The role of PR 10 proteins and molecular components of moulds and yeast in atopic dermatitis patients

ABSTRACT

This study aims to analyse the results of specific IgE to molecular components of PR 10 proteins, mould and yeast in atopic dermatitis (AD) patients, to find significant sets of molecular components in pairs and triplets in the relation to the severity of atopic dermatitis and the occurrence of asthma bronchiale and allergic rhinitis. The examination of the sensitization to molecular components with ALEX2 Allergy Explorer testing was performed. The level of specific IgE was recorded with Jittered box plots, the Kruskal–Wallis test was used to compare the level of specific IgE in different forms of AD. The relation between the results of specific IgE was evaluated with Coefficient of correlation (Kendall tau-b). The comparison of significant rules was performed with Fisher–Freeman–Halton exact test and with tests on proportions. Altogether 100 atopic dermatitis patients were examined. Molecular components Fra a 1 + 3, Mal d 1, Api g 1, Mala s 11 and Asp f 6 are recorded as a single molecular component with relation to the severe form of AD; these components are associated in pairs and triplets with molecular components Cor a 1.0401, Cor a 1.0103, Fag s 1, Alt a 1 and with the occurrence of allergic rhinitis. Results are presented in static and interactive tables and plots.

KEYWORDS:

• Atopic dermatitis
• ALEX2 Allergy explorer
• molecular components
• severity of atopic dermatitis
• disturbed epidermal barrier
• asthma bronchiale
• allergic rhinitis

Introduction

The longitudinal analysis of the course of IgE-associated allergy in birth cohorts has shown that atopic dermatitis together with food allergy are the first signs and symptoms of allergic sensitization in early childhood which are followed by respiratory forms of allergy, such as allergic rhinitis (AR) and asthma bronchiale (AB) (Matricardi et al., 2016). IgE sensitization to different allergens such as airborne, food-derived, microbial allergens and autoallergens is associated with atopic dermatitis (AD).

The Pathogenesis-related protein group 10 (PR-10 proteins) is one of the 11 subfamilies of the Bet v 1 family. PR-10 proteins are the major allergens in Fagales pollen and are recognized by virtually all allergic patients, thus representing the major cause of clinical allergy. The major role of PR-10 proteins is reported in response to biotic and abiotic stresses and defend plants against fungi and other microorganisms (Biedermann et al., 2019, July; Matricardi et al., 2016; McKenna et al., 2016). Their homologs are also present in a large number of plant-derived foods and thus frequently cause cross-sensitization and consequently plant-food allergy (oral allergy syndrome, in most cases) (Biedermann et al., 2019, July; Matricardi et al., 2016; McKenna et al., 2016). Fungi are potent sources of allergenic molecules covering a vast variety of molecular structures including enzymes, toxins, cell wall components and phylogenetically highly conserved cross-reactive proteins (Crameri et al., 2014; Matricardi et al., 2016). In contrast to other allergenic sources, fungi are very common in the environment, and exposure to airborne spores is almost constant throughout the year. Fungi may colonize the human body, and they may damage airways by the production of toxins, proteases, enzymes and volatile organic compounds and they develop rapidly on different kinds of foods (Biedermann et al., 2019, July; Crameri et al., 2014). Thus moulds have a far greater impact on the patients’ immune system than pollen or other allergenic sources. The spectrum of allergic symptoms caused by these hypersensitivity reactions to fungi is very broad, including rhinitis, asthma, atopic dermatitis (Crameri et al., 2014; Matricardi et al., 2016).

Multi-allergen tests containing micro-arrayed allergen molecules are useful for the identification of culprit allergens in atopic dermatitis and may improve the management of atopic dermatitis by allergen-specific immunotherapy, allergen avoidance, and IgE-targeting therapies in a personalized medicine approach. Molecular components are used in both single-parameter and multiplex analyses. In the case of patients with severe clinical manifestations or unclear examination findings, it is appropriate to use the multiplex method. The ALEX® system (Macro-Array Diagnostics, Vienna, Austria) is a multiplex test designed for solid phase immunoassay. The method allows the simultaneous determination of total IgE (semi-quantitative) and specific IgE (quantitative) in the ALEX2® version against 295 allergens, of which 178 molecular components and 117 extract allergens, in human serum or plasma (Heffler et al., 2018; Steering Committee Authors; Review Panel Members, 2020). In our previous studies, we evaluated in patients suffering from AD the occurrence of sensitization to molecular components with the use of ALEX2 Allergy Explorer testing. The high and very high levels of specific IgE were reached in 75.0% of patients with 18 molecular components (Čelakovská et al., 2021, May 17).

Aim of the study

Our study aims to perform the detailed statistical analysis of the results of specific IgE to molecular components of PR 10 proteins, moulds and yeast in patients suffering from atopic dermatitis. The goal of the analysis is to detect the most frequent sets of positive allergen reagents as a single rule or in pairs and triplets in association with the severity of atopic dermatitis patients (AD), the occurrence of asthma bronchiale (AB) and allergic rhinitis (AR).

