Reproduction and development of laboratory and wild house dust mites (Acari: Pyroglyphidae) and their relationship to the natural dust ecosystem.

Life histories of "wild" house dust mites, Dermatophagoides pteronyssinus (Trouessart) (Acari: Pyroglyphidae), were compared with laboratory cultures by using a diet consisting of skin and dust or a laboratory diet consisting of dried liver and yeast. Under constant conditions of 25 degrees C and 75% RH, fecundity and rate of reproduction were higher in laboratory cultures on both diets compared with wild mites. There were also trends for a shorter prereproductive period and more rapid egg development of laboratory mites compared with wild mites. Overall, there was little effect of diet on either strain of mites at 75% RH. At low RH (64%), fecundity was significantly lower (for both strains on both diets), and there were also trends for longer prereproductive period, reduced rate of reproduction, reduced adult survival, prolonged egg and juvenile development, or a combination compared with 75% RH. Additionally egg and juvenile mortality were significantly higher on the liver and yeast diet. Overall, the skin and dust diet favored both strains of mites at 64% RH. On the liver and yeast diet at 64% RH, wild mite adults performed significantly better than laboratory mites, and egg mortality was lower. These results suggest that laboratory mites have stronger reproduction and development than wild mites, except when under environmental stress and that diet is a significant factor, particularly in suboptimal conditions. This could have important implications for predictive models of house dust mite populations in their natural habitat. Ideally, such models should be developed using data from wild dust mite populations reared on a natural diet.

Right ventricular oxygen supply/demand balance in exercising dogs.

This is the first investigation of right ventricular (RV) myocardial oxygen supply/demand balance in a conscious animal. A novel technique developed in our laboratory was used to collect right coronary (RC) venous blood samples from seven instrumented, conscious dogs at rest and during graded treadmill exercise. Contributions of the RV oxygen extraction reserve and the RC flow reserve to exercise-induced increases in RV oxygen demand were measured. Strenuous exercise caused a 269% increase in RV oxygen consumption. Expanded arteriovenous oxygen content difference (A-V(Delta)O2) provided 58% of this increase in oxygen demand, and increased RC blood flow (RCBF) provided 42%. At less strenuous exercise, expanded A-V(Delta)O2 provided 60-80% of the required oxygen, and increases in RCBF were small and driven by increased aortic pressure. RC resistance fell only at strenuous exercise after the extraction reserve had been mobilized. Thus RC resistance was unaffected by large decreases in RC venous PO2 until an apparent threshold at 20 mmHg was reached. Comparisons of RV findings with published left ventricular data from exercising dogs demonstrated that increased O2 demand of the left ventricle is met primarily by increasing coronary flow, whereas increased O2 extraction makes a greater contribution to RV O2 supply.

Hyperlipidemia with hypoglycemia reduces myocardial oxygen utilization efficiency but not contractile function during coronary hypoperfusion.

This study was designed to determine changes in myocardial contractile function and fuel selection during moderate coronary hypoperfusion in the presence of elevated plasma free fatty acid (FFA) at normal and reduced blood glucose concentrations. Coronary perfusion pressure (CPP) was sequentially lowered from 100 to 60, 50, and 40 mmHg in the left anterior descending coronary artery (LAD) of anesthetized, open-chest dogs. Regional glucose uptake (GU), fatty acid uptake (FAU), percentage segment shortening (%SS), and oxygen consumption (MV O(2)) were determined with normal arterial plasma FFA concentrations (Group 1) or with elevated FFA concentrations (Groups 2 and 3). In Group 3, glucose in the coronary perfusate blood was reduced from 3.53+/-0.36 to 0.15+/-0.03 m M by hemodialysis. In Group 1, FAU fell by 85% as CPP was lowered to 60 mmHg and remained depressed as CPP was reduced further; GU did not fall significantly. Hyperlipidemia in Group 2 did not alter GU at any CPP, but maintained FAU at baseline levels until CPP was lowered to 40 mmHg. At 40 mmHg CPP, myocardial function and metabolic variables were similar in Groups 1 and 2. In Group 3 at 40 mmHg, FAU increased four-fold and MV O(2)doubled v Groups 1 and 2, and GU fell to zero. Despite these metabolic changes, %SS in Group 3 was unchanged relative to Group 2. Addition of glucose to the dialysate prevented the effects of dialysis on FAU, GU, and MV O(2). Thus, preferential glucose oxidation sustains myocardial oxygen utilization efficiency [(heart rate x %SS x maximum left ventricular pressure)/MV O(2)] during hypoperfusion. Blocking preferential glucose oxidation by combined hyperlipidemia and hypoglycemia lowers oxygen utilization efficiency, but does not compromise myocardial contractile function.

