This document discusses advances in molecular diagnostics for mite sensitization. It begins with a brief history of allergy to house dust mites and an overview of the major allergenic molecules from mites, including Der p 1, Der p 2, and Der p 23. It describes the concept of "molecular spreading" where the IgE response spreads from initial sensitization to major allergens to include other milder allergens over time. The document then covers classical and molecular diagnostic techniques for mite allergy. It concludes by discussing the potential predictive role of antibody patterns to different mite allergens.

1. Novità in ambito di diagnostica
molecolare nella
sensibilizzazione ad acari
Danilo Villalta
Immunologia e Allergologia
Ospedale «S. Maria degli Angeli»
Pordenone

2. OUTLINE
1. Note storiche e generalità sull’allergia agli
acari
2. Le molecole allergeniche degli acari:
gerarchia e proprietà
3. Lo spreading molecolare della risposta IgE
agli acari
4. La diagnostica classica e molecolare
nell’allergia ad acari
5. Possibile ruolo predittivo del pattern
anticorpale

3. OUTLINE
1. Note storiche e generalità sull’allergia agli
acari
2. Le molecole allergeniche degli acari:
gerarchia e proprietà
3. Lo spreading molecolare della risposta IgE
agli acari
4. La diagnostica classica e molecolare
nell’allergia ad acari
5. Possibile ruolo predittivo del pattern
anticorpale

4. Historical milestones
- As early as 1921 Kern RA reported that many asthmatics had strongly positive
skin test to house dust (Med Clin North Am 1921)
- In 1928 dr Dekker suggested that mites played an important role in allergy, but
this evidence was unconvincing (Munch Med Wochenschr 1928)
- In 1964 Woorhorst et al. identified Dermatophagoides pteronissinus as house
dust allergen (J Allergy 1967)
- In 1968 Brown described the first extract for dignostic skin test (Br Med J 1968)
- In 1971 extracts were available for immunotherapy (Smith AP. Br Med J 1971)
- In 1980 the first allergen of Dermatofagoides pteronissinus was described by
Chapman & Platts-Mills (J Immunol 1980)
- In 2013 was identified the molecular structure of the new major HDM allergen,
Der p 23 (Weghofer M, J Immunol 2013)

5. - The normal life span of a HDMs is approximately
65-100 days
- Proliferate maximally at a relative umidity of 75%
- Complete the egg-to-adulte cycle in 35 days at
23°C, but in only half time at 30°C
- Females lay approximately 50-80 eggs
during their lifetime
- Each immature mite stage need to shed
its exoskeleton before passing onto the
next stage

6. - During their lifetime mites produce
approximately 1000 solid fecal waste
particle (about 25 μ in diameter)
surrounded by a peritrophic membrane
containing proteolytic digestive enzymes

7. OUTLINE
1. Generalità sull’allergia agli acari
2. Le molecole allergeniche degli acari:
gerarchia e proprietà
3. Lo spreading molecolare della risposta IgE
agli acari
4. La diagnostica classica e molecolare
nell’allergia ad acari
5. Possibile ruolo predittivo del pattern
anticorpale

9. Schematic and still debated localization of the 12 examined
allergenic molecules of Dermatophagoides pteronyssinus.
1 15
18
11
21
14
23 7 5
4
2
1
22
Adapted and extended from
Weghofer M. J Immunol. 2013; 190:3059-67.
Banerjee S. J Invest Dermatol. 2015; 135:102-9.
Thomas WR. Allergol Int 2015; 64:304-11
Thomas WR. Chang Gung Med J. 2004; 27:563-9.
O'Neil SE. Clin Exp Allergy. 2006; 36:831-9.
Courtesy of P. Matricardi