Statistical analysis

The binary coding and association rules detected the most frequent groups of positive allergen reagents, molecular components and attributes such as AB, AR associated with AD subgroups (mild, moderate, severe forms of AD). The p-value of rules is based on Fisher’s Exact Test. Rules are presented in static and interactive tables and plots. We used for comparisons of significant rules Fisher–Freeman–Halton exact test and tests on proportions (Hauck–Anderson), p-values were adjusted for multiple testing (Benjamini–Hochberg). For the evaluation of positive results of specific IgE in mild, moderate and severe forms of atopic dermatitis we used Kruskal–Wallis test.

The relation between the results of specific IgE was evaluated with Coefficient of correlation (Kendall tau-b). We show the level of specific IgE with Jittered box plots and we use Kruskal–Wallis test to compare the level of specific IgE in mild, moderate and severe forms of AD. Software IBM SPSS Statistics v. 28 and several R packages (arules, igraph, ggpubr, dunn and associated packages) were used (Hahsler, 2017; Hahsler et al., 2005; NCSS 12 Statistical Software, 2018; Webb, 2007).

Patients and methods

In the period 2018–2020, 100 patients suffering from atopic dermatitis at the age of 14 years and older were examined. All these patients were examined in the Department of Dermatology, Faculty Hospital Hradec Králové, Charles University, Czech republic. The diagnosis of atopic dermatitis was made with the Hanifin–Rajka criteria (Hanifin & Rajka, 1980). Exclusion criteria were systemic therapy (cyclosporin, systemic corticoids, biological therapy), pregnancy and breastfeeding. Patients with atopic dermatitis having other systemic diseases were excluded from the study as well. The complete dermatological and allergological examination was performed in patients included in the study. This study was approved by Ethics Committee of Faculty Hospital Hradec Králové, Charles University of Prague, Czech Republic.

Dermatological examination

A complete dermatological examination was performed in patients included in the study. The severity of atopic dermatitis was scored in agreement with SCORAD, with an assessment of topography items (affected skin area), intensity criteria and subjective parameters. To measure the extent of atopic dermatitis, the rule of nines was applied on a front/back drawing of the patient’s inflammatory lesions. The extent was graded 0–100 points. The intensity part of the SCORAD index consists of six items: erythema, oedema/papules, excoriations, lichenification, crusts and dryness. Each item was graded on a scale 0–3. The subjective items included daily pruritus and sleeplessness. Both subjective items were graded on a 10-cm visual analog scale and the maximum subjective score was 20 points. All items were filled out in the SCORAD evaluation form. The SCORAD index formula was A/5 + 7B/2 + C. In this formula, A is defined as the extent (0–100 points), B is defined as the intensity (0–18 points) and C is defined as the subjective symptoms (0–20 points). The severity of atopic dermatitis is evaluated with SCORAD as a mild form to 20 points, as moderate over 20–50 points, as a severe form over 50 points (European Task Force on Atopic Dermatitis, 1993). The evaluation of SCORAD score was performed every 2 months during the study (European Task Force on Atopic Dermatitis, 1993).

Examination of specific IgE to allergen reagents

The serum level of the sIgE was measured by the components resolved diagnostic microarray-based sIgE detection assay ImmunoCAP ALEX2 Allergy Explorer (Hahsler, 2017; NCSS 12 Statistical Software, 2018; Webb, 2007). It is based on a state-of-the-art proprietary nano-bead technology. The ALEX2 Allergy Explorer measuring range for specific IgE is 0.3–50 kU_A/L (quantitative) and for total IgE is 1–2500 kU/L (semiquantitative). The sample requirement is 100 µL serum or plasma. The results are expressed as Class 0 (≤0.3 kU_A/L) – negative, or Class 1 (0.3 > 1 kU_A/L) – positivity. The ALEX2 Allergy Explorer is commercially available, having attained CE certification, which assures that the quality of the assay, regarding LoD, precision and repeatability as well as specificity and linearity, is in line with in vitro diagnostic features. There is no significant interference from high total IgE, haemoglobin, bilirubin or triglycerides.

Control group

When introducing the test into the clinical examination, we also examined 15 healthy volunteers – blood donors (equivalent to age, male and female representation). All of these blood donors had in the multiplex examination (ALEX 2–Allergy Explorer) the specific IgE negative – expressed as Class 0 (<0.3 kUA/L). This study was approved by Ethics Committee of Faculty Hospital Hradec Králové, Charles University of Prague, Czech Republic.

Bronchial asthma (AB)

The diagnosis of bronchial asthma (AB) was determined according to the guidelines of the Global Initiative for Asthma (GINA) at allergy outpatient clinic of the Institute of Clinical Immunology and Allergology, Faculty Hospital Hradec Kralove, Czech Republic (Global Initiative for Asthma. Global Strategy for asthma management and prevention – Update 2015. www.ginasthma.com).