High-molecular-weight allergens of the house dust mite: an apolipophorin-like cDNA has sequence identity with the major M-177 allergen and the IgE-binding peptide fragments Mag1 and Mag3.

BACKGROUND: A 349-residue recombinant polypeptide of Dermatophagoides farinae, Mag 3, has been shown to represent part of a larger 177-kD (M-177) allergen with very high IgE-binding activity. METHODS: Cloning and sequencing of cDNA from the house dust mites Dermatophagoides pteronyssinus and Euroglyphus maynei was used to characterise the polypeptide containing the Mag 3 sequence. RESULTS: cDNA clones containing the complete sequence of the E. maynei homologue of the M-177 allergen were isolated and analysed. The translation contained not only an amino acid sequence with 90% identity to the 349-residue Mag-3 fragment but also a further sequence with 90% identity to another IgE-binding recombinant D. farinae polypeptide designated Mag 1. The complete sequence encoded a mature polypeptide of 1,650 residues and a molecular mass of 189.5 kD. cDNA clones from D. pteronyssinus also encoded sequences equivalent to the Mag 1 and 3 polypeptides. The M-177 sequence showed strong similarity to the lipid transport apolipophorins found in insect lipophorins. CONCLUSIONS: cDNA sequence data show that the D. pteronyssinus and E. maynei homologues of the M-177 high-molecular-weight D. farinae allergen contain sequences equivalent to both the Mag 1 and Mag 3 recombinant IgE-binding fragments. The N-terminal sequence of the full-length 1,650 amino-acid allergen showed strong similarity to the insect apolipophorins which are poorly soluble in aqueous extracts and exist in the lipid transport particles in haemolymph. It is proposed that presentation in lipid particles could be a factor which enhances the immunogenicity of this group of allergens.

Molecular characterization of the group 4 house dust mite allergen from Dermatophagoides pteronyssinus and its amylase homologue from Euroglyphus maynei.

BACKGROUND: Of the ten recognised groups of Dermatophagoides pteronyssinus allergens, the group 4 is the only group that has not been characterised at the molecular level. METHODS: Primers were designed to PCR amplify Der p 4 (D. pteronyssinus) and Eur m 4 (Euroglyphus maynei) cDNA. These fragments were used to screen the corresponding cDNA libraries and the cDNA clones obtained were subsequently sequenced. The coding regions of Der p 4 and Eur m 4 were cloned into the pET expression vector and recombinant histidine-tagged proteins expressed in Escherichia coli. RESULTS: cDNA clones which included the mature protein coding sequence for Der p 4 and Eur m 4 were sequenced. The Der p 4 and Eur m 4 genes were found to code for 496 amino acid mature proteins with residues important for the function of alpha-amylase highly conserved. Der p 4 and Eur m 4 were calculated to be 90% identical and a BLAST search of the GenBank database found these sequences to be approximately 50% identical to insect and mammalian alpha-amylases. The calculated molecular weights of Der p 4 and Eur m 4 were approximately 57,000, although recombinant Der p 4 and Eur m 4 migrate on SDS-PAGE at about 60,000. Der p 4 recombinant protein was found to bind specific IgE in 3 of the 10 house dust mite allergic patients tested. CONCLUSIONS: This paper describes the first cDNA sequence of Der p 4 and Eur m 4 confirming that this allergen is house dust mite alpha-amylase.

A method for producing regional hypoglycemia in blood perfused tissue.

Numerous studies have focused on the metabolic contributions of glucose and other substrates in isolated tissue preparations by examining the effects of eliminating glucose from the physiologic perfusate or bath solution. To date, however, an effective method of glucose removal from the blood supply to selected tissue in the whole animal model has not been available. We have developed a method for blood glucose removal by continuous flow dialysis. This method was used to generate isolated coronary hypoglycemia for an investigation of myocardial metabolic substrate selection during hypoperfusion in open-chest, anesthetized dogs. Arterial blood was passed through the dialysis system against an isotonic and physiologic dialysate solution prior to controlled coronary perfusion. During normal perfusion pressure (100 mmHg), with a coronary blood flow of 32+/-4 ml/min, arterial blood glucose was reduced from 3.26+/-0.31 to 0.54+/-0.14 mM. When blood flow was reduced to 12+/-3 ml/min with lower perfusion pressure (40 mmHg), dialysis reduced arterial glucose from 3.53+/-0.36 to 0.15+/-0.03 mM. We conclude that this is an effective method for producing regional hypoglycemia.

