Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

Aspergillus fumigatus Cell Wall Promotes Apical Airway Epithelial Recruitment of Human Neutrophils.

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.

Production of a specific monoclonal antibody for 1-naphthol based on novel hapten strategy and development of an easy-to-use ELISA in urine samples.

1-naphthol (1-NAP) is the main metabolite of pesticide carbaryl and naphthalene, and is also a genotoxic and carcinogenic intermediate in the synthesis of organic compound, dyes, pigment and pharmaceutical industry. In this work, two novel haptens were designed and synthesized for developing a competitive indirect enzyme-linked immunosorbent assay (ciELISA) method for 1-NAP in urine samples. The assay showed a limit of detection of 2.21 ng/mL and working range from 4.02 ng/mL to 31.25 ng/mL for 1-NAP in optimized working buffer. The matrix effect of samples was eliminated via 15-fold dilution of optimized working buffer. Good average recoveries (102.4%-123.4%) with a coefficient of variation from 11.7% to 14.7% was obtained for spiked urine samples. Subsequent instrument verification test showed good correlation between the results of ciELISA and high-performance liquid chromatography. The developed ciELISA is a high-throughput tool to monitor 1-NAP in urine, which can provide technical support for the establishment of biological exposure level for the exposure to carbaryl, naphthalene and other related pollutants.

Immunoassay of red dyes based on the monoclonal antibody of ß-naphthol.

The objective of the present study was to develop a multi-analyte immunoassay for the determination of eight red dyes in food samples. Two dye intermediates (2-hydroxy-1-naphthoic acid and 1-amino-2-naphthol) were used as the haptens to produce the monoclonal antibodies. The obtained monoclonal antibodies recognized Sudan 1-4, Para red, Sudan red G, Sudan red B and Acid orange II simultaneously. After evaluation of different antibody/coating antigen combinations, a heterologous indirect competitive enzyme linked immunosorbent assay was developed to determine the eight red dyes in food samples (chili oil, chili powder, tomato sauce, hotpot seasoning). The crossreactivities to the eight analytes were in the range of 61%-79% (with ß-naphthol as 100%), and the limits of detection were in the range of 1.3-1.9 ng/mL. The recoveries of the eight analytes from the fortified blank samples were in the range of 84.2%-115% with coefficients of variation lower than 18.3%. Therefore, this method could be used as a rapid and simple tool to detect the residues of the eight red dyes in foods.

A sensitive and selective enzyme-linked immunosorbent assay for the analysis of Para red in foods.

Para red is a synthetic dye and a potential genotoxic carcinogen. A hapten mimicking Para red structure was synthesized by introducing a carboxyl to the naphthol part of Para red and coupled to carrier protein to form an immunogen for the production of specific antibodies. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Para red in food samples. The limit of detection and inhibition half-maximum concentrations of Para red in phosphate buffered saline with 10% methanol were 0.06 and 2.2 ng mL(-1), respectively. Cross-reactivity values of the ELISA with the tested compounds including Sudan red I, II, III, IV, and G, sunset yellow, 2-naphthol, and 4-nitroaniline were ≤0.2%. This assay was used to determine Para red in tomato sauce, chilli sauce, chilli powder and sausage samples after ultrasonic extraction, cleanup and concentration steps. The average recoveries, repeatability (intraday extractions and analysis), and intra-laboratory reproducibility (interday extractions and analysis) were in the range 90-108%, 4-12% and 8-17%, respectively. This assay was compared to a high-performance liquid chromatographic method for 28 samples, displaying a good correlation (R(2) = 0.95). Para red residues in 53 real world samples determined by ELISA were below the limit of detection.

Ultrasensitive detection of Sudan I in food samples by a quantitative immunochromatographic assay.

An immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for ultrasensitive determination of Sudan I in food samples was reported. Gold-silver core-shell bimetallic nanorods (referred to as Au@Ag NRs) were synthesized, characterized and used as the substrate for preparation of the ICA. Polyclonal antibody against Sudan I was immobilized on the surface of the Au@Ag NRs carrying the Raman reporter 5,5'-dithiobis (2-nitrobenzoic acid). The Raman scattering intensity on the test line was used for quantitation of Sudan I. The assay was completed in 15 min. IC50 and limit of detection (LOD) were 30 pg mL-1 and 0.2 pg mL-1, respectively. There was no cross-reactivity (CR) of the assay with Sunset Yellow, Lemon Yellow and Brilliant blue FCF, but only 3.53%-9.74% CR with Sudan II, III and IV. The recoveries of Sudan I from spiked food samples were in the range of 88.9-107.6% with relative standard deviation of 3.7-8.7% (n = 3).