10. Reithofer M & Jahn-Schmid B. Int J Mol Sci 2017.18,1368

11. Group 2
MD-2 like lipid binding protein
TSAI JJ PLOS 2011
«auto-adjuvant» properties

12. Miller JD. Clin Rev Allergy Immunol 2018, June 23

13. The «classical»
mite molecules
Cysteinprotease
Der p 1
Der p 2
Lipid binding protein
Der p 10
Tropomyosin

14. Trombone APF et al
CEA 2001;32:1323-8
- 95% HDM positive
- Accounting for 60%
of antibodies

15. J Immunol. 2013 April 1; 190(7): 3059–3067
Chitin-binding protein

16. Der p 23 is a protein of the peritrophic matrix that lines the midgut and surrounds
the digested food stuff and the fecal pellets. In mites, as in most Arthropods, fecal
pellets are surrounded by the peritrophic matrix. The peritrophic matrix is
synthesized along the whole midgut of the HDMs and functions as a permeability
barrier between the ingested food and midgut epithelium. It enhances digestive
processes and protects the midgut epithelium from mechanical disruption as well as
from attacks by toxins and pathogens. The peritrophic matrix is thought to be
secreted by the microvilli of gut epithelial cells and is composed of a meshwork of
chitin fibrils embedded in a matrix of proteins, proteoglycans, and glycoproteins. In
fact, the in situ localization suggests that Der p 23 is indeed synthesized in the
midgut epithelial cells where the allergen is localized in vesicles.

17. It reacted with IgE Abs from 74% of D.
pteronyssinus allergic patients (n = 347) at
levels comparable to the two major HDM
allergens, Der p 1 and Der p 2. Thus, Der p 23
represents a new major D. pteronyssinus
allergen. Furthermore, rDer p 23 exhibited
high allergenic activity… (test basofili)

18. 2016n. 416n. 503

19. Batard T et al Allergy 2015
Sero-prevalence

20. Sero-dominance
Batard T et al Allergy 2015

21. 2015
Serodominat allergens

22. 2015
Sero-prevalence: ~ 40 of HDM sensitized
Sero-dominance: 10-15% of total binding when present
Mild tier allergens

23. Batard T et al Allergy 2015
Sero-prevalence

24. Sero-dominance
Batard T et al Allergy 2015

25. 2015
5% in asthma
60% in AD
Dominant in
atopic dogs
High binding in
patient with active
scabies
Minor allergens

26. 2015
Allergens of unknown importance

27. OUTLINE
1. Generalità sull’allergia agli acari
2. Le molecole allergeniche degli acari:
gerarchia e proprietà
3. Lo spreading molecolare della risposta IgE
agli acari
4.La diagnostica classica e molecolare
nell’allergia ad acari
5. Possibile ruolo predittivo del pattern
anticorpale

28. 2017
prevalence
>40%
15-30%
<10%

29. time
A
94
AB
25
ABC
10
ABC
42
AB
39
A
4848 (51%)
26 (74%)
10 (100%)
AB
35
Figure 2
One or more of
Der p 1
Der p 2
Der p 23
One or more of
Der p 4
Der p 5
Der p 7
Der p 21
One or more of
Der p 11
Der p 14
Der p 15
Der p 18
Clone 16
One or more of
Der p 1
Der p 2
Der p 23
One or more of
Der p 1
Der p 2
Der p 23
+ +
One or more of
Der p 4
Der p 5
Der p 7
Der p 21
+
Molecularspreading
Posa D, et al. JACI 2017
Progressive IgE sensitization to D.pt. molecules of groups A, B, C

30. Some patients remain “blocked” at stage “A” of IgE sensitization
In others, the IgE molecular response spreads up to the level “ABC”
WHICH DETERMINANTS?

31. Earlier sensitization onset = stronger molecular spreading
Posa D, et al. JACI 2017
MORE
SPREADING
LESS
SPREADING

32. heredity AND environment affect
the spreading of the IgE response to mite allergen molecules
Posa D, et al. JACI 2017

33. OUTLINE
1. Generalità sull’allergia agli acari
2. Le molecole allergeniche degli acari:
gerarchia e proprietà
3. Lo spreading molecolare della risposta IgE
agli acari
4. La diagnostica classica e molecolare
nell’allergia ad acari
5. Possibile ruolo predittivo del pattern
anticorpale

34. Allergy 2010; 65: 184–190
Commercially available mite extracts

35. Values are expressed as μg protein/ml extract.
SPT
++
neg
++
neg
neg
neg
neg neg
neg
(cut-off < 7 mm2)

36. IAAI 2012

37. Casset A et ak Int Arch Allergy Immunol 2012

38. 2016

39. Der p 1

40. Der p 2

42. OUTLINE
1. Generalità sull’allergia agli acari
2. Le molecole allergeniche degli acari:
gerarchia e proprietà
3. Lo spreading molecolare della risposta IgE
agli acari
4. La diagnostica classica e molecolare
nell’allergia ad acari
5. Possibile ruolo predittivo del pattern
anticorpale

