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Atopic dermatitis (AD) patients often demonstrate positive skin prick test results and serum IgE antibodies to a range of different yeasts. This has been thought to be due to cross-reactivity. In this study, the cross-reactivity of IgE and IgG antibodies between mannan and crude antigens of Pityrosporum ovale, Candida albicans, and Saccharomycescerevisiae and crude antigens of Cryptococcus albidus and Rhodotorula rubra was examined by RAST and ELISA inhibition with two serum pools of AD patients. We found cross-reacting IgE and IgG antibodies. In the IgE response, the main cross-reacting pattern was the mannan region, although inhibition could be achieved also with crude antigens of C. albicans, S. cerevisiae, and, to some extent, C. albidus. P. ovale was the most potent inhibitor of IgE-binding components, and against it the highest IgE antibody levels were detected in AD serum pools. In contrast, C. albicans was found to be the most important inducer of IgG antibodies, since the IgG level against P. ovale mannan in both AD serum pools was very low. Cross-reacting antibodies were also seen in ELISA inhibition with both crude and mannan antigens, but since the IgG antibody level of P. ovale mannan in AD serum pools was low, further studies are needed to confirm the IgG results.
Atopic dermatitis (AD) patients with immediate skin prick test (SPT) reactivity to different yeasts are exposed to yeast allergens from different sources ( 1). Pityrosporum ovale is a common saprophytic yeast of the normal human skin flora, but can act as an allergen in AD patients, especially in patients with dermatitis of the head, neck, and shoulder (HNS) region ( 2–7). A strong association between the severity of AD and specific IgE antibodies to P. ovale has been found ( 8). Candida albicans, another common saprophytic yeast colonizing the gastrointestinal tract, has been supposed to play a role in asthma and allergic rhinitis ( 9, 10). Elevated IgE levels against C. albicans have also been found among AD patients ( 11). Exposure to yeasts can also take place through food containing Saccharomyces cerevisiae, commonly known as baker's yeast, and through airborne contacts, since several yeasts are aeroallergens; e.g., Cryptococcus and Rhodotorula ( 12).
Patients with allergies to fungi often demonstrate broad patterns of positive SPT results and specific serum IgE antibodies to a range of different yeasts ( 1, 13, 14). These results suggest extensive cross-reactions between yeast antibodies, as indicated by limited studies so far undertaken. Previously, a combination of IgE immunoblotting and RAST inhibition has revealed cross-reacting allergenic components in P. ovale and C. albicans crude extracts ( 15). Binding patterns suggested that the cross-reacting allergen is mannan. Similar results were obtained in a study which showed nearly complete reciprocal inhibition in the IgE ELISA (enzyme-linked immunosorbent assay) with crude extracts of P. ovale and C. albicans ( 16). It was also demonstrated that the cross-reactive components belonged to the pool of Con A-binding glycoproteins and/or polysaccharides ( 17).
So far, the cross-reactivity of yeast antibodies has not been studied with P. ovale mannan. Therefore, we have studied in more detail cross-reacting IgE and IgG antibodies to purified P. ovale mannan and the following other yeasts: P. ovale crude, C. albicans mannan and crude, S. cerevisiae mannan and crude, Rhodotorula rubra crude, and Cryptococcus albidus crude. RAST-inhibition and ELISA-inhibition methods were used in this study.
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- Material and methods
Fig. 1 shows inhibition of anti-P. ovale mannan IgE by fluid-phase incubation with extracts of other yeasts ( Fig. 1a) and inhibition of other yeasts by fluid-phase incubation with P. ovale mannan extract ( Fig. 1b) with serum pool I. P. ovale crude antigen had a strong capacity to inhibit P. ovale mannan (autoinhibition of P. ovale mannan was 100%). Other yeast extracts did not reveal any inhibition ( Fig. 1a). Fig. 1b shows that P. ovale mannan inhibited about 60% of IgE binding to C. albicans and S. cerevisiae mannans. A low concentration of P. ovale mannan extract could reach the 50% inhibition level. C. albicans crude and C. albidus crude were also inhibited by P. ovale mannan, but P. ovale could not inhibit S. cerevisiae crude and R. rubra.
Figure 1. a) Inhibition of IgE-binding to solid-phase P. ovale mannan by fluid-phase incubation of other yeast extracts. □: P. ovale mannan (Pom), ▪: P. ovale crude antigen (Poc), ○: C. albicans mannan (Cam), ●: C. albicans crude antigen (Cac), Δ: S. cerevisiae mannan (Scm), ▴: S. cerevisiae crude antigen (Scc), ◆: R. rubra (Rh), and ⋄: C. albidus (Cr). Inhibition was seen only with P. ovale crude and mannan extracts. b) Inhibition of IgE-binding to solid-phase extracts of other yeasts by fluid-phase incubation of P. ovale mannan. Nitrocellulose was used as solid-phase immunosorbent matrix, and serum pool I as reacting serum.
