.87, respectively) between the dust and air samples compared to those of the allergens Der f 1 and Der p 1 (R ² = 0.5, 0.7, respectively). This research contributes to the development of robust biological exposure assessment and the elaboration of airborne and dust-borne bio-mass in the living environments.     

Use of electroblotting as a method for preparing samples of proteins and their fragments for microsequencing

Gel electrophoresis in the presence of sodium dodecyl sulphate followed by electroblotting was employed in sample preparation for microsequencing proteins and protein fragments. Three types of solid supports were compared: glass fiber filters modified by aminopropyltriethoxysilane or covered with polybrene, and polyvinylidenedifluoride membranes. N-Terminal amino acid sequences of several proteins (Mr 14-140 kDA were determined on a gas-phase sequencer with the standard programme; 20-200 pmoles of the protein can be assayed by this method.     

Structural genes of the mouse major urinary protein are on chromosome 4

The major urinary proteins (MUPs) of mouse are a family of at least three major proteins which are synthesized in the liver of all strains of mice. The relative levels of synthesis of these proteins with respect to each other in the presence of testosterone is regulated by the Mup-a locus located on chromosome 4. In an effort to determine the mechanism of this regulation in molecular terms, a cDNA clone containing most of the coding region of a MUP protein has been isolated and identified by partial DNA sequence analysis. Using a combination of hybridization analysis and somatic cell genetics, the structural gene family has been unambiguously mapped to mouse chromosome 4. These data suggest that Mup-a regulation operates in a cis fashion and that models proposing trans regulation of MUP protein synthesis are unlikely.     

Immunochromatographic direct on sampling filter test for aeroallergens

An immunochromatographic method for qualitative and quantitative determination of aeroallergens direct on sampling (ADOS) filters has been developed. In this method, a porous polytetrafluoroethylene filter carrying adsorbed allergens is fixed by double-coated adhesive tape to a supporting filter paper matrix. Following addition of antibodies specific for the relevant allergens and washing and staining reagents via a reagent applicator an immunochromatogram is developed resulting in a 5-10 mm wide area of the sample filter covered with blue-violet-stained spots appearing on a faintly pink or white background. The method takes 30 to 90 min, depending on the nominal porosity (1.2-5 microm) and the defined reaction area (5-10 mm) of the sample filter. Application experiments with birch and grass pollen, soluble Bet v 1, Phl p 5 and mould allergens as well as cat allergen carried by airborne dust revealed a limit of detection of a few picograms of allergen as stained spots. The specificity of the new method to evaluate the type of allergen is a function of the selected antibodies. The concentrations of the allergen in an air sample are related to the number and intensity of stained spots.     

Monitoring of bacterial sugars and hydroxy Fatty acids in dust from air conditioners by gas chromatography-mass spectrometry

Bacterial levels in dust collected from hospital air-conditioning filters were examined by chemical analysis (without prior culture). The dust was analyzed by gas chromatography-mass spectrometry after hydrolysis and derivatization. l-Glycero-d-mannoheptose and hydroxy fatty acids (3-OH 12:0 and 3-OH 14:0) (primarily found in lipopolysaccharide) and muramic acid (a chemical marker for bacterial peptidoglycan) were present at higher levels in dust collected from filters primarily contacting outdoor (as opposed to indoor) air. The ratio of l-glycero-d-mannoheptose to muramic acid in dust (compared with those of a group of gram-positive and gram-negative bacteria) suggested that both dust types contained appreciable numbers of gram-negative bacteria. There is potential for the chemical assessment of the microbial content of airborne dust.     

The gene family for major urinary proteins: Expression in several secretory tissues of the mouse

The major urinary proteins (MUPs) of the mouse are encoded by a multigene family located at the Mup a locus on chromosome 4. Previous investigations have shown that the MUPs are synthesized in the liver, secreted and then excreted in the urine. We have found significant levels of MUP mRNA in several secretory tissues: the liver and the submaxillary, lachrymal and mammary glands. There are striking differences in hormonal and developmental regulation of MUP gene expression in these tissues. Furthermore, each tissue appears to express a characteristic pattern of MUP mRNAs. In particular, the lachrymal glands appear to express an entirely different set of MUP mRNAs. These results are discussed in relation to the organization of the MUP gene cluster and a possible function of the MUPs.     

Identification and characterization of functional genes encoding the mouse major urinary proteins.

Mouse Ltk- cells were stably transfected with cloned genes encoding the mouse major urinary proteins (MUPs). C57BL/6J MUP genomic clones encoding MUP 2 (BL6-25 and BL6-51), MUP 3 (BL6-11 and BL6-3), and MUP 4 (BL6-42) have been identified. In C57BL/6J mice, MUP 2 and MUP 4 are known to be synthesized in male, but not female, liver, and MUP 3 is known to be synthesized in both male and female liver and mammary gland. A BALB/c genomic clone (BJ-31) was shown to encode a MUP that is slightly more basic than MUP 2 and was previously shown to be synthesized in both male and female liver of BALB/c but not C57BL/6 mice. Comigration on two-dimensional polyacrylamide gels of the MUPs encoded by the transfecting gene provides a basis for tentative identification of the tissue specificity and mode of regulation of each gene. DNA sequence analysis of the 5' flanking region indicates that the different MUP genes are highly homologous (0.20 to 2.40% divergence) within the 879 base pairs analyzed. The most prominent differences in sequence occur within an A-rich region just 5' of the TATA box. This region (from -47 to -93) contains primarily A or C(A)N nucleotides and varies from 15 to 46 nucleotides in length in the different clones.