Cellular and molecular mechanisms of fungal ß-(1→6)-glucan in macrophages.

Over the last 40 yr, the majority of research on glucans has focused on ß-(1→3)-glucans. Recent studies indicate that ß-(1→6)-glucans may be even more potent immune modulators than ß-(1→3)-glucans. Mechanisms by which ß-(1→6)-glucans are recognized and modulate immunity are unknown. In this study, we examined the interaction of purified water-soluble ß-(1→6)-glucans with macrophage cell lines and primary peritoneal macrophages and the cellular and molecular consequences of this interaction. Our results indicate the existence of a specific ß-(1→6)-glucan receptor that internalizes the glucan ligand via a clathrin-dependent mechanism. We show that the known ß-(1→3)-glucans receptors are not responsible for ß-(1→6)-glucan recognition and interaction. The receptor-ligand uptake/interaction has an apparent dissociation constant (KD) of ∼ 4 µM, and was associated with phosphorylation of ERK and JNK but not IκB-α or p38. Our results indicate that macrophage interaction with ß-(1→6)-glucans may lead to modulation of genes associated with anti-fungal immunity and recruitment/activation of neutrophils. In summary, we show that macrophages specifically bind and internalize ß-(1→6)-glucans followed by activation of intracellular signaling and modulation of anti-fungal immune response-related gene regulation. Thus, we conclude that the interaction between innate immunity and ß-(1→6)-glucans may play an important role in shaping the anti-fungal immune response.

Comparison of the potency of a variety of ß-glucans to induce cytokine production in human whole blood.

ß-Glucans are components of fungal cell walls and potent stimulants of innate immunity. The majority of research on biological activities of glucans has focused on ß-(1→3)-glucans, which have been implicated in relation to fungal exposure-associated respiratory symptoms and as important stimulatory agents in anti-fungal immune responses. Fungi-and bacteria and plants-produce a wide variety of glucans with vast differences in the proportion and arrangement of their ß-(1→3)-, -(1→4)- and -(1→6)-glycosidic linkages. Thus far, the pro-inflammatory potential of different ß-glucans has not been studied within the same experimental model. Therefore, we compared the potency of 13 different glucan preparations to induce in vitro production of IL-1ß, IL-6, IL-8 and TNF-α in human, whole blood cultures. The strongest inducers of all cytokines were pustulan [ß-(1→6)-glucan], lichenan [ß-(1→3)-(1→4)-glucan], xyloglucan [ß-(1→4)-glucan] and pullulan [α-(1→4)-(1→6)-glucan]. Moderate-to-strong cytokine production was observed for curdlan [ß-(1→3)-glucan], baker's yeast glucan [ß-(1→3)-(1→6)-glucan] and barley glucan [ß-(1→3)-(1→4)-glucan], while all other glucan preparations induced very low, or no, detectable levels of cytokines. We therefore conclude that innate immunity reactions are not exclusively induced by ß-(1→3)-glucans, but also by ß-(1→6)- and ß-(1→4)-structures. Thus, not only ß-(1→3)-glucan, but also other ß-glucans and particularly ß-(1→6)-glucans should be considered in future research.

Measurement of ß-(1,3)-glucan in household dust samples using Limulus amebocyte assay and enzyme immunoassays: an inter-laboratory comparison.

