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Keywords:

  • (1[RIGHTWARDS ARROW]3)-β-D-glucan;
  • Review;
  • Respiratory health;
  • Fungi;
  • Damp;
  • Epidemiology

Abstract

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

Abstract

(1[RIGHTWARDS ARROW]3)-β-D-glucan are non-allergenic structural cell wall components of most fungi that have been suggested to play a causal role in the development of respiratory symptoms associated with indoor fungal exposure. This review describes the currently available epidemiological literature on health effects of (1[RIGHTWARDS ARROW]3)-β-D-glucan, focusing on atopy, airway inflammation and symptoms, asthma, and lung function. In addition to population studies, studies in human volunteers experimentally exposed to (1[RIGHTWARDS ARROW]3)-β-D-glucan are described as well as relevant animal studies. Furthermore, the review discusses exposure assessment methods, the potential for exposure control and it concludes with identifying research needs. The observational and experimental studies reviewed suggested some association between (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure, airway inflammation and symptoms, however, results were mixed and specific symptoms and potential underlying inflammatory mechanisms associated with exposure could not be identified. Large observational studies using well validated exposure assessment methods are needed to further our knowledge regarding the potential health effects of indoor (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure.


Introduction

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

Exposure to indoor fungi has widely been recognized as a plausible cause of dampness-related respiratory morbidity (Peat et al., 1998; Zock et al., 2002). However, the potential mechanisms for fungal-related airway diseases caused by indoor exposures are not clear. Some studies have shown associations between fungal exposure, sensitization and asthma (Black et al., 2000; Halonen et al., 1997; Zureik et al., 2002), but the evidence that fungal allergens and IgE allergic responses play a major role in indoor related respiratory symptoms is still very limited (Douwes and Pearce, 2003). In addition to allergic mechanisms, non-allergic responses to fungal exposures have been reported mainly in relation to fungal (1[RIGHTWARDS ARROW]3)-β-D-glucans. The first reports suggesting a potential role for (1[RIGHTWARDS ARROW]3)-β-D-glucans in the development of indoor air-related health effects appeared in the late 1980s and since then, a limited number of population studies have been published, mainly because of the fact that commercially available methods to analyze (1[RIGHTWARDS ARROW]3)-β-D-glucan were not widely available until some years ago.

(1[RIGHTWARDS ARROW]3)-β-D-glucan are non-allergenic water-insoluble structural cell wall components of most fungi, some bacteria, most higher plants and many lower plants (Stone and Clark, 1992). Glucans may account for up to 60% of the dry weight of the cell wall of fungi, of which the major part is (1[RIGHTWARDS ARROW]3)-β-D-glucan (Klis, 1994). They consist of glucose polymers with variable molecular weight and degree of branching i.e. triple helix, single helix or random coil structures (Williams, 1997). In the fungal cell wall, (1[RIGHTWARDS ARROW]3)-β-D-glucans are linked to proteins, lipids and carbohydrates such as mannan and chitin, and they contain (1[RIGHTWARDS ARROW]6)-β-glucan side-branches which may connect with adjacent (1[RIGHTWARDS ARROW]3)-β-D-glucan polymers (Klis, 1994). The (1[RIGHTWARDS ARROW]3)-β-D-glucan content of fungal cell walls has been reported to be relatively independent of growth conditions (Foto et al., 2004; Rylander, 1997a).

(1[RIGHTWARDS ARROW]3)-β-D-glucans can initiate a wide range of biological responses in vertebrates including stimulation of the reticuloendothelial system (Di Luzio, 1979), activation of neutrophils (Zhang and Petty, 1994), macrophages (Adachi et al., 1994; Lebron et al., 2003) complement (Saito et al., 1992) and possibly eosinophils (Ramani et al., 1988), resulting in enhancement of host-mediated induced resistance to infections and antitumor activity (Stone and Clark, 1992). (1[RIGHTWARDS ARROW]3)-β-D-glucans thus have potent biological properties, some of which may also play a role in the adverse health effects associated with indoor mold exposure. These biological properties are not dependent on viability and (1[RIGHTWARDS ARROW]3)-β-D-glucans from dead organisms may thus be equally relevant in causing potential health effects.

Sampling

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

In many studies reservoir dust from carpets or mattresses is collected and concentrations are usually expressed either in weight units per gram of sampled dust or per meter square. Although both measures are generally accepted, the latter measure may better reflect actual exposure [Institute of Medicine (IOM), 2004]. The advantage of settled dust sampling is the presumed time-integration that occurs in the deposition of fungal (1[RIGHTWARDS ARROW]3)-β-D-glucan on surfaces over time (IOM, 2000). Fungi may also proliferate in carpets provided there is sufficient access to water. However, surface sampling is a crude measure that is most likely only a poor surrogate for airborne concentrations.

