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BIBLIOGRAPHY UPDATES IN ALLERGOLOGY
May 2011

Claude MOLINA* & Jacques GAYRAUD**

1. Microflora and allergy: “Language” for Aesop
2. Blood biomarkers for asthma and chronic bronchitis
3. Resolution to well cooked egg allergy: the tolerance mechanism
4. “Intrinsic” asthma: the come-back

1.    Microflora and allergy :
Theme: Asthma
Key words: Atopy, exacerbation, asthma, virus, ribosomal RNA, microbial diversity, bronchial hyperresponsiveness, eurotium, aspergillus, penicillium, gram (-) germs, hygienic theory, macrolides

Like Aesop’s language,  Microflora may be for Allergy, the best and the worst”of things
1.1.    “The worst of things”
It is well known that infection is strongly associated with asthma exacerbations. A timely reminder is published in an European review with recommendations (N.G.Papadopoulos et al :Allergy 2011 66 458-468). The main responsible agents are respiratory viruses (Rhinovirus, Syncitial virus, type-C-virus, as well as metapneumovirus in children, influenza virus in adults) and bacteria such Chlamydophila and Mycoplasm, more often involved with asthma persistence.
But the mode of action of this microbiota , relatively unknown, is the subject of a very interesting scientific study. In some cases, improvement of respiratory symptoms, after macrolide therapy, has incited a group of American researchers (Yvonne J.Huang et al JACI 2011 127 2 372-381) to detect, through modern, culture-independent, techniques, one or more characteristic microbial profiles in asthmatics’ distal airways, and compare them with clinical features of asthma. In a pilot multicenter study among 65 asthmatic adults, more or less under control thanks to claritromycine treatment (vs 10 healthy control subjects) bronchial epithelial brushings were collected and 16S ribosomal RNA (present in all bacterial cell walls) was sequenced, amplified by PCR and parallel clone library-sequencing analysed.
They first observed the great diversity of the microbial population, statistically higher among asthmatic patients than controls. Moreover, comparing patients’ microbiota composition to corresponding clinical criteria, they observed a significant correlation between bacterial diversity and bronchial hyerresponsiveness (BHR). It is difficult to assign this association to a particular group of germs, (about 100 families were analysed and identified, including Comamonadaceae, Sphingomonadaceae, Oxalobacteraceae and other taxons). But it could very well account for the severity and treatment difficulties of certain forms of asthma.

1.2.    “The best of things”
The commensal bacterial flora may be beneficial for allergic and asthmatic patients. Two very interesting studies prove it.
One is an elegant experiment, by N.Harris’s Swiss team (T.Herbst et al. AJRCCM ahead of print 2011 25 Mars) which makes use of a model of allergic airway inflammation in transgenic germ-free mice, then sensitized by ovalbumin, and shows that the induced Th2-type humoral and cellular allergic response is strongly reduced with re-colonization by commensal bacterial flora, such as that detected on human intestinal, pharynx, or bronchial mucosa. It also ensures the recruitment and maturation of immune cells in the lung.
The second excellent study is published by the German-European group, led for many years by Erika Von Mutius and devoted to the beneficial role of farm life in preventing and protecting children against asthma. It appears that exposure to the various microorganisms of agricultural environment accounts for this effect (M.J Ege et al NEJM 2011 364 701-9).
Two groups of children, living on farms and going to school, were the targets of this bacteriological and epidemiological survey :
In the 1st group (over 6000 subjects) samples of mattress dust were screened for bacteria (16S ribosomal DNA) using modern genetic identification techniques; in the 2nd group (over 9000 children), samples of settled dust from children’s rooms taken by electrostatic collectors were evaluated by means of traditional germs and fungus culture.
In both groups the prevalence of asthma and atopy was significantly lower in farm children than in controls and the former are exposed to a larger number of germs.
In the 1st group the microbial diversity was inversely and significantly related to the prevalence of asthma (but not atopy). In the 2nd group bacterial detection revealed also a large variety of bacteria, including Listeria Monocytogenes, Corynebacterium but mostly two predominant species : Eurotium, a sexual form of Aspergillus, and Penicillium : their protective role against asthma is consistently suggested, whereas on the other hand,  Gram(-) germs could protect against atopy.
Thus the beneficial role of the environmental bacterial population is a striking confirmation of the hygienic theory of allergy.

2.    Blood biomarkers for asthma and chronic bronchitis
Theme: Asthma
Key words: Asthma, chronic bronchitis (COPD), biomarkers, ceruloplasmin, haptoglobin, hemopexin,  ?2 macroglobulin