A method for collecting right coronary venous blood samples from conscious dogs.

This report describes for the first time a technique to collect right coronary venous blood samples from conscious dogs. Catheters, prepared from Micro-Renathane tubing, were surgically implanted in right ventricular superficial veins of three anesthetized dogs. Also implanted were an arterial catheter, a right coronary flow transducer, and a right coronary artery constrictor. The coronary catheter was introduced at a venous bifurcation so that its side holes were positioned above the bifurcation; both ends of the catheter were exteriorized. Heparinized saline was continuously infused through the venous catheter by a battery-powered pump. The dogs were maintained for 10-13 days after surgery, and all catheters remained patent. Multiple right coronary venous samples were collected from each dog. These samples were analyzed for venous oxygen tension (PvO2) under baseline conditions, with right coronary pressure reduced to 50 mmHg, and during the reactive hyperemia after release of the right coronary artery constriction. PvO2 was 27.7 +/- 1.0 mmHg at baseline, 23.4 +/- 1.0 mmHg during coronary artery constriction, and 34.3 +/- 1.5 mmHg during reactive hyperemia. These data and the position of the catheter at autopsy demonstrated that coronary venous blood had been sampled.

Life cycle and reproduction of house-dust mites: environmental factors influencing mite populations.

An understanding of the life cycle of house-dust mites, as well as environmental factors influencing mite populations, can be exploited in mite control. The most important limiting factor for house-dust-mite populations is air humidity. House-dust mites osmoregulate through the cuticle and therefore require a high ambient air humidity to prevent excessive water loss. In addition, the supracoxal glands actively take up ambient water vapour, and the protonynph stage of the life cycle is resistant to desiccation. Larger house-dust-mite populations are found when the absolute indoor air humidity is above 7 g/kg (45% relative humidity at 20 degrees C). Consequently, ventilation by air-conditioning systems is being developed as a means of control. A number of other aspects of the domestic environment are also being manipulated in an integrated approach to render the habitat less suitable for mites. The potential exists for developing models for house-dust mite populations, environmental characteristics, and the effects of various approaches to control.

Mite allergens: significance of enzymatic activity.

Ten years ago, the cloning and sequencing of a cDNA encoding the group I allergen of house-dust mites unequivocally determined that protein allergens may have biochemical functions in addition to their ability to bind IgE. Since this discovery, several groups have speculated that the biochemical activities of allergens, or substances associated with allergens, may be involved in their immunogenicity or allergenicity. This paper will focus on just one biochemical function, proteolytic activity, and will be illustrated by examples of our own work that we believe support the hypothesis that this category of molecules are endowed with the properties of proallergic adjuvants.

Comparison of an ion-trap and a quadrupole mass spectrometer using diazepam as a model compound.

Recent innovations in mass spectrometry (MS) have led to the development of instruments with increased capabilities, smaller footprints, and relatively low cost. The traditional MS in most toxicology laboratories is a quadrupole system equipped with electron impact ionization. Recently, an ion trap with electron impact, positive chemical ionization, negative chemical ionization, and tandem MS capabilities was introduced by Finnigan MAT. This paper compares the sensitivity and precision of ion-ratio measurements between a Finnigan GCQ ion-trap mass spectrometer (ITMS) and a Hewlett Packard quadrupole mass spectrometer (QMS) using electron impact ionization with diazepam as the model compound. Additionally, the sensitivity and precision of ion ratio measurements are evaluated for the ITMS using positive chemical ionization, negative chemical ionization and tandem MS modes of analysis. In the full scan mode (m/z 50-650, 1 Hz), the ITMS had an average signal-to-noise ratio (S/N) of 1400 for a 2-ng injection of diazepam (10 injections per day for 5 days), within-run ion ratio precision had coefficients of variation from 5 to 11%. Using similar full scan conditions, a 10-ng injection of diazepam on the QMS had an average S/N ratio of 160, and precision of ion ratio measurements varied from 5 to 13%. In the selected ion mode (SIM) of analysis (three ions, 2 Hz), the ITMS had an average S/N of 14,000 for a 2-ng injection and ion-ratio precision ranging from 6 to 15%. Using similar SIM conditions, a 2-ng injection in the QMS had an average S/N of 3000 with ion ratio standard deviations of 0.67 to 2.9%. Overall, the ITMS provided at greater S/N, equivalent precision in full scan, but was 5- to 10-fold less precise in measuring ion ratios in the SIM mode as compared with the QMS.