43. 2015

44. 2015
OR
Der p 2 = 2.79
Der p 5 = 4.21
Der p 7 = 2.54

45. 2017
AR (oct-jan)
Asthma
ABC
AB
A
neg
ABC
AB
A
neg
Population size
n=722

46. IgE response ≤ 5y of age % OR p-value OR p-value
Der p 1 sensitization 4.5 4.4 <0.001 3.0 0.031
Der p 2 sensitization 5.4 2.8 0.005 1.9 0.175
Der p 23 sensitization 2.8 4.3 0.002 5.3 0.009
Group "A" 6.9 4.0 <0.001 3.2 0.004
OR, Odds ratio
Dependent variable: at least 1 years with asthma between 6 and 20 years ofage in 709 children (binary variable),
in particular 580 observations for level 1 and 129 for level 2.
¹ Each line refers to an independent model, adjusted for hay fever parents, exposure, breast-feeding after one
month, mother smoking in pregnancy, older siblings, German nationality. Reference category is participants with
no IgEsensitization.
(1.3 - 5.5) (0.7 - 4.8)
(1.7 - 11.0) (1.5 - 19.5)
(2.1 - 7.5) (1.4 - 7.0)
(2.0 - 9.2) (1.1 - 8.2)
Table 5 - Association of an IgE response to group "A" D.pt. molecules (Der p 1, Der p 2, Der p 23) at
preschool age (≤5y) with the onset of asthma at school age (6-20y), in 709 participants of the MAS
birth cohort.
Univariate analysis Multivariable analysis¹
95% CI 95% CI
Der p 1 and Der p 23 at pre-school age
predict asthma at school age
Posa et al. JACI 2017

47. 17 centri
519 soggetti SPT +
Sept 2019

48. TEST IN VITRO CON ESTRATTI DI ACARI
• Dosate IgE specifiche in 457 pazienti
• 23 (5%) sono risultati negativi sia per D1 che per D2.
• 411 (89,9%) sono risultati positivi per entrambi gli estratti.
• 16 (3,5%) monosensibili per D1
• 5 (1,1%) monosensibili per D2.
• Livello IgE specifiche 0,35-100 kU/L nei positivi.
• Livello medio IgE specifiche: D1= 22,5 kU/L; D2: 20,7 kU/L.
• La severità della rinite non correla con il livello delle IgE
specifiche.
• La presenza di asma correla significativamente con il titolo
di IgE specifiche (p< 0,0001 per D1 e D2).

49. Correlazione tra livello di IgE specifiche per
acari e presenza/severità di asma allergico

50. INDAGINI IN VITRO CON MOLECOLE
ALLERGENICHE

51. Prevalenze
• Positività per Der p 1: 58,6%
• Positività per Der p 2: 67,9%
• Positività per Der p 23: 59,8%
• Positività per Der p 10: 11,9%
67 (12,9%) NON SONO RISULTATI REATTIVI CON ALCUNA MOLECOLA.
17 (3,3%) MONOSENSIBILI A Der p 1
48 (9,3%) MONOSENSIBILI A Der p 2
42 (8,1%) MONOSENSIBILI A Der p 23
13 (2,5%) PAZIENTI SONO RISULTATI MONOSENSIBILI A Der p 10.
Allergeni maggiori

52. Influenza della polisensibilizzazione a molecole degli
acari sul livello di IgE specifiche per estratti
Il livello medio di IgE specifiche per estratto (D1) è
strettamente correlato al numero di molecole
riconosciute al paziente (p< 0,0001)
0
10
20
30
40
50
60
0 1 2 3 4
num molecole
Media RAST d1 kU/L

53. Significato clinico della
polisensibilizzazione ad allergeni di acari
Il rischio di asma bronchiale aumenta
con il numero di molecole a cui il
paziente è sensibilizzato.
La severità della sintomatologia
asmatica è in funzione del numero
di molecole a cui il paziente è
sensibile
0,00%
10,00%
20,00%
30,00%
40,00%
50,00%
60,00%
70,00%
0 1 2 3 4
num molecole
%ASMATICI
r: 0,844; p < 0.05

54. Prevalenza di sensibilizzazione alle
singole molecole in asmatici e non
Tutti i pazienti Monosensibili acari
La presenza di asma si associa con la
sensibilizzazione a Der p 2, Der p 10 e
Der p 23.
Nei pazienti monosensibili la presenza di
asma si associa con la sensibilizzazione al
solo Der p 23.