Airborne sampling requires very sensitive analytical methods. In addition, for an accurate assessment potentially large numbers of samples need to be collected as temporal variation in airborne concentrations is most likely very high (IOM, 2004). Airborne sampling after agitation of settled dust – as has been employed in several studies (Rylander, 1997b; Rylander et al., 1992, 1998; Thorn and Rylander, 1998a) – may overcome this problem. In addition, it may have the advantage (over settled dust sampling) that the more appropriate dust fraction i.e. airborne or inhalable particles will be sampled. However, these assumptions have not been confirmed, and procedures have not been standardized and/or validated. Therefore, uncertainty in exposure assessment of (1[RIGHTWARDS ARROW]3)-β-D-glucan is generally large which may result in obscured exposure-response relationships in epidemiological studies.

Analytical methods

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

Most studies use the glucan-specific limulus amebocyte lysate (LAL) assay [available from Associates of Cape Cod, Falmouth, MA, USA (a subsidiary of Seikagaku Corporation, Tokyo, Japan)] which is based on the same principles as the LAL assay described for endotoxin measurements (Aketagawa et al., 1993). However, rather than activating factor C, glucans activate factor G leading to a series of enzymatic reactions resulting in a color or turbidimetric response. The glucan-specific LAL assay does not cross-react with endotoxin as factor C has either been removed or disabled from the LAL preparation. Recently a modification of the conventional LAL assay (containing both factor C and G) has been described for the analysis of (1[RIGHTWARDS ARROW]3)-β-D-glucan (Foto et al., 2004). The modification comprises pre-treatment of samples with 0.5 N NaOH which is known to destroy endotoxin and increase the response of factor G to (1[RIGHTWARDS ARROW]3)-β-D-glucans. Only little experience is available with the modified LAL assay and a comparison with the glucan-specific assay has not been conducted. In addition, NaOH treatment does not destroy all endotoxin in complex samples containing a mixture of both endotoxin and (1[RIGHTWARDS ARROW]3)-β-D-glucan (Foto et al., 2004). False positive results can therefore not be excluded.

(1[RIGHTWARDS ARROW]3)-β-D-glucan-specific immunoassays have also been developed (Douwes et al., 1996; Milton et al., 2001). In the inhibition enzyme-linked immunoassay (ELISA), (1[RIGHTWARDS ARROW]3)-β-D-glucans in the test sample inhibit the binding of affinity-purified rabbit anti-glucan antibodies to the (1[RIGHTWARDS ARROW]3)-β-D-glucans coated to the microtiter plate (Douwes et al., 1996). Quantification is achieved by labeling the rabbit antibodies with an enzyme-linked anti-rabbit antibody. In the sandwich ELISA, galactosyl ceramide is used as the capture reagent and a monoclonal antibody specific for (1[RIGHTWARDS ARROW]3)-ß-D-glucans is used as the detector reagent (Milton et al., 2001). The ELISA methods are considerably cheaper than the LAL assays but are (as yet) not commercially available, and extensive experience in the indoor environment is only available for the inhibition ELISA. In addition, the ELISA methods are significantly less sensitive than LAL methods. No comparison studies using environmental samples have been conducted and results obtained using different analytical methods may therefore not be directly comparable.

Most (1[RIGHTWARDS ARROW]3)-β-D-glucans present in nature are not water-soluble at room temperature. Therefore, two alternative methods of extraction of environmental samples have been described in the literature: (i) alkaline extraction using either 0.3 m NaOH (Rylander et al., 1992) or 0.5 N NaOH (Foto et al., 2004); and (ii) heat extraction by autoclaving samples at 120°C (1 bar) for 1 h (Douwes et al., 1996). The extraction efficiency of both methods is not known. Alkaline extraction is most often employed when samples are analyzed using the LAL assay, whereas heat extraction is commonly used in combination with the ELISA method(s). No differences in (1[RIGHTWARDS ARROW]3)-β-D-glucan content were seen between both extraction methods with the inhibition ELISA method (Douwes et al., 1996). However, large differences are likely for the LAL assay i.e. heat extraction will most likely underestimate the glucan concentration as it favors the formation of triple helix structures and these structures are severely underestimated in the LAL assay. Alkaline treatment on the other hand, transforms triple helix to single helix or random coil formations which are associated with increased factor G activation. Therefore, alkaline extraction may be the most optimal extraction method to be used in combination with the LAL assay. Apart from one small comparison (Douwes et al., 1996), no validation work has been conducted and large differences in exposure assessment because of differences in extraction procedures may thus occur.