Environmental levels of ß-(1,3)-glucan, an inflammatory fungal cell wall component, have been suggested to be related to respiratory symptoms. However there is currently little data comparing ß-(1,3)-glucan detection methods and/or results obtained in different laboratories. The aim of this study was to compare levels of ß-(1,3)-glucans detected in household dust samples (n = 40) using different extraction/detection methods (Limulus amebocyte assay (LAL), inhibition enzyme immunoassay (EIA) and sandwich EIA) in five different laboratories. Dust sample aliquots were sent to participating centres, extracted and analysed for ß-(1,3)-glucan according to standard in-house procedures. Significant differences in the levels of ß-(1,3)-glucan were observed between all laboratories (geometric mean levels ranging from 15.4 µg g (-1) to 4754 µg g(-1) dust; p < 0.0001) with the exception of those using a similar LAL method. The inhibition EIA used in laboratory D produced mean ß-(1,3)-glucan measurements 80-100 times higher than the LAL assays, 4 times higher than the sandwich EIA in the same lab, 17.6 times those obtained with the EIA in lab E and 363 times those obtained in the EIA in laboratory C. Pearson's correlations generally showed significant associations between methods and laboratories, particularly those using similar methodology (R ranging from 0.5 to 0.8; p < 0.001), although some poor and even inverse correlations were observed. Bland-Altman analyses showed moderate to good agreement between most assays, although clear absolute differences were observed. In conclusion, although results obtained with different methods were often significantly correlated and therefore comparable in relative terms, direct comparison of results between laboratories and assays may be inappropriate.

IgG to various beta-glucans in a human adult population.

BACKGROUND: Fungal ß-(1,3)-glucans are pro-inflammatory agents, and exposures to ß-(1,3)-glucans are associated with respiratory tract symptoms. IgG anti-(1,3)-glucan titers are measured in diagnosis of fungal infections. Although other ß-glucan structures exist, like ß-(1,6)-glucans, little is known about their antigenic or pro-inflammatory properties. We aimed to investigate IgG titers and specificities in human sera against different ß-glucans with varying structures. METHODS: IgG anti-ß-glucan was measured by enzyme immunoassay in a random sample of 40 sera from healthy adults, with a panel of 8 differently structured glucans. In a subsequent larger series, IgG anti-ß-(1,6)-glucan was measured in a random sample of 667 sera from three occupational populations with different organic dust exposures. Possible determinants of IgG anti-ß-(1,6)-glucan titers were explored with linear-regression analysis. RESULTS: We found wide variation in anti-ß-glucan IgG levels. The highest titers were found for pure ß-(1,6)-glucan pustulan. Moderate to strong reactions with other ß-(1,6)-containing structures appeared to be due to cross-reacting anti-ß-(1,6)-glucan antibodies. Surprisingly, the mean IgG anti-ß-(1,6)-glucan titer was significantly lower in agricultural workers - with highest organic dust exposure - than in spray painters and bakery workers. Smoking status was associated with lower IgG anti-ß-(1,6)-glucan titers in all populations. CONCLUSIONS: IgG to ß-(1,3)- and ß-(1,6)-glucans can be found in normal human sera. ß-(1,6)-glucans appear to be much more potent antigens. The health impact of high anti-ß-(1,6)-glucan antibody levels remains unclear and further investigations are needed.

C. albicans increases cell wall mannoprotein, but not mannan, in response to blood, serum and cultivation at physiological temperature.

The cell wall of Candida albicans is central to the yeasts ability to withstand osmotic challenge, to adhere to host cells, to interact with the innate immune system and ultimately to the virulence of the organism. Little is known about the effect of culture conditions on the cell wall structure and composition of C. albicans. We examined the effect of different media and culture temperatures on the molecular weight (Mw), polymer distribution and composition of cell wall mannan and mannoprotein complex. Strain SC5314 was inoculated from frozen stock onto yeast peptone dextrose (YPD), blood or 5% serum agar media at 30 or 37°C prior to mannan/mannoprotein extraction. Cultivation of the yeast in blood or serum at physiologic temperature resulted in an additive effect on Mw, however, cultivation media had the greatest impact on Mw. Mannan from a yeast grown on blood or serum at 30°C showed a 38.9 and 28.6% increase in Mw, when compared with mannan from YPD-grown yeast at 30°C. Mannan from the yeast pregrown on blood or serum at 37°C showed increased Mw (8.8 and 26.3%) when compared with YPD mannan at 37°C. The changes in Mw over the entire polymer distribution were due to an increase in the amount of mannoprotein (23.8-100%) and a decrease in cell wall mannan (5.7-17.3%). We conclude that C. albicans alters the composition of its cell wall, and thus its phenotype, in response to cultivation in blood, serum and/or physiologic temperature by increasing the amount of the mannoprotein and decreasing the amount of the mannan in the cell wall.

