Efficacy of occupant-collected dust samples in the evaluation of residential allergen and fungal levels.

This study evaluated the ability of a resident to evaluate their home for allergens and mold using a settled dust test kit compared with evaluation and collection of settled dust by an industrial hygienist. Forty-three home residents were provided with a kit containing written instructions and a vacuum cleaner attachment for collecting a settled dust sample. Within 2 weeks of receiving the occupant-collected sample, an industrial hygienist evaluated these homes, including a visual inspection, collection of settled dust, and collection of spore trap samples. Settled dust samples were analyzed for major dog, cat, dust mite, and cockroach allergens using immunoassay methods, and for mold spore equivalents using quantitative polymerase chain reaction methods for the 13 mold species or species groups comprising the American Relative Moldiness Index (ARMI). Allergen concentrations and ARMIs were compared between the resident- and industrial hygienist-collected samples. Linear regression between the two sets of samples showed strong correlations for dog allergen (r(2) = 0.92) and cat allergen (r(2) = 0.90). Correlations for dust mite (r(2) = 0.57) and cockroach allergens (r(2) = 0.22) were lower, likely due to most samples being near the limit of detection. ARMIs were highly correlated (r(2) = 0.68) and were in categorical (high, medium, or low) agreement for 76% of residences. These results show that residents can reliably follow directions and collect settled dust samples, providing an efficient method to remotely screen homes for elevated allergen levels and to identify homes with a potential mold or moisture problem that may need further evaluation.

Contribution of Ara h 2 to peanut-specific, immunoglobulin E-mediated, cell activation.

BACKGROUND: Ara h 2 is a potent peanut allergen but its contribution to the ability of a crude peanut extract (CPE) to cross-link IgE and activate mast cells has not been rigorously evaluated. OBJECTIVE: To measure the contribution that Ara h 2 makes to the effector function of a CPE. METHODS: Ara h 2 was specifically removed from a CPE as demonstrated by immunoblots, 2D gels, and an inhibitory ELISA. Functional assays of sham-treated and Ara h 2-depleted CPEs were performed with RBL SX-38 cells sensitized with IgE from highly peanut-allergic subjects and with naturally sensitized basophils. RESULTS: Depletion of approximately 99% of the Ara h 2 from the CPE led to an increase in the concentration of the CPE necessary to give 50% of maximal degranulation (EC50) of the SX-38 cells following sensitization with sera that contain anti-Ara h 2 IgE. Assays with a pool of 10 sera showed a small but significant increase in the EC50 following depletion of Ara h 2 (1.65+/-0.15-fold; P<0.05) and assays of seven individual sera showed a similar increase in the average EC50 (1.7+/-0.2-fold; P<0.02). The percent of the anti-peanut IgE that binds Ara h 2 correlated with an increase in the EC50 of the CPE following depletion of Ara h 2 (r=0.83; P<0.02). On the other hand, data from three of these patients studied with a basophil histamine release assay did not show a significant effect of depletion of Ara h 2. CONCLUSION: Based on its ability to cross-link IgE effectively, Ara h 2 is clearly an important peanut allergen. Its ability to cross-link IgE effectively from a specific serum is related to the proportion of anti-Ara h 2 in that serum but Ara h 2 does not account for a majority of the effector activity of the CPE for any of the sera studied.

Airway inflammation and bronchial hyperresponsiveness after Mycoplasma pneumoniae infection in a murine model.

The interaction between chronic infection and chronic asthma is receiving increased investigation as a factor in the pathophysiology of asthma. To further understand this interaction, we used an animal model (BALB/c mice) with a Mycoplasma pneumoniae respiratory infection. Mice were studied 3, 7, 14, and 21 d after infection. Bronchial hyperresponsiveness (BHR) was assessed by methacholine challenge and was significantly heightened in the infected mice compared with saline controls at Days 3, 7, and 14. The associated inflammatory response was mainly neutrophils. The tissue inflammatory score significantly correlated to BHR (r = 0.78, P < 0.0001). Additionally, tissue interferon (IFN)-gamma was significantly suppressed at Days 3 and 7 in the infected group compared with controls; and at Days 3, 7, and 14 compared with Day 21 in the infected group. There was a significant negative correlation between lung tissue messenger RNA levels of IFN-gamma corrected for beta-actin and BHR (r = -0.50, P = 0.022). Thus, M. pneumoniae respiratory infection is associated with BHR in this murine model. It appears that acute mycoplasma infection suppresses IFN-gamma, which may be a pivotal factor in the control of BHR.

Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.

A 5-week-old male infant presented with severe bacterial infections and poor wound healing, suggesting a neutrophil defect. Neutrophils from this patient exhibited decreased chemotaxis, polarization, azurophilic granule secretion, and superoxide anion (O(2)(-)) production but had normal expression and up-regulation of CD11b. Rac2, which constitutes >96% of the Rac in neutrophils, is a member of the Rho family of GTPases that regulates the actin cytoskeleton and O(2)(-) production. Western blot analysis of lysates from patient neutrophils demonstrated decreased levels of Rac2 protein. Addition of recombinant Rac to extracts of the patient neutrophils reconstituted O(2)(-) production in an in vitro assay system. Molecular analysis identified a point mutation in one allele of the Rac2 gene resulting in the substitution of Asp57 by an Asn (Rac2(D57N)). Asp57 is invariant in all defined GTP-binding proteins. Rac2(D57N) binds GDP but not GTP and inhibits oxidase activation and O(2)(-) production in vitro. These data represent the description of an inhibitory mutation in a member of the Rho family of GTPases associated with a human immunodeficiency syndrome.

Binding of rat and human surfactant proteins A and D to Aspergillus fumigatus conidia.

Surfactant proteins A (SP-A) and D (SP-D) are thought to play important roles in pulmonary host defense. We investigated the interactions of rat and human SP-A and SP-D with Aspergillus fumigatus conidia. Rat SP-D but not rat SP-A bound the conidia, and the binding was inhibited by EDTA, mannose, glucose, maltose, and inositol. Binding studies using a mutant recombinant rat SP-D with altered carbohydrate recognition but normal structural organization clearly established a role for the carbohydrate recognition domain in binding to conidia. However, neither rat SP-A nor SP-D increased the association of fluorescein isothiocyanate-labeled conidia with rat alveolar macrophages as determined by flow cytometry. Both human SP-A (isolated from normal and alveolar proteinosis lungs) and SP-D (recombinant protein and protein isolated from alveolar proteinosis lungs) bound the conidia. These data indicate that important differences exist between rat and human SP-A in binding to certain fungi. Human SP-A and SP-D binding to conidia was also examined in the presence of hydrophobic surfactant components (HSC), containing both the phospholipid and hydrophobic proteins of surfactant. We found that HSC inhibited but did not eliminate human SP-A binding to Aspergillus conidia. In contrast, the SP-D binding to conidia was unaffected by HSC. These findings indicate that SP-D plays a major role in the recognition of Aspergillus conidia in alveolar fluid.

Staphylococcus aureus isolated from Kawasaki disease patients hyper-releases extracellular protein A.

S. aureus isolates from patients with Kawasaki disease (KD) release high levels of extracellular protein A (SpA), as compared to S. aureus in other diseases. The molecular weight of this released protein A is about 70 kDa. Extracellular KD SpA purified by affinity chromatography possessed the same amino acid sequence at the NH2-terminal IgG binding region and the same antigenic specificity as recombinant and cell-wall-bound SpA preparations. The size of DNA fragments containing the spa gene from S. aureus KD strains was 160-165 kb. All of these DNA fragments contained the igb portion encoding the IgG-binding region of KD SpA. Significantly higher molecular size of the SpA molecules hyper-released in the stationary-phase culture and the lack of production of other exo-proteins allow us to speculate that S. aureus isolated from patients with KD have mutations occurring in the agr locus.

Prediction of early-onset asthma in genetically at-risk children.