Epidemiological studies

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

An overview of population studies published in the scientific literature is presented in Table 1. A large number of health effects has been evaluated including lung function [forced expiratory volume in 1 s (FEV1) and peak flow (PEF) variability], nasal congestion, airway hyperreactivity, atopy, symptoms (upper and lower respiratory symptoms, eye irritations, head ache, fatigue/tiredness, joint pains, skin symptoms, flu-like symptoms, nausea, gastro-intestinal symptoms), inflammation characterized by inflammatory cells (T-lymphocytes, neutrophils, eosinophils, macrophages), and cytokines and other inflammatory markers [interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-10, Interferon (INF)-γ, Tumour necrosis factor (TNF)-α, Eosinophil cationic protein (ECP), Myeloperokidase (MPO), C-reactive protein (CRP), albumin] in blood, sputum and nasal lavage. Most studies included a range of these outcomes generally resulting in only a few significant associations (see Table 1). Although the focus has been on these positive findings (Rylander and Lin, 2000) it is clear that the results were not always consistent (see below).

Table 1.  Overview of epidemiological studies, a case study and human challenge studies regarding ß(1[RIGHTWARDS ARROW]3)-D-glucan exposure and associated health effects
Reference/environmentExposure measurementsStudied health effects
nAssayConcentration (range or means)No. subjectsAssociated with exposureNot associated with exposure
  1. a Analyses were not adjusted for other risk factors; bAn association was only found for specific sub groups; cNo association was found for the whole population, or when other exposure categories were used; dAnalyses were adjusted for endotoxin and dust-mite allergen levels; eAnalyses were adjusted for endotoxin exposure; fGlucan levels were very strongly correlated with endotoxin; analyses were not adjusted for endotoxin.

  2. * Same study population as Thorn and Rylander (1998a).

  3. ** Subjects were selected from Heldal et al. (2003a).

  4. *** Subjects were selected from Thorn and Rylander (1998a).

  5. † This study comprised a comparison of only two exposure situations, therefore no direct association between glucan and symptoms could be assessed.

  6. ‡ ELISA results were below LOD.

  7. [UPWARDS ARROW] positive association; [DOWNWARDS ARROW]negative association; [DOWNWARDS ARROW][UPWARDS ARROW]both negative and positive associations were found.

  8. LAL, Limulus Amebocyte Lysate; ELISA, Enzyme-linked immunosorbent assay; MPO, Myeloperoxidase; FEV1, Forced expiratory flow in 1 s; PEF, Peak flow; IFN, Interferon; IL, Interleukin; TNF, Tumour necrosis factor; MPO, Myeloperoxidase; BMNCs, Blood mononuclear cells; AH, Airway hyperreactivity; ECP, Eosinophil cationic protein; ns, non-significant; IgE, Immunoglobulin E; LOD, Limit of detection.