Allergic rhinitis (AR)

The evaluation of allergic rhinitis (AR) was made according to the allergy testing and personal history of the Institute of Clinical Immunology and Allergology, Faculty Hospital Hradec Kralove, Czech Republic (Wang et al., 2018). AR was defined as a process that included three cardinal symptoms: sneezing, nasal obstruction and mucus discharge. Symptoms occur with allergen exposure in the allergic patient (Wang et al., 2018).

Results

100 atopic dermatitis patients were included in the study (48 men and 52 women with an average age 40.9 years: min. age 14 years, max. age 67 years and with an average SCORAD 39, s.d.13.1 points). The mild form of AD was recorded in 14 patients (14. 0%), moderate form in 61 patients (61. 0%), severe form in 25 patients (25. 0%); 55 patients (55. 0%) suffer from bronchial asthma and 74 patients (74. 0%) suffer from allergic rhinitis. The characteristics of patients are shown in Table 1. In Bart Chart No. 1, we show the relative frequency of positive results to molecular components of PR 10 proteins, moulds and yeast and the frequency of mild (AD1), moderate (AD2) and severe (AD3) forms of AD, the frequency of asthma bronchiale (AB) and allergic rhinitis (AR).

Graph No. 1. The relative frequency of positive results to molecular components of PR 10 proteins, moulds and yeast, the frequency of mild (AD 1), moderate (AD 2) and severe forms of AD (AD 3), the frequency of asthma bronchiale (AB) and allergic rhinitis (AR).

Table 1. The characteristic of patients.

The characteristic of patients, number of patients
Patients suffering from atopic dermatitis	100 patients
Sex	48 men, 52 women
The average age	40.9 years (min. age 14 years, max. age 67 years)
The average SCORAD	39 points, s.d. 13.1 points
Severity of AD	AD1 – mild form 14 (14%)
	AD2 – moderate form 61 (61%)
	AD3 – severe form 25 (25%)
Family history about atopy	Positive family history 48 patients (48%)
	Negative family history 52 patients (52%)
Onset of AD	Under 5 years of age 61 patients (61%)
	After 5 years of age 39 patients (39 %)
Eczematic lesions	Persistent 57 patients (57%)
	Occasional 43 patients (43%)
Bronchial asthma	55 (55%)
Allergic rhinitis	74 (74%)

In Bar Chart No. 2, we show relative frequencies of positive specific IgE to molecular components of PR 10 proteins and moulds and yeast in mild (AD 1), moderate (AD 2) and severe (AD 3) forms of AD (descending according to the largest representation). In Complement to Bar Chart No. 2, we show the relative frequencies of positive specific IgE to molecular components of PR 10 proteins, moulds, yeast and 95% Exact (Clopper–Pearson) Confidence Interval. The highest sensitization rate in mild and moderate forms of AD is confirmed to molecular component Bet v 1 (in 50% of patients with mild form and in 50.8% of patients with moderate form). In patients with severe form, the highest sensitization rate is confirmed to molecular component Fra a 1 + 3 in 64%, to Bet v 1 in 60%.

Graph No. 2. The relative frequencies of positive specific IgE to molecular components of PR 10 proteins and moulds, yeast in mild (AD 1), moderate (AD 2) and severe forms (AD 3) of AD.

Complement to Bar Chart No. 2

Table

Molecular components	Proportions of positive and 95% exact (Clopper–Pearson) confidence interval
	AD mild form (n = 14)			AD moderate form (n = 61)			AD severe form (n = 25)
	Proportion	Lower	Upper	Proportion	Lower	Upper	Proportion	Lower	Upper
Betv 1	0.500	0.230	0.770	0.508	0.377	0.639	0.600	0.387	0.789
Fags1	0.357	0.128	0.649	0.475	0.346	0.607	0.600	0.387	0.789
Cora1.0103	0.429	0.177	0.711	0.443	0.315	0.576	0.600	0.387	0.789
Fraa1 + 3	0.214	0.047	0.508	0.426	0.300	0.559	0.640	0.425	0.820
Cora1.0401	0.286	0.084	0.581	0.410	0.286	0.543	0.600	0.387	0.789
Alng1	0.357	0.128	0.649	0.377	0.256	0.510	0.600	0.387	0.789
Mald1	0.143	0.018	0.428	0.393	0.271	0.527	0.600	0.387	0.789
Glym4	0.214	0.047	0.508	0.328	0.213	0.460	0.480	0.278	0.687
Arah8	0.214	0.047	0.508	0.311	0.199	0.443	0.440	0.244	0.651
Apig1	0.071	0.002	0.339	0.180	0.094	0.300	0.400	0.211	0.613
Dauc1	0.071	0.002	0.339	0.148	0.070	0.262	0.360	0.180	0.575
Alta1	0.214	0.047	0.508	0.262	0.158	0.391	0.280	0.121	0.494
Malas11	0.000	0.000	0.232	0.180	0.094	0.300	0.520	0.313	0.722
Aspf6	0.000	0.000	0.232	0.164	0.082	0.281	0.400	0.211	0.613
Aspf3	0.000	0.000	0.232	0.131	0.058	0.242	0.280	0.121	0.494
Clah8	0.000	0.000	0.232	0.115	0.047	0.222	0.320	0.149	0.535
Malas6	0.000	0.000	0.232	0.098	0.037	0.202	0.320	0.149	0.535
Sacc	0.000	0.000	0.232	0.098	0.037	0.202	0.320	0.149	0.535
Alta6	0.000	0.000	0.232	0.098	0.037	0.202	0.240	0.094	0.451
Malas5	0.000	0.000	0.232	0.082	0.027	0.181	0.200	0.068	0.407
Clah	0.000	0.000	0.232	0.016	0.000	0.088	0.200	0.068	0.407
Aspf1	0.000	0.000	0.232	0.016	0.000	0.088	0.120	0.025	0.312
Aspf4	0.071	0.002	0.339	0.000	0.000	0.059	0.080	0.010	0.260
Pench	0.000	0.000	0.232	0.016	0.000	0.088	0.080	0.010	0.260