The W.T. Grant Foundation Asthma Risk Study was designed to prospectively examine children who were considered at a genetically increased risk for the development of asthma. The respective contributions of 11 potential risk factors, both environmental and biological, were assessed in order to determine their relative roles in affecting the early onset of asthma. This is a report of an inception cohort of children born to asthmatic mothers and followed for a 3-year period. All 150 families were recruited from the general community and living within 2 h of the National Jewish Center for Immunology and Respiratory Medicine (Denver, CO). Mothers in the index risk sample had been previously diagnosed with asthma and were recruited during their pregnancy through physician referrals and media solicitation. The index sample of 150 families was 92% Caucasian and predominantly middle class. The mean age of mothers was 29.3 years, and of fathers, 31.1 years. The main outcome was the determination of the early onset of asthma and its association with quantified risk factors. By age 3 years, 14 of the 150 children had developed asthma. Frequent illness, IgE levels at age 6 months, parenting difficulties, and early eczema were significantly associated with the onset of asthma (P = 0.003, P = 0.006, P = 0.01, and P = 0.03, respectively). Only frequent illness, elevated serum IgE levels, and parenting difficulties entered a predictive model where they were independently related to the development of asthma.

Staphylococcal toxins augment specific IgE responses by atopic patients exposed to allergen.

Microbial agents are known to play a significant role in aggravating allergic diseases. Recently described viral and bacterial superantigens represent one important strategy by which infectious agents can stimulate the immune response. In previous work, we reported that the staphylococcal toxin toxic shock toxin-1 (TSST-1), a prototypic superantigen, induces in vitro total IgE synthesis after cross-linking T and B cells. This study was carried out to establish a potential link between superantigens and the enhanced IgE response to specific allergens in allergic patients. Peripheral blood mononuclear cells from atopic patients were isolated during and outside the pollen allergen season and stimulated with TSST-1, a prototypic superantigen. Total IgE and interferon-gamma production were measured in supernatants of these cultures. Outside the pollen season, TSST-1 significantly increased total IgE production only in the presence of exogenous interleukin-4, whereas during the pollen season IgE production was significantly enhanced without the need of exogenous interleukin-4. This increase in the absence of exogenous interleukin-4 was associated with significantly lower interferon-gamma production by peripheral blood mononuclear cells stimulated by TSST-1 during the pollen season. Moreover, TSST-1 stimulation of peripheral blood mononuclear cells from inhalant allergic patients was followed by an increased production of allergen-specific IgE that was restricted to the allergen to which the patient was allergic and recently exposed. In addition, TSST-1 induced on B cells the expression of B7.2, a molecule that has recently been demonstrated to enhance T helper 2 responses and to be involved in IgE regulation. This study, by demonstrating that superantigens can augment allergen-specific IgE synthesis and B7.2 expression, provides a mechanism by which microbial superantigens may modulate allergic responses.

Effect of pH on the stability of methacholine chloride in solution.

Methacholine chloride bronchoprovocation challenges are performed for the diagnosis and investigation of hyperreactive airways. Over the last 20 yrs various formulations and pH values for the preparation of solutions of methacholine have been described. To determine the stability of methacholine chloride solutions prepared in a variety of buffers with differing pH values and under varying storage temperatures, we measured methacholine concentrations at intervals from 1 to 5 weeks. It was found that methacholine chloride solutions rapidly decompose if the pH is greater than 6 and that decomposition is more rapid as the pH is raised; solutions at pH 9, i.e. bicarbonate buffer, and stored at 27 degrees C have degradation up to 36% after only one week. Solutions of the same pH but prepared in different buffers can have both varied rates of deterioration and different absolute amounts of methacholine hydrolysed, e.g. solutions prepared in pH 9 borate buffer and stored at 27 degrees C have up to 60% degradation after 1 week. Solutions prepared in saline are stable probably because methacholine solutions are weakly acidic. The results emphasise the importance of preparing methacholine chloride in the proper buffers for use in the accurate assessment of airway responsiveness.

A twin and family study of the association between immune system dysfunction and dyslexia using blood serum immunoassay and survey data.