Indoor environment
Rylander et al., 199246LALProblem buildings: 0.2–0.55 ng/m339Dry cough [UPWARDS ARROW]; skin rashes [UPWARDS ARROW]aNose and eye irritations; chest tightness; head ache; tiredness; joint pains, etc.
 Schools, post office, day care36Control building: <0.1 ng/m3405
Rylander, 1997a24LALBefore renovation: 11.4 ng/m311Airway hyperreactivity [UPWARDS ARROW]aLung function; symptoms
 Day care center13After renovation: 1.2 ng/m3
Thorn and Rylander, 1998a75LAL0–19 ng/m3129Atopy [UPWARDS ARROW]b; serum MPO [UPWARDS ARROW]; FEV1[DOWNWARDS ARROW]bAtopyc; Airway hyperreactivity; ECP; C-reactive protein; FEVinline image; symptoms
 Row houses
Rylander et al., 19986LALProblem school: 15.3 ng/m365Cough [UPWARDS ARROW]; cough with phlegm [UPWARDS ARROW], hoarseness [UPWARDS ARROW]Atopy
 Schools11Control school: 2.9 ng/m3141
Wan and Li, 1999?LALDay care centers: 5.7 ng/m340Lethargy/fatigue [UPWARDS ARROW]Eye and nose irritations, skin and respiratory symptoms
 Day care, Offices, homesOffice buildings: 3.2 ng/m369
 Homes: 3.7 ng/m322
Douwes et al., 2000a69ELISANon-symptomatic children: 126 ng/m269PEF variability in symptomatic children [UPWARDS ARROW]dNo other health effects were studied
 Homes74Symptomatic children: 169 ng/m274
Beijer et al., 2003* Row houses17LALHigh exposed: 6 ng/m317 high expCytotoxic CD8+ T-cells [UPWARDS ARROW]; IFN-γ/IL-4 ratio after in vitro stimulation of BMNCs [UPWARDS ARROW]bBMC secretion of IL-10 and Il-1β, serum ECP, MPO, IFN-γ and IL-4; differential cell counts in blood; symptoms
 18Low exposed: 0.9 ng/m318 low exp
Occupational environment
Mandryk et al., 199954LALSawmill: 1.4 ng/m3168Base line lung function [UPWARDS ARROW]; cross-shift lung function [UPWARDS ARROW][DOWNWARDS ARROW];No other health effects were studied
 Sawmill, wood chipping, joineries39Joineries: 0.6 ng/m3
Mandryk et al., 200036LALGreen mills: 3 ng/m387Base line lung function [DOWNWARDS ARROW]; cross-shift lung function; respiratory symptoms [UPWARDS ARROW]; these effects were observed only in green mill workerseNo other health effects were studied
 Saw mills18Dry mills: ∼0.3 ng/m3
Rylander et al., 199920LALPaper mill: 2.0–97.7 ng/m383Throat and nose irritation [UPWARDS ARROW] (not significant); cough with phlegm [UPWARDS ARROW] (ns); joint pains[UPWARDS ARROW]; tiredness [UPWARDS ARROW]; flu-like symptoms [UPWARDS ARROW]; airway hyperreactivity (AH)[UPWARDS ARROW], ECP in blood [UPWARDS ARROW]; results for AH and ECP were not shown in the paper fLung function
 Paper industry8Controls: 0.1 ng/m344
Thorn and Rylander, 1998b House hold waste collectors20LALCompostable waste: 19.1 ng/m325 collectorsBlood lymphocytes [UPWARDS ARROW]Respiratory and other symptoms; lung function; airway hyperreactivity; inflammatory markers in induced sputum and serum/blood
 Unsorted waste: 9.2 ng/m320 controls
 Controls: 1.1 ng/m3 
Douwes et al., 2000b60ELISAFirst survey: 0.54–4.85 μg/m314No significant associations were foundInflammatory markers in nasal lavage
 Compost industry43Second survey: 0.36–4.44 μg/m315
Wouters et al., 2002118ELISA1.3 μg/m347No significant associations were foundInflammatory markers in nasal lavage
 Waste collectors
Heldal et al., 2003a93LAL40 ng/m331IL-8 in nasal lavage [UPWARDS ARROW]; nasal congestion [UPWARDS ARROW]Neutrophils; MPO and ECP in nasal lavage
 Waste handlers
Heldal et al., 2003b**25LAL52 ng/m325IL-8 in sputum [UPWARDS ARROW]; analyses were not adjusted for endotoxin whereas endotoxin was associated with IL-8 and other inflammatory markersNeutrophils; MPO and ECP in sputum
 Waste handlers
Gladding et al., 2003 Waste recycling156LAL4.8–40.1 ng/m3159Cough with phlegm [UPWARDS ARROW]; hoarse/parched throat [UPWARDS ARROW]; stomach problems [UPWARDS ARROW]; other respiratory symptoms [UPWARDS ARROW] (ns); skin rash [UPWARDS ARROW] (ns); nausea [UPWARDS ARROW] (ns); analyses were not adjusted for endotoxin whereas endotoxin was associated with symptoms.Blood lymphocytes, neutrophils, eosinophils and serum IgE
Blood monocytes and erythrocyte sedimentation rates were significantly lower in subjects working in one plant with elevated glucan levels compared with two other plants with lower glucan levelsf
Eduard et al., 200190ELISA0.82 μg/m3106No significant associations were foundRespiratory, eye and nose symptoms
 Farming
Case study
Rylander et al., 1994?LAL41.9 ng/m32Airway inflammation [UPWARDS ARROW]; cough [UPWARDS ARROW]; wheeze [UPWARDS ARROW]; tiredness [UPWARDS ARROW]; symptoms disappeared after moving out of the mold infested houseNo other health effects were studied
Human challenge studies
Rylander, 1996210 ng/m3 (aerosolized and particulate curdlan)26Nose and throat irritations [UPWARDS ARROW]; FEV1[DOWNWARDS ARROW]b; this was only observed for particulate curdlanFEVinline image; Airway hyperreactivity
Thorn et al., 2001210 ng/m3 (grifolan suspended in saline)21Blood eosinophil levels [DOWNWARDS ARROW]; TNF-α secretion by stimulated BMNCs [DOWNWARDS ARROW]; FEV1[UPWARDS ARROW]Sputum ECP, MPO, TNF-α, IL-8, IL-10, eosinophil, lymphocyte, macrophage, and neutrophils; Blood ECP, TNF-α, IL-10, eosinophil, lymphocyte, monocyte and neutrophils; lung function parameters other than FEV1
Beijer et al., 2002***28 ng/m3 (grifolan suspended in saline)17 high expTNF-α secretion by stimulated BMNCs [DOWNWARDS ARROW]; blood lymphocytes [UPWARDS ARROW]; These associations were only significant for the 17 subjects living in high exposure environmentsIL-1β, IL-4, IFN-γ secretion by stimulated BMNCs; serum ECP and MPO
18 low exp
Sigsgaard et al., 20001 mg/ml (nasal instillation of solubilized curdlan)5Albumin and IL-1β in nasal lavageTNF-α, IL-1β, IL-6 and IL-8 in nasal lavage