A major house dust mite allergen disrupts the immunoglobulin E network by selectively cleaving CD23: innate protection by antiproteases.

Asthma is a chronic life-threatening disease of worldwide importance. Although allergic asthma and related atopic conditions correlate strongly with immune sensitization to house dust mites, it is unclear why antigens from mites provoke such powerful allergic immune responses. We have characterized the protease activity of Der p I, the group I protease allergen of the house dust mite Dermatophagoides pteronyssinus, and here report that it cleaves the low-affinity immunoglobulin (Ig) E Fc receptor (CD23) from the surface of human B lymphocytes. Der p I selectively cleaves CD23 and has no effect on the expression of any other B cell surface molecules tested. We speculate that this loss of cell surface CD23 from IgE-secreting B cells may promote and enhance IgE immune responses by ablating an important feedback inhibitory mechanism that normally limits IgE synthesis. Furthermore, since soluble CD23 is reported to promote IgE production, fragments of CD23 released by Der p I may directly enhance the synthesis of IgE. alpha 1-Antiprotease, a pulmonary antiprotease, is also shown to inhibit the cleavage of CD23 by Der p I. This may be significant in the etiopathogenesis of asthma, because other indoor pollutants associated with asthma are known to potently inhibit this antiprotease. These data suggest that the proteolytic activity of Der p I, the group I allergen of the house dust mite D. pteronyssinus, is mechanistically linked to the potent allergenicity of house dust mites. Furthermore, inhibition of Der p I by alpha 1-antiprotease suggests a mechanism by which confounding factors, such as tobacco smoke, may act as a risk factor for allergic asthma.

Effects of the fungus Aspergillus penicillioides on the house dust mite Dermatophagoides pteronyssinus: an experimental re-evaluation.

In this report the widely-held view that house dust mites benefit from fungal contamination of the dietary substratum is re-examined. The performance of Dermatophagoides pteronyssinus (Acari: Pyroglyphidae) is documented over two successive generations in the presence or absence of the xerophilic fungus Aspergillus penicillioides (Hyphomycetales: Moniliaceae). This fungus reduced survival, development rate, adult length and fecundity of D. pteronyssinus. Detrimental effects of A. penicillioides were proportional to the fungal density. Despite the antagonistic effects of A. penicillioides, a requirement for the fungus was indicated by the poor performance of fungus-free mites in the second generation; sustained culture of D. pteronyssinus in the absence of fungi is probably not possible. It is suggested that fungi may alter the particulate nature of the substratum to the detriment of house dust mites, but also provide micronutrients deficient in the diet.

Comparative IgE responses to extracts of five species of house dust mite, using western blotting.

IgE responses towards extracts of Dermatophagoides pteronyssinus, D. farinae, D. microceras, Euroglyphus maynei and Gymnoglyphus longior have been evaluated with sera from a mite sensitive population and the international reference pool of sera, NIBSC 82/528, using Western blotting. Using mite body and faecal extracts 21 of 29 mite protein bands responded to by sera were common to all five species of mite. All sera were unique with respect to proteins bound and species recognized. Mite proteins bound by more than 40% of sera included the Group II and III main allergens and protein bands 14 (MW 40.4 kD) and 21 (MW 27.8 kD), all of which were found in every mite species studied. The Group I allergen, found in all species, was bound by 29.4% of sera. Similar response profiles between mite species suggest that human specific IgE may bind predominantly to crossreactive determinants on mite allergens.

How relevant are house dust mite-fungal interactions in laboratory culture to the natural dust system?

Both house dust and house dust mites Dermatophagoides pteronyssinus contained a wider range of fungi than laboratory mite cultures. In total, nine species of fungi were isolated from D. pteronyssinus in house dust, and these included three xerophilic species (Eurotium amstelodami, Aspergillus penicillioides and Wallemia sebi) commonly found in laboratory cultures of D. pteronyssinus. It is concluded that mites do interact with a similar range of fungi in natural dust and in laboratory culture, but that the diversity of fungal species in the laboratory is reduced and the density of individual fungal species in culture exceeds that of house dust. In a second experiment, dust samples were incubated at room temperature with 75% relative humidity. The diversity of fungi invariably declined from up to 13 genera to the few species recorded in laboratory culture. This suggests that the dominance of xerophilic fungi in laboratory mite rearings is mediated primarily by low relative humidity, and the exclusion of air-borne spores.