[Synthesis of Sudan I artificial antigen].

OBJECTIVE: To prepare Sudan I-HS conjugates with the carrier protein for developing a new immune assay for Sudan I. METHODS: Sudan I was actived by succinic anhydride and converted into Sudan I hemisuccinate (Sudan I-HS) as the hapten, then Sudan I-HS was linked to carrier protein bovine serum albumin (BSA) as immunogen, and ovalbumin (OVA) as coating antigen all by the active ester method. RESULTS: The results of IR and HPLC-MS indicated that the hapten was synthesized successfully. The conjugation ratios of Sudan I-HS-BSA and Sudan I-HS-OVA confirmed by ultraviolet spectrum were 11 : 1 and 5 : 1, respectively. CONCLUSION: Antigens Sudan I-HS-BSA and Sudan I-HS-OVA were prepared successfully.

Highly specific polyclonal antisera against estriol: cross-reactivity restriction following affinity chromatography.

An immunosorbent technique was developed to attenuate cross-reactivity of a polyclonal antiserum against a 4(2) (rho-carboxyphenylazo)-1,3,5[10]-estratrien-3,16 alpha,17 beta-triol-bovine serum albumin conjugate. The chromatographic separation of antiserum through stationary phases having either rho(carboxymethyl)phenylazo-phenol or rho(carboxymethyl)-phenylazo-2-naphthol side residues reduced the antiserum avidity, while increasing the apparent antiserum affinity and decreasing the residual cross-reactivities against heterologous ligands. The highly specific antiserum obtained allowed the development of a competitive binding assay over an extended analytical range, which opens up the possibility of direct measurement of estriol from the early pregnancy to delivery. The significance of the attenuation of antiserum cross-reactions after affinity chromatography is discussed with reference to epitope-paratope interaction in the case of small endogenous molecules like estrogens.

A monoclonal antibody-based immunosensor for detection of Sudan I using electrochemical impedance spectroscopy.

Sudan I monoclonal antibodies (Mabs) were prepared by hybridoma technique and firstly used to develop a Sudan I immunosensor by immobilizing the Mabs on a gold electrode. o-Mercaptobenzoic acid (MBA) was covalently conjugated on the gold electrode to form a self-assembled monolayer (SAM). The immobilization of Sudan I Mabs to the SAM was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies reproducibly and densely on the SAM. The changes of the electrode behavior after each assembly step were investigated by cyclic voltammetric (CV) technique. The Sudan I concentration was measured through the increase of impedance values in the corresponding specific binding of Sudan I and Sudan I antibody. A linear relationship between the increased electron-transfer resistance (Ret) and the logarithmic value of Sudan I concentrations was found in the range of 0.05-50 ng mL(-1) with the detection limit of 0.03 ng mL(-1). Using hot chili as a model sample, acceptable recovery of 96.5-107.3% was obtained. The results were validated by conventional HPLC method with good correlation. The proposed method was proven to be a feasible quantitative method for Sudan I analysis with the properties of stability, highly sensitivity and selectivity.

Rôle of a metabolite in allergenicity of Sudan I.

A metabolite of Sudan I (1-phenylazo-2-naphthol) was isolated after in vitro incubation with the supernatant of guinea pig liver homogenate (S-9). The metabolite was found to be 4'-hydroxy-1-phenylazo-2-naphthol by gas chromatography-mass spectrometry (GC-MS), and proton and carbon-13 nuclear magnetic resonance (NMR) analyses. It elicited positive reactions in guinea pigs sensitized to Sudan I. It was also shown to be allergenic. The results suggest that para-hydroxylation of the phenyl group of Sudan I may play an important rôle in its allergenicity.

Pattern of specific airway response in asthma due to western red cedar (Thuja plicata): relationship with length of exposure and lung function measurements.