We conducted a study of the association between developmental reading disability (DRD) and immune disorders (ID) using both survey and immunoassay data in two separate samples of families. One sample was made up of twins and their parents and was ascertained through a population-based sampling scheme. The other sample was a set of extended pedigrees selected for apparent autosomal dominant transmission of DRD. We failed to find an association between DRD and ID in either sample, regardless of the method used to assess immune system function. Even though our twin sample provided evidence that both DRD and immune conditions were significantly heritable, there was no evidence for a genetic correlation between ID and DRD nor was there any clear indication that a special subgroup of individuals may be comorbid for these conditions because of genetic reasons. How these negative findings can be reconciled with the developmental hypothesis of Geschwind, Behan, Galaburda, and colleagues, and how they may relate to the gene locus influencing DRD that has been recently located in the HLA region of the short arm of chromosome 6 is discussed.

The immune system can affect learning: chronic immune complex disease in a rat model.

Evidence is presented that the immune system can affect central nervous system functioning, leading to changes in learning. Immune complex disease is induced in rats and their behavior tested using a Lashley maze. Significant differences in behavior were found between the animals with high disease activity and those with low disease activity and the non-disease controls. These changes were not due to uremia and are most likely due to the immune response. There is some evidence immune complex deposits in the choroid plexus may play some role, but not the sole or major role in the behavioral changes. This provides a model by which immunologic processes can cause neuropsychiatric manifestations in autoimmune diseases like lupus, as well as showing that immune processes can affect behavioral functioning.

Early preferential stimulation of gamma delta T cells by TNF-alpha.

Although recent findings indicate that gamma delta T cells influence both early innate and Ag-specific adaptive host responses, it has remained unclear what triggers gamma delta T cell reactivity. Investigating very early T cell activation in mouse and human models of bacterial infection, we measured CD69 expression as an indicator of early cellular activation. Both murine alpha beta and gamma delta T cells responded polyclonally to systemic bacterial infections, and to LPS. However, gamma delta T cells responded more strongly to the bacteria and to LPS. In vitro LPS-stimulated human T cells showed a similar differential response pattern. We identified TNF-alpha as mediator of the early differential T cell activation, and of differential proliferative responses. The stronger response of gamma delta T cells to TNF-alpha was correlated with higher inducible expression levels of TNF-Rp75. Among unstimulated splenocytes, more gamma delta T cells than alpha beta T cells expressed CD44 at high levels. The data suggest that TNF-Rp75 determines the differential T cell reactivity, and that most gamma delta T cells in the normal spleen are present in a presensitized state. As TNF-alpha stimulates activated T cells, it may early preferentially connect gamma delta T cell functions with those of cells that produce this cytokine, including activated innate effector cells and Ag-stimulated T lymphocytes.

Clinically relevant concentrations of ethanol attenuate primed neutrophil bactericidal activity.

BACKGROUND: Acute alcohol intoxication is associated with an increased risk of infection in the injured patient. The impact of clinically relevant levels of ethanol (ETOH) on neutrophil (PMN) bactericidal activity remains ill-defined. PMN priming optimizes microbicidal activity by enhancing oxygen radical production, degranulation, and adhesion molecule up-regulation. We hypothesized that clinically relevant levels of ETOH attenuate these primed PMN responses critical to eradicate infection. METHODS: After incubation with ETOH (0-1.0%), isolated human PMNs were primed with beta-acetyl-gamma-O-alkyl and activated with N-formyl-methionyl-leucyl-phenylalanine. Superoxide generation was measured by cytochrome c reduction, elastase release was measured by cleavage of methoxysuccinyl-ala-ala-pro-val-p-nitroanilide, and CD11b was measured by fluorescent monoclonal antibody staining. Bactericidal activity was assessed by Staphylococcus aureus killing. RESULTS: ETOH attenuated superoxide production dose-dependently with significance at 0.3% ETOH. Elastase release was attenuated starting at 0.2% ETOH, and CD11b expression was reduced starting at 0.4% ETOH. S. aureus killing was impaired dose-dependently with significance at 0.3% ETOH. CONCLUSION: Clinically relevant concentrations of ETOH attenuate PMN functions critical in host defense against invading pathogens. These results provide direct in vitro evidence consistent with previous in vivo data that acute alcohol intoxication is important in the pathogenesis of trauma-related infections.

Phenotypic features of selective T cell deficiency characterized by absence of CD8+ T lymphocytes and undetectable mRNA for ZAP-70 kinase.