Airborne cultivable microflora and microbial transfer in farm buildings and rural dwellings.

OBJECTIVES: Exposure to environments rich in microorganisms such as farms has been shown to protect against the development of childhood asthma and allergies. However, it remains unclear where, and how, farm and other rural children are exposed to microbes. Furthermore, the composition of the microbial flora is poorly characterised. We tested the hypothesis that farm children are exposed indoors to substantial levels of viable microbes originating from animal sheds and barns. We also expected that environmental microbial flora on farms and in farm homes would be more complex than in the homes of rural control children. METHODS: Dust samples were collected using passive samplers in the bedrooms of the following groups of children in rural Bavaria, Germany: (i) those living on farms (n=144), (ii) those regularly exposed to farm environments but not living on farms (n=149) and (iii) those never visiting farms (n=150). For farm children, additional samples were collected in animal sheds and barns. All samples were subjected to fungal and bacterial culturing. RESULTS: Detectable levels of microorganisms were more often found in samples taken from farm dwellings than from other homes. Farm dwellings also showed higher microbial levels. Microbial counts of farm dwelling samples correlated with the counts in corresponding animal sheds and barns. CONCLUSIONS: Microorganisms are transported from animal sheds and barns into farm dwellings. Therefore, children living in these environments are exposed when indoors and when visiting animal sheds and barns. Indoor exposure may also contribute to the protective effect of the farm environment.

Development of a sandwich ELISA to measure exposure to occupational cow hair allergens.

BACKGROUND: Cow hair and dander are important inducers of occupational allergies in cattle-exposed farmers. To estimate allergen exposure in farming environments, a sensitive enzyme immunoassay was developed to measure cow hair allergens. METHODS: A sandwich ELISA was developed using polyclonal rabbitantibodies against a mixture of hair extracts from different cattle breeds. To assess the specificity of the assay, extracts from other mammalian epithelia, mites, molds and grains were tested. To validate the new assay, cow hair allergens were measured in passive airborne dust samples from the stables and homes of farmers. Dust was collected with electrostatic dust fall collectors (EDCs). RESULTS: The sandwich ELISA was found to be very sensitive (detection limit: 0.1 ng/ml) and highly reproducible, demonstrating intra- and interassay coefficients of variation of 4 and 10%, respectively. The assay showed no reactivity with mites, molds and grains, but some cross-reactivity with other mammalian epithelia, with the strongest reaction with goat. Using EDCs for dust sampling, high concentrations of bovine allergens were measured in cow stables (4,760-559,400 µg/m²). In addition, bovine allergens were detected in all areas of cattle farmer dwellings. A large variation was found between individual samples (0.3-900 µg/m²) and significantly higher values were discovered in changing rooms. CONCLUSION: The ELISA developed for the detection of cow hair proteins is a useful tool for allergen quantification in occupational and home environments. Based on its low detection limit, this test is sensitive enough to detect allergens in passive airborne dust.

Passive airborne dust sampling with the electrostatic dustfall collector: optimization of storage and extraction procedures for endotoxin and glucan measurement.

We recently introduced a passive dust sampling method for airborne endotoxin and glucan exposure assessment-the electrostatic dustfall collector (EDC). In this study, we assessed the effects of different storage and extraction procedures on measured endotoxin and glucan levels, using 12 parallel EDC samples from 10 low exposed indoor environments. Additionally, we compared 2- and 4-week sampling with the prospect of reaching higher dust yields. Endotoxin concentrations were highest after extraction with pyrogen-free water (pf water) + Tween. Phosphate-buffered saline (PBS)-Tween yielded significantly (44%) lower levels, and practically no endotoxin was detected after extraction in pf water without Tween. Glucan levels were highest after extraction in PBS-Tween at 120 degrees C, whereas extracts made in NaOH at room temperature or 120 degrees C were completely negative. Direct extraction from the EDC cloth or sequential extraction after a preceding endotoxin extraction yielded comparable glucan levels. Sample storage at different temperatures before extraction did not affect endotoxin and glucan concentrations. Doubling the sampling duration yielded similar endotoxin and only 50% higher glucan levels. In conclusion, of the tested variables, the extraction medium was the predominant factor affecting endotoxin and glucan yields.