In Graph No. 3 (box plots with dots jittered plot IgE measurements), we show the comparisons of sensitization to PR 10 proteins, moulds and yeast according to the level of specific IgE in patients suffering from mild, moderate and severe forms of AD. In Complement to Graph No. 3, we show the difference in the level of specific IgE in mild, moderate and severe forms of AD. In patients suffering from a severe form of AD, we confirmed the significantly higher occurrence of positive results of specific IgE to molecular components (allergen reagents) Api g 1, Ara h 8, Asp f 3, Cla, Cla h 8, Cor a 1.0103, Cor a 1.0401, Dau c 1, Fag s, Fra a 1 + 3, Mal d 1, Mala s 11, Mala s 6, Sacc in comparison to patients with mild form. In patients suffering from a moderate form of AD, we confirmed the significantly higher occurrence of positive results of specific IgE to molecular components (allergen reagents) Api g 1, Cla h and Mal d 1 in comparison to patients with mild form. When comparing sensitization to molecular components in groups of patients with moderate and severe forms of atopic dermatitis, we confirmed the significant difference in the level of specific IgE to molecular components Cla h 8, Dau c 1, Mala s 11, Mala s 6 and Sacc.

Graph No. 3. The scatter plots. A detailed comparison of sensitization to PR 10 proteins, moulds and yeast according to the level of specific IgE in patients suffering from mild (AD 1), moderate (AD 2) and severe form (AD 3). Box plots with jittered dots IgE measurements. Tests of equality in distributions (Kruskal–Wallis) following pair-wise comparisons (pwc) Dunn test, p-value Benjamini–Hochberg adjusted, ⁎ (0.01 ≤ p < 0.05), ⁎⁎ (p < 0.01), NS or blank (p ≥ .0.05).

Complement to Graph No. 3

The statistical evaluation of differences in the level of specific IgE to molecular components of PR 10 proteins, moulds, yeast in mild, moderate and severe forms of AD. The significant difference is recorded extra bold with *.

Table

	Monte Carlo significance – p-values
Molecular component	Mild × moderate form AD	Mild × severe form AD	Moderate × severe form AD
Aln g 1	0.213	0.052	0.258
Alt a 1	0.530	0.392	0.490
Alt a 6	0.461	0.469	0.771
Api g 1	0.024*	0.029*	0.448
Ara h 8	0.293	0.047	0.109
Asp f 1	0.232	0.167	0.671
Asp f 3	0.155	0.025*	0.059
Asp f 4	0.574	0.313	0.481
Asp f 6	0.214	0.081	0.155
Bet v 1	0.523	0.108	0.106
Cla h	0.014*	0.004*	0.096
Cla h 8	0.138	0.000*	0.000*
Cora 1.0103	0.358	0.042*	0.072
Cor a1.0401	0.140	0.007*	0.083
Dau c 1	0.301	0.016*	0.010
Fag s 1	0.582	0.045*	0.073
Fra a 1 + 3	0.219	0.012*	0.131
Gly m 4	0.443	0.077	0.111
Mal d 1	0.041*	0.004*	0.112
Mala s 11	0.433	0.002*	0.002*
Mala s 5	0.288	0.073	0.126
Mala s 6	0.524	0.046*	0.045*
Pen ch	0.280	0.060	0.087
Sacc	0.143	<.001*	<.001*

In Table 2, we show simultaneous occurrences of positive results of specific IgE in mild, moderate and severe forms of AD. Atopic dermatitis patients with a mild form are on average sensitized to the number of 2.8 molecular components of PR 10 proteins, with a moderate form to 4.0, with a severe form to 5.9; the difference is not significant. Patients with a mild form are on average sensitized to the number of 0.2 molecular components of moulds (yeast), with a moderate form to 1.28, with a severe form to 3.3; the difference is significant. The sum of positive results of specific IgE to moulds (yeast) per patient increases with AD severity, this finding is not observed for PR 10 proteins.