In order to investigate the relationship between the pattern of response (immediate, late and dual) to specific bronchial challenge test with plicatic acid or red cedar extract and the clinical features of asthma, 332 patients with asthma induced by western red cedar dust were examined at the time of diagnosis. Specific challenge test induced in thirty-one patients (9.3%) an isolated immediate reaction, in 144 patients (43.4%) an isolated late reaction and in 157 patients (47.3%) a dual reaction. Patients with a dual reaction had a longer period of exposure to red cedar dust between the onset of the respiratory symptoms and the time of the definitive diagnosis, a lower FEF 25-75% and a greater degree of non-specific bronchial hyperresponsiveness compared to patients with isolated immediate or isolated late reactions; the difference in bronchial hyperresponsiveness to methacholine among the three groups persisted when the values were adjusted for the different baseline value of FEV1. There was no difference in the prevalence of specific serum IgE antibodies to plicatic acid-human serum albumin conjugate among the three groups of patients with different type of response to red cedar. Except for the greater degree of non-specific bronchial hyperresponsiveness, patients with isolated late reactions were not different from those with isolated immediate reactions in other clinical findings. These findings indicate that a dual reaction in patients with occupational asthma due to simple chemical agents is indicative of a greater severity of disease at diagnosis. The pathogenetic mechanism of various types of asthmatic reaction is unknown and it is likely to be different between isolated immediate and isolated late reactions.

Immunologic studies of the mechanisms of occupational asthma caused by western red cedar.

BACKGROUND: Occupational asthma caused by western red cedar (Thuja plicata) is a common problem in sawmill industries. The objective of this study was to examine the cellular and immunologic mechanisms of western red cedar asthma (WRCA) more closely. METHODS: Bronchial biopsy specimens, bronchoalveolar lavage (BAL) mast cells and peripheral blood basophils from patients with WRCA, patients with atopic asthma, and nonatopic control subjects were challenged in vitro with plicatic acid (PA), PA-human serum albumin conjugate (PA-HSA), grass pollen, or calcium ionophore. RESULTS: PA (100 micrograms/ml) released histamine from the basophils of 9 of 11 patients with WRCA, 1 of 7 patients with atopic asthma, and 2 of 7 normal subjects. PA triggered histamine release from 10 of 11 bronchial biopsy specimens and 8 of 8 BAL samples from patients with WRCA. Interestingly, PA released histamine from BAL cells and bronchial biopsy specimens from 3 of 7 normal subjects but in none of the patients with atopic asthma. PA-HSA-induced histamine release from basophils and biopsy specimens was confined to patients with WRCA. PA-specific IgE was not detectable in serum from most patients with WRCA, and their serum did not transfer PA sensitivity to human lung fragments or lactate-stripped basophils. After pretreatment with anti-IgE in the absence of calcium, basophils from 14 subjects with WRCA still responded to PA (mean 64% to 67% of pretreatment response), whereas responses to grass pollen or anti-IgE were abolished. CONCLUSIONS: This study confirms that PA releases histamine from bronchial mast cells of most patients with WRCA but not from those of patients with atopic asthma. The PA response of some normal subjects suggests that PA may have both specific and nonspecific actions on mast cells and basophils, whereas the serologic studies indicate histamine release in WRCA cannot simply be attributed to PA-specific IgE.

Relationship between types of asthmatic reaction, nonspecific bronchial reactivity, and specific IgE antibodies in patients with red cedar asthma.

We studied the relationship between specific IgE antibodies, nonspecific bronchial reactivity to methacholine, and the type of asthmatic reaction in patients with red cedar asthma. The level of circulating specific IgE antibodies (expressed as RAST ratios) was not related to the type of asthmatic reaction, the degree of nonspecific bronchial hyperreactivity [expressed by the provocative concentration of methacholine producing a 20% decrease in the forced expiratory volume in 1 sec (PC20)] or the index of reactivity to plicatic acid. On the other hand, methacholine PC20 was found to correlate with the index of reactivity to plicatic acid in the late asthmatic reaction (LAR) and both the immediate and late components of the dual asthmatic reaction (DAR). Development of the LAR is associated with increase in nonspecific bronchial hyperreactivity. Repeated inhalation challenge with plicatic acid in eight patients with LAR resulted in DAR in all. The results suggest that the mechanism responsible for the LAR is associated with an increase in nonspecific bronchial reactivity; furthermore, the immediate component of DAR could also be related to heightened bronchial hyperreactivity.

Plasma histamine after methacholine, allergen, and aspirin challenges.

Plasma histamine levels were measured by radio-enzymatic technique in seven patients following 10 challenges: five methacholine challenge tests, four antigen inhalation challenge tests, and one oral aspirin challenge test. Baseline plasma histamine was the same in all patients except in the aspirin-challenged patient, who had a higher baseline histamine level. There was no statistical change in the level of histamine throughout the test in either the methacholine-challenged or the antigen-challenged patients, whereas there was a marked increase in histamine levels in the aspirin challenged patient. A possible explanation is that methacholine and antigen are inhaled and therefore have primarily local effects on the lung, whereas oral aspirin has a systemic effect with consequently systemic changes in histamine which are detectable as changes in plasma level.