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Fig. 2 shows inhibition of anti-P. ovale mannan IgE by fluid-phase incubation with extracts of other yeasts ( Fig. 2a) and inhibition of other yeasts by fluid-phase incubation with P. ovale mannan extract ( Fig. 2b) with serum pool II. P. ovale mannan was inhibited by 60–70% by C. albicans and S. cerevisiae mannans, the 50% inhibition level being reached by 5–7 mg/ml of C. albicans and S. cerevisiae mannans (autoinhibition was 100%). Cross-reactivity was clearly seen between mannans. Among other inhibitor extracts, only P. ovale and C. albicans crude extracts reached the 50% inhibition level, others reaching only the 45% level with mild inhibition of P. ovale mannan. Fig. 2b shows that P. ovale mannan could inhibit C. albicans and S. cerevisiae mannans by up to 60–70%. In this case, P. ovale mannan, surprisingly, inhibited C. albicans crude antigen better than C. albicans mannan. S. cerevisiae, R. rubra, and C. albidus crude extracts did not reach the 50% inhibition level when inhibited by P. ovale mannan. As control, RAST inhibition with nonatopic sera showed no inhibition.
Figure 2. a) Inhibition of IgE-binding to solid-phase P. ovale mannan by fluid-phase incubation of other yeast extracts. □: P. ovale mannan (Pom), ▪: P. ovale crude antigen (Poc), ○: C. albicans mannan (Cam), ●: C. albicans crude antigen (Cac), Δ: S. cerevisiae mannan (Scm), ▴: S. cerevisiae crude antigen (Scc), ◆: R. rubra (Rh), and ⋄: C. albidus (Cr). b) Inhibition of IgE-binding to solid-phase extracts of other yeasts by fluid-phase incubation of P. ovale mannan. Nitrocellulose was used as solid-phase immunosorbent matrix, and serum pool II as reacting serum.
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Table 1 presents IgE nitrocellulose RAST indexes for different yeast antigens in the studied serum pools. Table 2 shows IgG levels measured with inhibition ELISA. IgG antibody level against C. albicans mannan was highest in both pools, but in contrast to IgE results, levels of IgG antibodies against P. ovale mannan were low. In general, the level of IgG antibodies in both AD pools was low. In pool II, only the response to C. albicans was elevated; others were near background levels. Pool I had more reacting IgG antibodies, anti-C. albicans mannan and crude antigen being strongest. In these serum pools, P. ovale mainly raised IgE antibodies and C. albicans IgG antibodies.
Table 1. RAST indexes ([sample – buffer]×100/[total activity – buffer]) of two AD serum pools and control serum against different yeast extracts without inhibition
| ||Pool I||Pool II||Control serum|
| Crude extract||25.4||14 ||<0.2|
| Crude extract||2.15||3.79|| 0.7|
| Crude extract||4.07||7.43||<0.2|
| Crude extract||0.31||4.02||<0.2|
| Crude extract||1.11||3.07||<0.2|
Table 2. IgG antibodies against different yeast extracts in serum pools analyzed by ELISA (OD) (mean background 0.08 OD is included to values presented)
| ||Pool I||Pool II|
| Crude extract||0.62||0.18|
| Crude extract||1.12||0.52|
| Crude extract||0.37||0.16|
| Crude extract||0.49||0.11|
| Crude extract||0.34||0.09|
Since IgG levels were low in two serum pools, we also wanted to test individual AD patient sera to learn more about the levels of IgG antibodies. We tested IgG levels against C. albicans and P. ovale mannans. In all eight AD sera tested, C. albicans mannan IgG-antibody levels (1.58–2.19 OD, mean background 0.08 OD) were 3–7 times higher than P. ovale mannan levels, which were low (0.21–0.71 OD, mean background 0.08 OD) (not shown).
Fig. 3a shows inhibition of anti-P. ovale mannan IgG by fluid-phase incubation in serum pool I with other yeasts, and Fig. 3b shows inhibition of other yeasts by fluid-phase incubation in serum pool I with P. ovale mannan. Fig. 3a shows P. ovale mannan inhibited by itself, P. ovale crude, R. rubra, C. albidus, and S. cerevisiae crude and mannan. C. albicans was different from the others, since its ELISA results seen in Table 2 were three times higher than P. ovale mannans, although it could not act as a clear inhibitor. As seen in Fig. 3b, P. ovale mannan inhibited all other yeasts. Cross-reacting IgG antibodies between P. ovale mannan and other yeasts seemed to exist. However, the low OD level of P. ovale mannan should be taken into consideration.