Table 2. Sum of positive allergens of PR 10 respectively mould, yeast per patient in AD 1 (mild form), AD 2 (moderate form) and AD 3 (severe form) groups. The significant relation is recorded extra bold*.

Characteristics of SUM of positive components per patient		Mean	95% Confidence interval for mean		Median	Maximum	Interquartile range	Test Kruskal–Wallis
			Lower bound	Upper bound
PR 10 SUM	AD 1	2.86	0.69	5.03	0.50	11	6	p = 0.124
	AD 2	4.00	2.84	5.16	1.00	11	9
	AD 3	5.92	3.87	7.97	9.00	11	11
Mould and yeast SUM	AD 1	0.29	0.02	0.56	0.00	1	1	p < 0.001*
	AD 2	1.28	0.82	1.73	1.00	7	2
	AD 3	3.36	2.39	4.33	3.00	8	5

The binary coding and association rules allowed us to detect the most frequent sets (rules, groups of positive allergen reagents), associated with AD subgroups. In total, 3838 rules were extracted, 93 from them were nonredundant (unique), 31 rules were significant. Table 3 presents the set of 31 significant rules. These rules are sorted in decreasing order by support. We found the set of 8 significant rules (bold, it is limited to the rules with Fisher Exact Test p < 0.01) and the set of 23 rules (bold with *, significance 0.01 < p < 0.05). Rules are in significant association to the severe form of AD, exception the rule in italic, which is in association with moderate form. No significant rule was related to a mild form of AD. These results are presented in interactive plot and tables.

Table 3. The set of 31 significant rules. These rules are sorted in decreasing order by support.

	Nonredundant rules in ascending order by p-value. A rule represents positive result on all its components.		p-value
1	{Mala s 11}	{AD = 3}	0.0004
2	{Asp f 6, Mala s 11}	{AD = 3}	0.0021
3	{AR, Aln g 1, Fra a 1 + 3, Mal d1}	{AD = 3}	0.0056
4	{Asp f 6}	{AD = 3}	0.0063
5	{Aln g 1, Fra a1 + 3, Mal d 1}	{AD = 3}	0.0065
6	{AR, Aln g 1, Mal d 1}	{AD = 3}	0.0082
7	{AR, Aln g 1, Cor a 1.0401, Fra a 1 + 3}	{AD = 3}	0.0082
8	{Aln g 1, Cor a 1.0401, Fra a 1 + 3}	{AD = 3}	0.0092
9	{AR, Fra a 1 + 3, Mal d 1}	{AD = 3}	0.0116*
10	{AR, Aln g 1, Fra a 1 + 3}	{AD = 3}	0.0116*
11	{Aln g 1, Mal d 1}	{AD = 3}	0.0128*
12	{Fra a1 + 3, Mal d 1}	{AD = 3}	0.0128*
13	{Aln g 1, Fra a 1 + 3}	{AD = 3}	0.0128*
14	{Api g 1}	{AD = 3}	0.0153*
15	{AR, Mal d 1}	{AD = 3}	0.0161*
16	{AR, Aln g 1, Cor a 1.0401}	{AD = 3}	0.0161*
17	{AR, Cor a 1.0401, Fra a 1 + 3}	{AD = 3}	0.0161*
18	{Cor a 1.0401, Fra a 1 + 3}	{AD = 3}	0.0175*
19	{AR, Aln g 1}	{AD = 3}	0.0220*
20	{Mal d 1}	{AD = 3}	0.0235*
21	{Fra a 1 + 3}	{AD = 3}	0.0243*
22	{AR, Cor a 1.0401}	{AD = 3}	0.0295*
23	{AR, Cor a 1.0103, Fra a 1 + 3}	{AD = 3}	0.0295*
24	{Aln g 1, Cor a 1.0401}	{AD = 3}	0.0311*
25	{Cor a 1.0103, Fra a 1 + 3}	{AD = 3}	0.0311*
26	{AR, Alt a 1, Fag s 1}	{AD = 2}	0.0355*
27	{AR, Aln g 1, Cor a 1.0401, Gly m 4}	{AD = 3}	0.0383*
28	{AR, Cor a 1.0401, Fra a1 + 3, Gly m 4}	{AD = 3}	0.0383*
29	{AR, Fag s 1, Fra a 1 + 3}	{AD = 3}	0.0388*
30	{AR, Bet v 1, Fra a 1 + 3}	{AD = 3}	0.0388*
31	{Aln g 1}	{AD = 3}	0.0405*

The set of 8 significant rules (bold) – it is limited to the rules with Fisher Exact test p < 0.01, the set of 23 rules (marked bold with *) present significance 0.01 < p < 0.05. Rules are in significant association to severe AD form of AD (AD 3), exception the rule in italics, when we compared the significant relation to moderate form of AD (AD 2). No significant rule was related to mild AD. AR – allergic rhinitis, AB – asthma bronchiale.