Upper airway irritations, lung function and airway responsiveness

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

Several studies reported associations between (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure and upper airway irritations and fatigue/tiredness (Gladding et al., 2003; Heldal et al., 2003a; Mandryk et al., 2000; Wan and Li, 1999), however, these associations were not confirmed in other studies (Rylander, 1997b; Rylander et al., 1992; Thorn and Rylander, 1998b). Also, no clear associations with lung function were found i.e. some studies reported adverse effects on lung function (Douwes et al., 2000a; Mandryk et al., 2000; Rylander, 1996) whereas others found no (Thorn and Rylander, 1998a,b) or even an opposite association (Mandryk et al., 1999; Thorn et al., 2001). Similarly, some studies reported significant effects on airway responsiveness (Rylander, 1997b) whereas others failed to demonstrate such an association (Thorn and Rylander, 1998a,b).

Atopy

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

One study suggested that (1[RIGHTWARDS ARROW]3)-β-D-glucan was associated with an increased risk of atopy (Thorn and Rylander, 1998a), similar as has been observed in some animal studies (see below). However, this was not confirmed in a smaller study (Rylander et al., 1998). Also, an association between (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure and an increased T helper 1 cell (Th1) immune response was suggested (Beijer et al., 2003) which appears to be contradictive with the finding of a higher prevalence of atopy in subjects with high (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure (NB atopy is a Th2 driven immune response). An up-regulation of Th1 immune activity was, however, only shown in non-atopic subjects. Finally, some studies suggested that associations between (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure and symptoms and lung function changes were stronger in atopics than in non-atopics (Douwes et al., 2000a; Rylander et al., 1998).

Airway inflammation

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

The data with regard to the potential effects of glucan on airway inflammation is also mixed. In vitro studies have demonstrated the potential of (1[RIGHTWARDS ARROW]3)-β-D-glucans to induce inflammatory responses (see below). In addition, several studies have shown that (1[RIGHTWARDS ARROW]3)-β-D-glucan may cause airway inflammation in laboratory animals (see below). However, in most population studies (and human challenge studies; see below) no such association was found (Table 1). Heldal et al. (2003a,b), found a significant association between (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure and IL-8 in sputum and nasal lavage in waste handlers but that may have also been caused by highly correlated endotoxin exposures. Endotoxin levels in this study were, however, relatively low (7–180 EU/m3).

Some observational studies also focused on inflammatory mediators in blood; one study showed an association between (1[RIGHTWARDS ARROW]3)-β-D-glucan and MPO (Thorn and Rylander, 1998a) but this was not confirmed in another study (Rylander et al., 1999). Also, no association between (1[RIGHTWARDS ARROW]3)-β-D-glucan and MPO in sputum was found (Heldal et al., 2003a; Thorn et al., 2001). A positive association was demonstrated between (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure and lymphocytes in blood (Thorn and Rylander, 1998b). Another study showed an inverse association between (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure and the number of cytotoxic CD8+ T-cells (Beijer et al., 2003) and Phytohemagglutinin (PHA) and Lipopolysaccharide (or endotoxin) (LPS)-induced TNF-α production by blood mononuclear cells (BMNCs) (Beijer et al., 2002; Thorn et al., 2001). An inhibited endotoxin-induced TNF-α production by alveolar macrophages has also been demonstrated in a study in rats exposed to (1[RIGHTWARDS ARROW]3)-β-D-glucan (see below). The meaning of these findings is not clear and results require further confirmation from both human and animal studies.