Plicatic acid-specific IgE and nonspecific bronchial hyperresponsiveness in western red-cedar workers.

In a cross-sectional survey of 652 workers in a western red-cedar sawmill, we obtained data on symptoms, pulmonary function, immediate skin reactivity to common allergens, nonspecific bronchial responsiveness, total IgE level, and sensitization to plicatic acid conjugated with human serum albumin as measured by RAST. Dust exposure was estimated by personal and area sampling for total dust during a work shift and cumulative exposure by duration of employment. Seven percent of the workers had an elevated RAST, and 20% had nonspecific bronchial hyperresponsiveness. Elevation in RAST was associated with bronchial hyperresponsiveness. Almost half (46%) of the workers with RAST elevation had bronchial hyperresponsiveness compared to 18% in workers with no RAST elevation. The association was unaffected by total IgE level or by limiting the analysis to workers without respiratory symptoms and was most apparent in younger workers. Bronchial hyperresponsiveness was associated with increased prevalence of respiratory symptoms as well as with lower levels of pulmonary function. The likelihood of bronchial hyperresponsiveness increased with increasing age but was unrelated to the dust-exposure concentration. RAST elevation was unrelated to employment duration or dust exposure and was not associated with an increased prevalence of symptoms or lower levels of pulmonary function independent of bronchial hyperresponsiveness. We conclude that plicatic acid-specific IgE and nonspecific bronchial hyperresponsiveness are associated in western red-cedar workers and that this association may reflect a causal connection.

An enzyme immunoassay of phaseolinone and its application in estimation of the amount of toxin in Macrophomina phaseolina-infected seeds.

A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue.

T-lymphocyte responses to plicatic acid-human serum albumin conjugate in occupational asthma caused by western red cedar.

BACKGROUND: T cells are known to play a major role in the pathogenesis of atopic allergic asthma, but it is less clear whether they are involved in occupational asthma caused by low molecular weight chemicals such as plicatic acid. OBJECTIVES: We sought to determine whether peripheral blood T cells from patients with western red cedar asthma (WRCA) recognize plicatic acid (PA) conjugated to human serum albumin (HSA) as judged by proliferation or cytokine production and to analyze the response to PA inhalation with flow cytometry. RESULTS: Significant proliferative responses to PA-HSA were observed in eight of 33 patients with WRCA, none of 10 exposed nonasthmatic cedar workers, and one of 18 nonasthmatic control subjects. Two of 25 patients with WRCA also showed proliferative responses to unconjugated PA. All the WRCA responders were either currently exposed to cedar or had ceased exposure within the preceding 2 years. None of the four patients receiving oral steroids responded, but inhaled steroids did not seem to influence responsiveness. No correlations were found between the maximum stimulation response and any of the current FEV1 values, the current PC20 methacholine values, or the magnitude of the late asthmatic response to PA. Peripheral blood T-cell subset proportions and their degree of activation were similar in patients with WRCA and exposed control subjects. There was no change in T-cell phenotypes or their activation markers after PA inhalation challenge. In vitro, PA-HSA stimulation did not affect subset ratios but led to release of small amounts of IL-5 and IFN-gamma, with no detectable increase in IL-4. CONCLUSIONS: PA-HSA-specific T lymphocytes seem to be present in small numbers in the peripheral blood of patients with WRCA and may respond to antigenic exposure by producing IFN-gamma and IL-5. However, the proportion of responding cells would appear to be lower than in comparable studies of atopic asthma.

Occupational asthma caused by eastern white cedar (Thuja occidentalis) with demonstration that plicatic acid is present in this wood dust and is the causal agent.

A worker developed symptoms of work-related asthma a few weeks after starting to work in a sawmill where eastern white cedar (Thuja occidentalis) was transformed into shingles. The diagnosis of occupational asthma was confirmed by monitoring of peak expiratory flow rates and bronchial responsiveness to histamine off work and at work, and specific inhalation challenges in the laboratory that demonstrated an isolated late asthmatic reaction after exposure for 4 hours to the wood dust. Specific inhalation challenges with western red cedar (thuja plicata) for 2 hours and plicatic acid (PA) for 7 minutes also caused an isolated late asthmatic reaction. Elevated specific IgE levels to PA were present. Antiserum was produced in rabbits that were immunized with PA conjugated to human serum albumin. With this antiserum in inhibition experiments, cross-reactivity between western red cedar and eastern white cedar, both belonging to the family of arborvitae, was found. It is estimated that eastern white cedar contains approximately half the amount of PA present in western red cedar.