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In Table 4, we show the relative frequencies of these rules (groups of positive molecular components with the occurrence of AR, AB) in mild, moderate and severe forms of AD. Molecular components Fra a 1 + 3, Mal d 1, Api g 1, Mala s 11 and Asp f 6 are recorded as a single component with relation to the severe form of AD. The occurrence of allergic rhinitis was recorded in groups with Aln g 1, Fra a 1 + 3, Mal d 1, Cor a 1.0401, Cor a 1.0103, Gly m 4, Fag s and Bet v 1. We observe that the frequency of all these groups increases from mild to moderate and severe forms of AD, p-values indicate the significant increase.

Table 4. Comparisons of relative frequencies of 31 significant rules within AD groups (AD 1 – mild form, AD 2 – moderate form, AD 3 – severe form of atopic dermatitis, AR – allergic rhinitis, AB – asthma bronchiale); p- values were adjusted for multiple testing (Benjamini–Hochberg = B-H).

Allergens associated with AD3 and association rules	Relative frequencies			AD pair-wise comparisons
Positive rules (frequency min = 10)	AD1 (n = 14)	AD2 (n = 61)	AD3 (n = 25)	Only significant pairs are presented at the level (B-H) adjusted p < 0.05
Mala s 11	0.000	0.180	0.520	AD1_AD2 (0.006), AD1_AD3 (0.001), AD2_AD3 (0.001)
Asp f 6, Mala s 11	0.000	0.131	0.400	AD1_AD2(0.044), AD1_AD3 (0.001), AD2_AD3 (0.005)
AR, Aln g 1, Fra a 1 + 3, Mal d 1	0.071	0.295	0.560	AD1_AD3 (0.008), AD2_AD3 (0.032)
Asp f 6	0.000	0.164	0.400	AD1_AD2 (0.011), AD2_AD3 (0.019), AD1_AD3(0.001)
Aln g 1, Fra a 1 + 3, Mal d 1	0.071	0.344	0.600	AD1_AD3 (0.004), AD2_AD3 (0.043)
AR, Aln g 1, Mal d 1	0.071	0.311	0.560	AD1_AD3 (0.008), AD2_AD3 (0.047)
AR, Aln g 1, Cor a 1.0401, Fra a 1 + 3	0.143	0.295	0.560	AD1_AD3 (0.032), AD2_AD3 (0.032)
Aln g 1, Cor a 1.0401, Fra a 1 + 3	0.143	0.344	0.600	AD1_AD3 (0.017), AD2_AD3 (0.044)
AR, Fra a 1 + 3, Mal d 1	0.071	0.328	0.560	AD1_AD3 (0.008)
AR, Aln g 1, Fra a 1 + 3	0.214	0.295	0.560	AD1_AD3 (0.040)
Aln g 1, Mal d 1	0.143	0.361	0.600	AD1_AD3 (0.017)
Fra a 1 + 3, Mal d 1	0.071	0.377	0.600	AD1_AD2 (0.041), AD1_AD3 (0.004)
Aln g 1, Fra a 1 + 3	0.214	0.344	0.600	AD1_AD3 (0.044), AD2_AD3 (0.044)
Api g 1	0.071	0.180	0.400	AD1_AD3 (0.047), AD2_AD3 (0.047)
AR, Mal d 1	0.071	0.344	0.560	AD1_AD3 (0.008)
AR, Aln g 1, Cora1.0401	0.143	0.328	0.560	AD1_AD3 (0.033)
AR, Cor a 1.0401, Fra a 1 + 3	0.143	0.328	0.560	AD1_AD3 (0.033)
Cor a 1.0401, Fra a 1 + 3	0.143	0.377	0.600	AD1_AD3 (0.017)
AR, Alng 1	0.214	0.328	0.560	AD1_AD3 (0.049)
Mal d 1	0.143	0.393	0.600	AD1_AD3 (0.017)
Fra a 1 + 3	0.214	0.426	0.640	AD1_AD3 (0.032)
AR, Cora 1.0401	0.143	0.361	0.560	AD1_AD3 (0.033
AR, Cora1.0103, Fra a 1 + 3	0.143	0.328	0.560	AD1_AD3 (0.033)
Aln g 1, Cor a1.0401	0.286	0.377	0.600	Pair-wise NS, linear_by-linear 0.048
Cor a 1.0103, Fra a 1 + 3	0.214	0.393	0.600	AD1_AD3 (0.033) notAdj
AR, Alt a 1, Fag s 1 to AD2	0.000	0.197	0.080	NS
AR, Aln g 1, Cora1.0401, Gly m 4	0.071	0.262	0.440	AD1_AD3 (0.017) notAdj
AR, Cora1.0401, Fra a 1 + 3, Gly m 4	0.071	0.262	0.440	AD1_AD3 (0.017) notAdj
AR, Fag s 1, Fra a 1 + 3	0.214	0.361	0.560	AD1_AD3(0.037) notAdj
AR, Bet v 1, Fra a 1 + 3	0.214	0.361	0.560	AD1_AD3(0.037) notAdj

We evaluated the mutual relation between the results of specific IgE to molecular components of PR 10 proteins, moulds and yeast with Coefficient of correlation (Kendall tau-b). We show this relation in mild form of AD (Table 5a, AD 1), in moderate form (Table 5b, AD 2) and in severe form (Table 5c, AD 3). The correlation between the results of specific IgE to molecular components of PR 10 proteins, moulds and yeast increases significantly with the severity of AD.