Limitations of the epidemiological studies

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

The population studies described above (and summarized in Table 1) suggest an association between exposure and a wide range of health effects, however, only a small number of all tested variables showed significant associations and results were often inconsistent. The lack of consistency between studies may largely be because of the relatively small sample size of most of the reported studies; some studies in fact involved a comparison of only two exposure situations (Rylander, 1997b; Rylander et al., 1998). Also, often only relatively few exposure measurements were taken. Therefore, uncertainty in exposure assessment was large which may have resulted in obscured exposure-response relationships. Another limitation in many of the reported field studies was the lack of control for potential confounders such as other common bioaerosol exposures that may cause similar health effects (Douwes et al., 2003). A number of studies showed a strong correlation between endotoxin and (1[RIGHTWARDS ARROW]3)-β-D-glucan levels (Douwes et al., 2000a,b; Gladding et al., 2003; Rylander et al., 1999) suggesting that some of the findings may potentially have been caused by endotoxin. However, in some of the studies where analyses were adjusted for allergen and endotoxin no major changes in the relationship between (1[RIGHTWARDS ARROW]3)-β-D-glucan and health effects were observed after adjustment (Douwes et al., 2000a; Mandryk et al., 2000). Another potential weakness is that in several studies (Gladding et al., 2003; Rylander et al., 1999; Thorn and Rylander, 1998a) exposure groups were constructed using cut-off points that were not based on objective criteria, but in stead the reasons for choosing cut-off points were not clear and did not appear to be driven on an a priori hypothesis precluding a straight forward interpretation. Similarly, in some studies (Beijer et al., 2003; Thorn and Rylander, 1998a) associations were only found when part of the study population was excluded whereas reasons for exclusion were often unclear. For example, one study in the general population (Thorn and Rylander, 1998a) showed that baseline FEV1 values were inversely related to (1[RIGHTWARDS ARROW]3)-β-D-glucans, but only when analyses were restricted to those younger than 65 yr and with glucan exposures above 1 ng/m3 (exclusion criteria were not motivated). When the data were analyzed using three instead of two exposure groups (criteria for cut-off points were unclear) no association with FEV1 was found. As criteria for subgroup analyses and exposure grouping were not clearly described results from these studies should be interpreted with caution.

Human challenge studies

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

In addition to the population studies discussed above several experimental exposure studies have also been conducted (Table 1). The first study showed a small increase in the severity of symptoms of nose and throat irritations after a challenge of aerosolized (1[RIGHTWARDS ARROW]3)-β-D-glucan (Rylander, 1996). No effects on FEV1 or airway responsiveness were found. Exposure to particulate (1[RIGHTWARDS ARROW]3)-β-D-glucan resulted in a significant but very small decrease in FEV1 directly and 3 days after exposure; no significant association was found with airway responsiveness. Another study failed to show airway inflammation after a (1[RIGHTWARDS ARROW]3)-β-D-glucan challenge (Thorn et al., 2001). Seventy-two hours after the challenge both blood eosinophil levels (borderline significant, P < 0.06) and TNF-α secretion by PHA stimulated BMNC were lower. No significant differences in lung function values were observed at 24 h, however, the FEV1 value was significantly higher 72 h after glucan challenge. A third study showed that (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure decreased TNF-α production of BMNCs in subjects with previous high glucan exposures but not in those with low indoor exposures (Beijer et al., 2002). No significant differences were found in BMNC secretion of the other studied cytokines (Table 1). Also no differences in ECP and MPO in serum were observed.

Sigsgaard et al. (2000) demonstrated an increase in albumin and a slight increase in IL-1ß in the nasal mucosa in a small group of Danish garbage workers (n = 5) and controls (n = 5) who were exposed to solubilized (1[RIGHTWARDS ARROW]3)-β-D-glucan by nasal instillation. No increase in the other tested cytokines was observed (Table 1). However, in a whole blood assay measuring cytokine release after ‘ex vivo’ exposure to high concentrations of (1[RIGHTWARDS ARROW]3)-β-D-glucans, a significant increase in all measured cytokines (TNF-α, IL-1ß, IL-6 and IL8) was found. This was confirmed in several other studies in which blood was collected from healthy volunteers (Wouters et al., 2002), atopics and non-atopics (Kruger et al., 2004), and municipality and fish factory workers (Bønløkke et al., 2004).

Results of the whole blood experiments demonstrate the pro-inflammatory potential of glucans at very high levels. However, in vivo challenge studies as described above show only minor inflammatory effects and no clear effects on lung function. However, these mainly negative results should be interpreted with caution as only a few are available and experiments were conducted with only two types of (1[RIGHTWARDS ARROW]3)-β-D-glucan (curdlan and grifolan) which may not necessarily be the most biologically active.

Animal studies

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

Studies in rats have demonstrated the inflammatory potency of (1[RIGHTWARDS ARROW]3)-β-D-glucans using high concentrations of Zymosan. Intratracheal installation of Zymosan induced a dose-dependent neutrophil influx into the airways and an increase in pulmonary albumin, breathing frequency and nitric oxide production by alveolar macrophages (Young et al., 2001). Exposure to particulate (1[RIGHTWARDS ARROW]3)-β-D-glucan induced greater pulmonary toxicity than soluble (1[RIGHTWARDS ARROW]3)-β-D-glucans (Young et al., 2003a). Also, a partially opened triple helix conformation of (1[RIGHTWARDS ARROW]3)-β-D-glucans was more active than the closed formation (Young et al., 2003b). The inflammatory potency thus appears strongly dependent on the type and conformation of (1[RIGHTWARDS ARROW]3)-β-D-glucans. However, there is reason for caution as some of the inflammatory activity may be because of other components as Zymosan is a rather crude mixture of glucans and other yeast cell wall components. In addition, other studies in guinea pigs did not show an acute inflammatory effect of (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure (Fogelmark et al., 1992, 1994). Repeated exposures on the other hand did result in an increase in inflammatory cells in the airways (Fogelmark et al., 1992, 1994), particularly eosinophils (Fogelmark et al., 2001). This was not confirmed in mice, in which lung specimens were normal (i.e. no signs of inflammation or fibrosis) at histopathological examination, even after repeated exposures to aerosolized (1[RIGHTWARDS ARROW]3)-β-D-glucan (Korpi et al., 2003).