Figure 3. a) Inhibition ELISA of anti-P. ovale mannan IgG by fluid-phase incubation in serum pool I with other yeasts; b) inhibition of other yeasts by fluid-phase incubation with P. ovale mannan in serum pool I. Percent inhibition was counted from 100%– (sample with inhibition [OD] – buffer/sample without inhibition [OD] – buffer). Symbols are same as in Fig. 1.
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Inhibition ELISA was performed also with serum pool II, but OD levels were too low for interpretation of the inhibition results (shown in Table 2).
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Highly significant correlation has been found between anti-P. ovale and anti-C. albicans IgE antibody levels in AD patients ( 6, 8). This correlation has been claimed to be due to cross-reactivity of IgE-binding components of Con A-binding glycoproteins and/or polysaccharides by Doekes & van Ieperen-van Dijk ( 16). In our earlier study, we found positive P. ovale mannan RAST in 77% of AD patients with positive commercial P. orbiculare RAST, suggesting that mannan is an important allergen of P. ovale ( 18). In the present study, cross-reactivity was for the first time investigated with purified P. ovale mannan. We showed that there were cross-reacting antibodies between yeast mannans in both the IgE and the IgG response.
In pool I, there were a high IgE antibody level against P. ovale mannan and a low one against C. albicans and S. cerevisiae mannans. Pool II contained IgE antibodies against P. ovale mannan, but also far more antibodies against other mannans. Since the specific IgE level of P. ovale in serum pool I was higher than the indexes of other yeasts, strong P. ovale mannan could possible eliminate inhibition when P. ovale mannan disks were used, since it could itself inhibit many other yeasts. Alternatively, it had common epitopes with other yeasts, but also many of its own epitopes, which could not be inhibited by other yeasts. However, findings with serum pool II showed the cross-reacting antibodies between P. ovale mannan and other yeast mannans.
Savolainen & Broberg ( 15) found earlier that C. albicans crude extract could partly inhibit the IgE binding to P. ovale crude extract in two out of five sera tested. On the basis of immunoblotting and the RAST-inhibition pattern, they concluded that cross-reacting epitopes reside mainly in the mannan of C. albicans and in the mannan-rich high-molecular-weight diffuse band of P. ovale ( 15). The findings of Doekes & van Ieperen-van Dijk ( 16) are in agreement with these findings and with our new results.
Huang et al. ( 29) found weak inhibition of IgE binding to P. orbiculare crude extract with C. albicans crude antigens. Only a few of the P. orbiculare protein components in the highest molecular range exhibited a little inhibition, and no inhibition of the high-molecular-weight Candida mannans was observed with the P. orbiculare extract. They suspected that the P. orbiculare extract used was rich in protein allergens, but possibly did not contain carbohydrate-rich allergens ( 29). Several differences between their crude extract and our P. ovale crude extract could be found in the preparation of antigen; for example, culturing times (our 1 week compared to their 4 days), the culture medium used, and sonication compared to our X-press chamber for disruption of the cells. Huang et al. ( 29) pointed out that Doekes & van Ieperen-van Dijk ( 16) and Savolainen & Broberg ( 15) both used commercial P. ovale extracts, which seemed to contain only a few protein components, and that would explain some of the differences in the results ( 29). In contrast, in our P. ovale crude antigen, we earlier found 39 different IgE-staining protein bands with also high-molecular-weight mannan staining in immunoblotting ( 18). Thus, we believe that mannan is an important cross-reacting component of P. ovale, but, as Huang et al. ( 29) pointed out, there is also little inhibition between protein components, as seen also in the present study with the crude antigens.
P. ovale mannan seems to be an important cross-reacting antigen component in IgG binding too. This subject is more complex, since IgG levels against P. ovale mannan and crude antigens were clearly lower than against C. albicans. IgG levels of C. albicans were high, mannans being even higher than crude extracts. This indicates the importance of C. albicans as a trigger for IgG production and also indicates the importance of mannan. Koivikko et al. ( 1) reported a 6% level of cross-reactivity between sheep and rabbit anti-C. albicans IgG antibodies and P. ovale extract. Doekes & van Ieperen-van Dijk ( 16) found ±10% cross-reactivity with commercial rabbit anti-C. albicans IgG preparation. Thus, we were the first to use human sera and purified mannan to study the cross-reactivity of IgG-binding components. Our results were different from the two earlier mentioned ( 1, 16), since we found more cross-reactivity, mannan being important. However, more studies are needed to confirm the cross-reactivity of IgG-binding components.