Table 5. Coefficient of correlation (Kendall tau-b) between PR 10, mould, yeast allergens.

*Correlation is significant at the 0.05 level (2-tailed).

**Correlation is significant at the 0.01 level (2-tailed).

Note: A value of coefficient of correlation greater than 0.7 is considered a strong correlation, value between 0.5 and 0.7 is moderate.

Discussion

The purpose of our study was to perform the detailed statistical analysis of results of specific IgE to allergen reagents from PR 10 proteins, moulds and yeast in patients suffering from atopic dermatitis; the serum level of the specific IgE was measured by the components resolved diagnostic microarray – ImmunoCAP ALEX2 Allergy Explorer. The results of our study show the differences in sensitization in the mild, moderate and severe forms of AD and show how the molecular components of PR 10 proteins, mould and yeast are associated into groups in relation to the severity of AD and in relation to the occurrence of AR and AB.

From PR 10 proteins, positive specific IgE to Bet v 1 predominates in the mild and moderate forms of AD; as for the other components, we find significant differences in the level of specific IgE between mild, moderate and severe forms.

Statistical analysis showed that multiple sensitization to PR 10 proteins has no significant effect on the severity of AD. On the contrary, if the patients have multiple sensitization to molecular components of moulds and yeast, so they suffer significantly more often from a severe form of atopic dermatitis. The results of our study also show how molecular components are associated in groups in relation to the severity of AD. We found 31 groups with positive results of specific IgE (as a single rule or in pairs and triplets), where we detected the relation to the severity of atopic dermatitis and to the occurrence of allergic rhinitis. 30 rules are significantly related to severe form of AD, only one rule relates to moderate form of AD (AR, Alt a 1, Fag s 1). As a single significant rule, we found molecular components Fra a 1 + 3, Mal d 1, Api g 1 and Aln g 1 from PR 10 proteins and Mala s 11 and Asp f 6 from moulds and yeast; these molecular components play the important role in patients suffering from the severe form of AD. We find these components in groups in pairs and triplets with molecular components Cor a 1.0401, Cor a 1.0103, Fag s 1, Alt a 1 and with the occurrence of allergic rhinitis (AR). We observe that the frequency of all these rules increases significantly from mild to moderate and to severe forms of atopic dermatitis. Although the relative frequency of positive specific IgE to Bet v 1 is the most frequent of all components, the molecular component Bet v 1 was found only in one triplet (AR, Bet v 1, Fra a 1 + 3) and there is no difference in sensitization to Bet v 1 in the mild, moderate and severe forms of AD. The attribute asthma bronchiale is not found in significant pairs or triplets with molecular components of PR 10 proteins, moulds, yeast. In contrast, the occurrence of allergic rhinitis is significantly associated with the molecular components Fra a 1 + 3, Aln g 1, Mal d 1, Cor a 1.0103, Cor a 1.0401, Gly m 4, Alt a 1 and Fag s 1.

In patients with the severe form of AD, the highest relative frequency of positive specific IgE to molecular component Fra a 1 + 3 was recorded (in 64% of patients with severe form). The clinical symptoms after ingesting strawberries in our patients are rather exceptional, with manifestations of itchy skin and oral allergic syndrome. Although strawberries are highly appreciated fruits, their intake can induce allergic reactions in atopic patients as it happens with other members of the Rosaceae family, e.g. apples, cherries or peaches (Afrin et al., 2016, June 8; Amil-Ruiz et al., 2011, November; Giampieri et al., 2013, March; Orozco-Navarrete et al., 2020, September 30; Scheurer et al., 1997; Vanek-Krebitz et al., 1995). Allergy to Fra a 1 is a type I birch pollen-related food allergy. This allergy to the strawberry Fra a 1, and other related fruit proteins, is caused by a previous sensitization to the major birch pollen allergen Bet v 1 (Franz-Oberdorf et al., 2016; Valenta & Kraft, 1996). This cross-reactivity is the reason why more than 70% of the patients with birch pollen allergies in Central and Northern Europe, and in North America, develop allergies to fruits, nuts, vegetables, and legumes; and about 15–30% of them display allergic reactions after the intake of fresh strawberry fruits (Hofer et al., 2017). The symptoms are generally mild and in the form of oral allergy syndromes (OAS) coursing with itching and swelling, although, in rare occasions, systemic urticaria or even anaphylaxis has been reported (Ma et al., 2003). Patterns of sensitization vary depending on the geographical area (Biedermann et al., 2019, July). In our study, we confirmed the significant role of molecular components from PR 10 proteins Bet v 1 (Birch), Aln g 1 (Alder) and Cor a1.0103 (Corylus avelana). We compared the results of our study with the botanical characteristic of our region. Based on data from the nearest phenological station Běleč nad Orlicí (241 m above sea level), 50°12′ N, 15°56′ E), which is located near Hradec Králové (East Bohemia region in Czech Republic), the following species are present from important pollen allergens: owing to the sandy substrate, white birch (Betula pendula) and wood pine (Pinus sylvestris) predominate, as well as common hazel (Corylus avellana). There is also abundant sticky alder (Alnus glutinosa), willow (Salix caprea), hornbeam (Carpinus betulus), summer oak (Quercus robur), spruce (Picea excelssa), deciduous larch (Larix decidua), Heartworm (Tilia cordata). For trees flowering in the phenological pre-spring (hazel and alder) the shift is 11–29 days. Birch and other related trees of the families Betulaceae and Fagaceae (alder, hazel, oak, hornbeam, chestnut and beech) constitute the birch homologous group (Biedermann et al., 2019, July).