Several studies have explored the potential modifying effects of (1[RIGHTWARDS ARROW]3)-β-D-glucan on endotoxin toxicity. In guinea pigs it was shown that the inflammatory response to endotoxin was reduced by simultaneous (1[RIGHTWARDS ARROW]3)-β-D-glucans exposure (Fogelmark et al., 1992, 1994). An inhibitory effect of (1[RIGHTWARDS ARROW]3)-β-D-glucan (Zymosan) on pulmonary responsiveness has also been demonstrated in rats, but only when they were pre-treated with (1[RIGHTWARDS ARROW]3)-β-D-glucan, and only for certain indicators of airway inflammation such as lactate dehydrogenase activity, albumin level, and pulmonary TNF-α, and alveolar macrophage production of reactive oxidant species. (Young et al., 2002). The endotoxin-induced increased breathing rate and neutrophil infiltration were not affected. In contrast, other studies have suggested that (1[RIGHTWARDS ARROW]3)-β-D-glucan may enhance the toxicity of endotoxin (Bower et al., 1986; Cook et al., 1980). Also, in guinea pigs, repeated exposures to a combination of (1[RIGHTWARDS ARROW]3)-β-D-glucans and endotoxin resulted in a larger increase in inflammatory cells than for the two components separately, suggesting an enhanced rather than an inhibitory effect (Fogelmark et al., 1992, 1994).

Finally, several studies assessed the effects of (1[RIGHTWARDS ARROW]3)-β-D-glucan on the development of specific IgE sensitization. One study in mice showed that pre-exposure to inhaled (1[RIGHTWARDS ARROW]3)-β-D-glucans enhanced ovalbumin-induced IgE antibody responses and airway antigen-specific allergic reactions including eosinophil infiltration (Wan and Li, 1999). In addition, it appeared that spleen cells derived from (1[RIGHTWARDS ARROW]3)-β-D-glucan exposed mice produced significantly higher amounts of IL-4 (IL-4 stimulates B-cells to IgE production and naïve Th0 cells to differentiate toward an atopic Th2 state) (Wan and Li 1999, unpublished results). This suggests that (1[RIGHTWARDS ARROW]3)-β-D-glucans may enhance atopic Th2 immune responses (Rylander and Lin, 2000). A study in guinea pigs that were exposed daily to an aerosol of (1[RIGHTWARDS ARROW]3)-β-D-glucans for a period of 5 weeks showed an increase in eosinophil numbers in the airways of exposed animals (Fogelmark et al., 2001) confirming the previous study to a certain extent. In addition, two other studies demonstrated that (1[RIGHTWARDS ARROW]3)-β-D-glucans isolated from barley, the fungus Sclerotinia sclerotiorum, and baker's yeast significantly increased the IgE immune response in mice (Instanes et al., 2004; Ormstad et al., 2000). In contrast, another study in guinea pigs showed that (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure caused a decrease in ovalbumin induced eosinophil numbers (Rylander and Holt, 1998).

Thus, animal experiments suggest that (1[RIGHTWARDS ARROW]3)-β-D-glucan has the potential (in high concentrations) to (i) induce neutrophilic and possibly eosinophilic airway inflammation; (ii) modify endotoxin-induced airway inflammation; and (iii) modify the atopic immune response. However, there is considerable reason for caution as the results were not always consistent. This may be because of differences in solubility and conformation of the (1[RIGHTWARDS ARROW]3)-β-D-glucans used in these experiments (Young et al., 2003a,b). Differences in administration and timing of (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure may be another reason.

Exposure control

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

If environmental exposures to (1[RIGHTWARDS ARROW]3)-β-D-glucans cause health effects as suggested then control is essential. Specific measures to control exposure have not been developed but several studies investigated the association between exposure and housing characteristics and occupant behavior. These studies may help to develop procedures to manage exposure in the home environment.