In vitro evaluation of acaricidal and fungicidal activity of the house dust mite acaricide, Allerbiocid.

The acaricidal and fungicidal activities of a new acaricide Allerbiocid, formulated for the control of house dust mites, were investigated. The components of the Allerbiocid formula are: 3% benzyl benzoate (acaricidal and fungicidal), 1% tannic acid (protein denaturant) and 0.5% Tween (surface active agent) in 70% isopropyl alcohol (antiseptic). At application rates of both 150 and 250 ml/m2, the acaricidal activity appeared to work by both ingestion and contact and remained active upon contact with mites for up to 10 weeks. When Sabouraud agar was treated with Allerbiocid at a concentration of 5 ml/20 ml medium, the preparation was also found to have fungicidal and fungistatic activities on various species of fungi commonly found in house dust. Allerbiocid contains tannic acid which is a protein denaturant. Levels of Der p I in dust were found to be reduced by over 90% after treatment with Allerbiocid. The importance of denaturation of mite faecal allergens and allergens associated with dead fungal spores is discussed.

House dust mite sensitivity: interaction of genetics and allergen dosage.

Pairwise analysis of siblings from 21 families showed that house dust mite (HDM) sensitive children were exposed to higher concentrations of Der p I allergen in their mattress (P = 0.005) and bedding (P = 0.04), but not bedroom floor (P = 0.33), than their atopic sibling who was not sensitive to HDM antigens. There was no difference in the exposure to HDM numbers/100 mg of dust in the mattress (P = 0.61) or bedroom floors (P = 0.09). In contrast, pairwise analysis of siblings from 15 families showed that HDM sensitive children were not exposed to significantly different concentrations of Der p I in the mattress (P = 0.96), bedding (P = 0.11) or bedroom floor (P = 0.70) nor HDM numbers/100 mg of dust in the mattress (P = 0.12) and bedroom floor (P = 0.98) than their non-atopic siblings. These findings were identical when absolute allergen load was compared in these pairs. Genetic linkage studies in these families suggest the tendency to atopic IgE responses is conferred by a putative atopy locus on chromosome 11q. These results together suggest that differences in allergen levels in beds, among siblings with a comparable genetic tendency to atopy, play a significant role in determining the development of HDM allergy.

Molecular characterisation of group I allergen Eur m I from house dust mite Euroglyphus maynei.

Using the polymerase chain reaction (PCR) we have amplified and cloned genomic DNA encoding the secreted group I allergen proteins from the house dust mite species Euroglyphus maynei, Dermatophagoides pteronyssinus and D. farinae. Affinity chromatography using a monoclonal antibody to the allergen Der p I was used to purify the group I protein from E. maynei. We present the deduced amino acid sequence of a new member of the group I house dust mite allergen family Eur m I. The three proteins show a high level of primary structure similarity: Eur m I and Der p I show 85% amino acid identity, and the three allergen amino acid sequences taken together show 78% identity. A potential N-glycosylation site and residues of the cysteine protease active site are also conserved between the three proteins.

Evidence refuting the contribution of the fungus Aspergillus penicillioides to the allergenicity of the house dust mite Dermatophagoides pteronyssinus.

This communication demonstrates unequivocally that the fungi associated with house dust mites do not contribute to mite allergenicity. The evidence is twofold: first, larval mites which lack fungi have allergen profiles indistinguishable from fungus-bearing adult mites. Second, the allergen profile of experimentally-derived fungus-free adult mites and mites re-fed the fungus Aspergillus penicillioides are identical.

House dust mite sensitivity: a comparison of immune response with exposure to four species of Pyroglyphidae.

Twenty-five atopic children under 11 years of age were studied, using skin and RAST tests, for their specific IgE response to four species of pyroglyphid house dust mites, Dermatophagoides pteronyssinus, D. farinae, D. microceras and Euroglyphus maynei. All of the children were sensitive to D. pteronyssinus, 20 (80%) of these children were also sensitive to D. farinae and D. microceras, and 16 of the latter (64%) were also sensitive to E. maynei. Dust samples from various sites in the homes of the children revealed D. pteronyssinus in all homes studied but no D. farinae or D. microceras. E. maynei, although identified, was not present in significant numbers in any site. A control group of 20 atopic children of similar age who were not sensitive to house dust mite allergens had a similar exposure to the four mite species. These results suggest that factors in addition to mite exposure are important in the development of specific IgE responses to house dust mites.