Molecular components of apple Mal d 1 and of celery Api g 1 were also found as significant molecular components in relation to severe form of atopic dermatitis. In Northern Europe, Mal d 1 is the major allergen that causes allergic reactions to fruit. Mal d 1 is very similar to the main birch pollen allergen Bet v 1 and has similar epitopes for IgE antibodies, resulting in cross-reactions (Gao et al., 2008). In celery, the PR-10 protein Api g 1 is a major allergen, especially in Central Europe. Also, profilin is supposed to sensitize a relevant number of celeriac–allergic patients (Bauermeister et al., 2009). In our study, molecular component Mal d 1 is associated in significant pairs and triplets with other molecular components such as Fra a 1+3, Aln g 1, Cor a 1.0103, Cor a 1.0401, Gly m 4, Alt a 1 and Fag s 1. On the other hand, molecular component Api g 1 is recorded as a single component with a significant relation to the severe form of AD.

In our previous study, food allergy to apple was confirmed in 15.9% and to celery in 5.3% of atopic dermatitis patients (Celakovska et al., 2021).

From moulds and yeast, we confirmed the significant relation between the sensitization to Mala s 11 and Asp f 6 and the severe form of AD. In our previous study, we confirmed, that the sensitization to Mala s 6, Mala s 11, Sac c, Asp f 6, Cla h and Cla h 8 correlate to the severity of atopic dermatitis (Celakovska et al., 2021, March 4). The yeast Malassezia sympodialis, which is an integral part of the normal cutaneous flora, has been shown to elicit specific IgE- and T-cell reactivities in AD patients. Several possible pathogenic mechanisms enable Malassezia to trigger the development of AD. Malassezia spp. may release more allergens in a less acidic (pH <6), typical for AD, environment. The similarity between fungal thioredoxin and human proteins causes T-cell cross-reactivity. An interaction between Malassezia spp. and keratinocytes alters the profile of cytokine release, and yeast cells can survive when absorbed by keratinocytes. Dendritic cells of AD patients induced by Malassezia are less susceptible to lysis mediated by NK cells which exerts a pro-inflammatory effect. Despite the evidence that Malassezia spp. contribute to the development of AD, the pathogenetic mechanisms and relationship between Malassezia and immune defence remain partly unexplained and require further research (Nowicka & Nawrot, 2019, July; Vilhelmsson et al., 2008, December).

In our previous studies, we evaluated the occurrence of sensitization to food and inhalant allergens in patients suffering from AD, but the sensitization was determined according to extracting specific IgE, atopy patch test and skin prick tests without using molecular allergy diagnosis (Celakovska & Bukač, 2015; Celakovská et al., 2011, July; Čelakovská & Bukač, 2011). Results of our study show that molecular allergy diagnosis are typical methods of precision medicine and help clarify the influence of individual molecules on the severity of atopic dermatitis, which can be important in immunotherapy.

Conclusion

In patients with the severe form of atopic dermatitis, the important role plays molecular components Fra a 1 + 3, Mal d 1, Api g 1 and Aln g 1 from PR 10 proteins and Mala s 11 and Asp f 6 from moulds and yeast. We find the significant association of these components in pairs and triplets with molecular components Cor a 1.0401, Cor a 1.0103, Fag s 1, Alt a 1 and with the occurrence of allergic rhinitis. The relative frequency of positive specific IgE to Bet v 1 is the most frequent of all components both in the mild and moderate forms of atopic dermatitis; in the severe form, the highest sensitization rate was confirmed to Fra a 1 + 3. Multiple sensitization to the molecular components of PR 10 proteins does not affect the severity of AD, on the contrary, multiple sensitization to fungi leads to more severe forms of AD.

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Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This work was supported by Progres: Charles University, Medical Faculty Hradec Králové [Grant Number Q 40/10].