A group of researchers from 9 Australian universities suggests, as a diagnosis method,  identification, in the blood of adult subjects, of novel specific protein biomarkers of these respiratory diseases, through two-dimensional electrophoresis followed by mass spectrometry (N.M.Verrills et al AJRCCM 2011 18 March ahead of print).
In a first analysis, they detected 70 proteins of which 58 were validated, 20 considered as possible candidates and grouped in 3 categories : iron metabolism (ceruloplasmin (C), haptoglobin (H), hemopexin (Hx)), coagulation cascade (prothrombin (P), α-2 macroglobuline (α-2M), fibrinogen…) and Complement.
3 groups of adult patients were tested : 21 asthmatics (A), 5 suffering from chronic obstructive pulmonary disease (COPD) and 17 controls (T), plus , later on16 older subjects (7 A and 9 COPD).
After a first stage of major biomarker validation, proving that a panel containing several of them is necessary for an accurate diagnosis, and after a statistical comparison in subjects and controls of those markers’ respective sensitivity and specificity, the authors arrive at the following conclusion : 4 biomarkers are able to discriminate between the 3 groups tested : C, H, Hx and α2M.
Thus, H+Hx clearly discriminate between A and T, while C+Hx distinguish between COPD and T. In differentiating between A and COPD, which is often a problem for the clinician, the H+C+ α2M combination provides the highest sensitivity and H+Hx+ α2M the best specificity. Finally, the use of inhaled corticosteroids does not modify these observations.
This meticulous research demonstrates that while blood is easy to obtain for diagnostic purposes in an adult (as urine is in a child, cf. our April BUAs), these tests are not within the scope of all laboratories and are only useful in a small number of cases where clinical examination is unclear ; moreover, they are not decisive and are certainly costly.
However, they open new perspectives on physiopathology and biochemical mechanisms which support these diseases.

3.    Resolution to well-cooked egg allergy : the tolerance mechanism
Theme: Food allergy
Key words: Egg, uncooked egg, well-cooked egg, tolerance, ovalbumin, ovomucoid, protein degradation, intestinal digestion

Two recent articles, one clinical and the other experimental, evoke this problem. The British team of Cambridge Paediatricians (A.Clark et al  Clin. Exp.Allergy 2011 13 April ahead of print) undertook a longitudinal study between 2004 and 2010 on 95 children suffering from egg allergy (EA) with a median onset at 12 months. The disease was typically marked by cutaneous and gastrointestinal symptoms, with anaphylaxis in 7 cases, (needing to resort to adrenalin 5 times) and respiratory reactions in 11 cases.
The authors, aware of the lack of national or international recommendations on the issue, , performed repeat challenges at least once a year, with a total of 181 “open” trials, of which 77 with well-cooked egg and 104  uncooked.
Out of the 28 positive cases among the 77 (37%), 65% had cutaneous reactions, 68% intestinal and 39% rhinitis with no other respiratory reactions. Adrenalin was never required.
Tolerance was gained twice as rapidly to well-cooked than uncooked egg (an average of 5 vs 10 years) and continued to 13 years. Nearly one-third had resolved allergy to well-cooked egg at 3 years, and two-thirds at 6 years. The authors consequently suggest the initiation of home reintroduction of well-cooked egg into the child’s diet from age 2 or 3, particularly if  EA was moderate and without asthmatic reaction.

As to the tolerance mechanism, it is evoked in the interesting experimental work by the New York Mount Sinai Hospital paediatrician group (G.Martos et al JACI 2011.127 . 4  990-997) who have studied ovalbumin and egg white ovomucoid allergenicity. Thus, in the sensitised mouse, ingestion of cooked egg white causes no allergic reaction, whereas peritoneal administration induces anaphylaxis. Heating breaks down those proteins which, digested and absorbed in the intestinal tract, become unable to triggering basophil activation.
So, degradation of cooked egg white proteins, strengthened by gastro-intestinal digestion, is one of the explanatory mechanisms of egg tolerance in the majority of children allergic to that food.

4.    The come-back of  “intrinsic” asthma
Theme: Asthma
Key words: Intrinsic asthma, dust mites, total IgE, specific IgE

The term “intrinsic asthma” had been banished from recent medical literature, partly because its definition was based on negative criteria (presumed absence of allergy) and partly because it incited the over-worked clinician to shorten his/her questioning and examination of the patient, by-passing thorough etiological research and adopting the easy solution of corticoid prescription.
But a team of pneumologists and researchers from several Belgian universities has recently reopened the file by stressing the fact that in these allergy-free A (Asthma), IgEs were locally detected but that antigen-response specificity was elusive (J. Mouthuy et al. AJRCCM. April 2011 ahead of print).
They therefore submitted a group of 29 non-atopic A (negative skin and IgE tests), compared to 24 allergic As and 25 controls, to a classical series of biologic and functional respiratory investigations (looking for bronchial hyperresponsiveness),
Identification of dust mite – specific IgE in sputum (Der p and recombinant Der p1 and p2)was also performed  to define their clinical meaning and functional relevance (through both whole-lung challenge and basophil activation test).
Results were as follows :
The intrinsic asthmatics’ sputum (IA) contained, like that of the allergic asthmatics (AA), high and significant levels of total IgEs and dust mite - specific IgEs (Der p), as compared to controls (C). Those IgEs recognise in vitro major allergens Der p1 and Der p2 and are able to trigger activation of blood basophils from atopic donors.
However, dust mite (Der p) nasal provocative challenges performed in 12 IA vs 6 AA and 6 C, did not translate into any functional or cellular response from IAs, contrary to the bronchospastic and inflammatory reaction caused in AAs.
As a whole, for these authors, IAs do locally produce IgEs, including dust mite – specific IgEs which can be detected in sputum and are able to activate effector cells in vitro - albeit without clinical response in vivo.
It is certainly a valuable study, but is based on too few cases. The authors put forward the weak postulate of a second signal (viral ?) that promotes in atopic patients, IgE-mediated asthmatic responses through Fc€R1 which would not be found in IA. However, the definition of this entity still remains questionable.


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Comments and questions to :

Pr. Claude Molina    or    Dr Jacques Gayraud
* **