beta-(1,3)-Glucan exposure assessment by passive airborne dust sampling and new sensitive immunoassays.

Associations between house dust-associated beta-(1,3)-glucan exposure and airway inflammatory reactions have been reported, while such exposures in early childhood have been suggested to protect against asthma and wheezing. Most epidemiological studies have used reservoir dust samples and an inhibition enzyme immunoassay (EIA) for beta-(1,3)-glucan exposure assessment. The objective of this study was to develop inexpensive but highly sensitive enzyme immunoassays to measure airborne beta-(1,3)-glucans in low-exposure environments, like homes. Specificities of available anti-beta-(1,3)-glucan antibodies were defined by direct and inhibition experiments. Three suitable antibody combinations were selected for sandwich EIAs. beta-(1,3)-Glucans in passive airborne dust collected with an electrostatic dust fall collector (EDC) and floor dust from seven homes were measured with the three EIAs. Floor dust samples were additionally analyzed in the inhibition EIA. The sandwich EIAs were sensitive enough for airborne glucan measurement and showed different specificities for commercial glucans, while the beta-(1,3)-glucan levels in house dust samples correlated strongly. The feasibility of measuring glucans in airborne dust with the recently introduced EDC method was further investigated by selecting the most suitable of the three EIAs to measure and compare beta-(1,3)-glucan levels in the EDC and in floor and actively collected airborne dust samples of the previously performed EDC validation study. The EDC beta-(1,3)-glucan levels correlated moderately with beta-(1,3)-glucans in actively collected airborne dust and floor dust samples, while the glucan levels in the airborne dust and floor dust samples did not correlate. The combination of the newly developed beta-(1,3)-glucan sandwich EIA with EDC sampling now allows assessment in large-scale population studies of exposure to airborne beta-(1,3)-glucans in homes or other low-exposure environments.

Evaluation of a low-cost electrostatic dust fall collector for indoor air endotoxin exposure assessment.

Exposure to endotoxin in home environments has become a key issue in asthma and allergy research. Most studies have analyzed floor or mattress dust endotoxin, but its validity as a proxy for airborne exposure is unknown, while active airborne dust sampling is not feasible in large-scale population studies because of logistic and financial limitations. We therefore developed and evaluated a simple passive airborne dust collection method for airborne endotoxin exposure assessment. We explored an electrostatic dust fall collector (EDC), consisting of a 42- by 29.6-cm-sized folder with four electrostatic cloths exposed to the air. The EDC was tested during two 14-day periods in seven nonfarm and nine farm homes and in farm stables. In parallel, active airborne dust sampling was performed with Harvard impactors and floor dust collected by vacuuming, using nylon sampling socks. The endotoxin levels could be measured in all EDC cloth extracts. The levels (in EU/m(2)) between EDCs used simultaneously or in different sampling periods in the same home correlated strongly (r > 0.8). EDC endotoxin also correlated moderately to strongly (r = 0.6 to 0.8) with the endotoxin measured by active airborne dust sampling and living room floor dust sampling and-in farm homes-with the endotoxin captured by the EDC in stables. In contrast, endotoxin levels measured by floor dust sampling showed only a poor correlation with the levels measured by active airborne dust sampling. We therefore conclude that measuring endotoxin levels with the EDC is a valid measure of average airborne endotoxin exposure, while reproducibility over time is at least equivalent to that of reservoir dust analyses.