Gehring et al. (2001) showed that (1[RIGHTWARDS ARROW]3)-β-D-glucan levels in house dust were strongly associated with the amount of dust on the floor. Presence of carpets, mold growth and pets (particularly dogs) in the home, number of occupants, time spent indoors by the occupants, and low frequency of cleaning were also associated with higher levels of dust and (1[RIGHTWARDS ARROW]3)-β-D-glucan (Douwes et al., 1998; Gehring et al., 2001; Rylander et al., 1994, 1998). A (weak) positive association between indoor relative humidity and (1[RIGHTWARDS ARROW]3)-β-D-glucan levels in the home has also been demonstrated (Gehring et al., 2001). Also, one study reported that homes in which organic waste was separated and stored indoors in kitchen compost bins (as is becoming increasingly common in many European countries) had substantially higher levels of (1[RIGHTWARDS ARROW]3)-β-D-glucan indoors (Wouters et al., 2000). Therefore, (1[RIGHTWARDS ARROW]3)-β-D-glucan levels may be lowered by measures to reduce indoor dust, mold growth and humidity. In addition, avoiding the use of indoor compost bins for organic waste storage may also lower indoor (1[RIGHTWARDS ARROW]3)-β-D-glucan levels.

Conclusions

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

The observational and experimental studies described above suggest some association between (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure, airway inflammation and symptoms, however, results are mixed and specific symptoms associated with exposure can at this stage not be identified. Based on subgroup analyses, it has been speculated that atopics and/or subjects with pre-existing symptoms may be more susceptible (Douwes et al., 2000a; Rylander et al., 1998) but this requires further study. Ex vivo observations and animal experiments indicate that (1[RIGHTWARDS ARROW]3)-β-D-glucan at high concentrations have the potential to initiate airway inflammation, however, in inhalation experiments in human (at much lower doses) these effects could not consistently be reproduced. This may be because of the fact that inhalation experiments were conducted with soluble (1[RIGHTWARDS ARROW]3)-β-D-glucan that may not represent the most potent (1[RIGHTWARDS ARROW]3)-β-D-glucan fraction. Currently, a clear interpretation of the potential health effects of (1[RIGHTWARDS ARROW]3)-β-D-glucan is hampered because of the fact that (i) only a relatively small number of observational studies are available, most of which lacked statistical power; (ii) some of these studies have not appropriately controlled the analyses for other potential causal exposures and/or had weaknesses in the design and/or statistical analyses; and (iii) different methods to assess exposure were used and only relatively few samples were collected, potentially resulting in substantial exposure misclassification obscuring exposure – response relationships. Thus, the currently available epidemiological data do not permit conclusions to be drawn regarding the presence (or absence!) of an association between environmental glucan exposure and specific adverse health effects, nor is it clear from the currently available evidence which specific inflammatory mechanisms underlie the presumed health effects.

Recommendations for future research

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

In addition to more and larger observational studies there is a need for human challenge studies testing various conformations of (1[RIGHTWARDS ARROW]3)-β-D-glucans (triple, single helix and random coil) as well as soluble and non-soluble forms of glucan. Moreover, there is a clear need for validation and further development of existing methods to measure environmental (1[RIGHTWARDS ARROW]3)-β-D-glucan. This should include a comparison of extraction methods as well as a comparison between the currently commonly applied assays to measure (1[RIGHTWARDS ARROW]3)-β-D-glucan i.e. the LAL and the inhibition ELISA assay. Other important areas that require further research include: (i) issues regarding individual susceptibility for (1[RIGHTWARDS ARROW]3)-β-D-glucans; (ii) the potential interaction effects between (1[RIGHTWARDS ARROW]3)-β-D-glucans and other exposures such as allergens and endotoxin; (iii) more research into other health effects such as skin conditions, neurological and gastro-intestinal symptoms, all of which have been speculated to be related to (1[RIGHTWARDS ARROW]3)-β-D-glucan exposure; (iv) the assessment of the biological properties of non-fungal (1[RIGHTWARDS ARROW]3)-β-D-glucan; and (v) the assessment of determinants of exposure to allow more specific control measures to be developed.

Acknowledgements

  1. Top of page
  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References

The author would like to thank Professor Neil Pearce and Dr Wijnand Eduard for their valuable comments on the draft manuscript. Jeroen Douwes is supported by a sir Charles Hercus Research Fellowship from the Health Research Council (HRC) of New Zealand. The Centre for Public Health Research is supported by a Program Grant from the HRC of New Zealand.

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  2. Introduction
  3. Exposure assessment
  4. Sampling
  5. Analytical methods
  6. Epidemiological studies
  7. Upper airway irritations, lung function and airway responsiveness
  8. Atopy
  9. Airway inflammation
  10. Limitations of the epidemiological studies
  11. Human challenge studies
  12. Animal studies
  13. Exposure control
  14. Conclusions
  15. Recommendations for future research
  16. Acknowledgements
  17. References
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