Selective T cell deficiency is a rare immune deficiency characterized by the absence of CD8+ T lymphocytes and depressed/absent T cell function. This syndrome has been associated with mutations in the gene for ZAP-70, a tyrosine kinase that has profound effects on signaling via the T cell receptor. In this paper we describe a patient with selective T cell deficiency and certain phenotypic features that are unique among the small number of patients described. The patient had virtually absent T cell function, hypogammaglobulinemia, and no response to vaccination. The T lymphocytes failed to respond to mitogenic stimuli, even in the presence of exogenous interleukin 2. Similar to other patients with this disorder, the T cells were capable of proliferating when stimulated by pharmacologic agents such as phorbol ester and ionomycin. While peripheral blood T cells had limited capability to increase cytosolic Ca2+ levels in response to mitogenic stimulation, thymocytes responded to a large panel of antibodies and mitogens. This report broadens the spectrum of clinical presentations associated with selective T cell deficiency and, for the first time, compares the responses of both peripheral T cells and thymocytes. The data support the concept that the defect in signal transduction resulting from the absence of ZAP-70 is primarily manifested following export of T lymphocytes from the thymus and that selection of CDS-positive T cells is dependent on the presence of ZAP-70.

Comparison of exhaled nitric oxide, serum eosinophilic cationic protein, and soluble interleukin-2 receptor in exacerbations of pediatric asthma.

The hypotheses tested in this study were that during acute asthma exacerbations (1) exhaled nitric oxide concentrations [eNO] are a more sensitive, noninvasive indicator of asthma disease activity than serum markers of inflammation such as eosinophil cationic protein (ECP) or soluble interleukin 2 receptor (sIL2R), and (2) elevated [eNO] are reduced after treatment with glucocorticoids (GC). Peak eNO levels were measured by chemiluminescence during slow expiration. Seven asthmatic subjects (mean age 11 yrs; mean morning FEV1 65% predicted) receiving inhaled GC, and with no radiographic evidence of acute sinusitis, were studied before and after a course of oral GC. Measurements of [eNO], ECP and sIL2R levels, and FEV1% were obtained before and after a course of GC. Six atopic nonasthmatic subjects (mean age 12 years; mean FEV1 94% predicted) and seven normal subjects (mean age 13 years; mean FEV1 100% predicted) were studied. The mean peak [eNO] level (parts per billion: ppb) for the asthma subjects before treatment (52 +/- 5 ppb SEM) was greater than the value for both nonasthmatic atopic and normal subjects (16 +/- 2 ppb and 14 +/- 2 ppb SEM, respectively; P < 0.0001). There was no significant difference in ECP or sIL2R values between asthmatic subjects and either atopic or normal subjects (P > 0.05). Baseline pre-GC treatment ECP levels in the asthmatic subjects were significantly higher (P < 0.002) than post-GC treatment values. The mean peak [eNO] level in the asthmatic subjects declined after oral GC treatment to 14 +/- 1 ppb (P < 0.0002) and was less than 2 ppb different from either control group (P > 0.75). We conclude that [eNO] is a more sensitive marker of asthma disease activity than ECP and sIL2R levels. In addition, [eNO] appears to be a more useful indicator of the beneficial response to GC therapy than these other measurements in pediatric asthma.

The potential role of gastroesophageal reflux in the pathogenesis of food-induced wheezing.

Severe reactive airways disease (RAD) in children is frequently associated with gastroesophageal reflux or food allergy. However a relationship between these two confounding factors has yet to be investigated. We postulate that, in certain patients with micro-aspiration of gastric contents into the airways, food allergens sensitize T cells in the peribronchial lymphoid tissue and induce the production of food-specific IgE antibodies that sensitize airway cells. Subsequent exposure to these food allergens might then induce IgE dependent mediator release from mast cells as well as T cell and eosinophil activation, thus contributing to airway inflammation and RAD. In the current report, we describe the case of a patient with severe asthma who had food allergy and gastroesophageal reflux whose clinical findings support this hypothesis. We also provide additional evidence for a high rate of food sensitization in patients with bronchopulmonary dysplasia (BPD), RAD and GER. We conclude that additional studies are warranted to examine the possibility that patients who have RAD and GER require an evaluation for food allergy.