Omnia non pariter rerum sunt omnibus apta
Propertius, Elegies III,9,7
PART 1 – NOT EVERY ALLERGY HAS THE SAME ROOTS
Introduction – not everybody's cup of tea
Over decades, noncommunicable diseases (NCDs) have become an emergency problem for the developed world. Obesity, cardiovascular diseases, chronic nephropathies, psychiatric disorders and neoplastic diseases top the list of the major global threats and are also increasing in the developing world . These include metabolic diseases, neurodegenerative conditions, autoimmune disorders (type 1 diabetes, chronic inflammatory bowel diseases, thyroiditis, rheumatoid disease) as well as allergic conditions and asthma . Inflammation and immune dysregulation are common features of all these different conditions and they highlight the central multisystem role of the immune system .
The increase of respiratory allergy in westernized countries, started 50 years ago, seems to have plateaued at the beginning of this century , but a new wave of food allergies has emerged in the last 10–15 years [5,6], in particular in preschool children [7–10]. This ‘second wave’ is particularly evident in countries where respiratory allergy had increased, for example United Kingdom, Australia and United States [5,7,11]. Peanut allergy has more than doubled in the last 15 years and has recently been recorded in 1–2% of children in Australia, Canada, United Kingdom and United States . The reasons for the differences in temporal presentation of various allergic conditions and the intergenerational dissimilarities in the disease profile have not been elucidated; however, as the increase in allergic disease has occurred too rapidly (within one to two generations) to be attributed to genetic changes in the population, it is likely related to environment.
The search for environmental causes of allergies and epidemiological trends is a good opportunity to explain the complexity of allergic disease. Whatever the cause(s) of this increase are, they do not act in the same way in different populations, socioeconomic environments and cultural habits; this reflects the situation at the individual level, in which the paediatric allergist knows perfectly that every child has his/her specific allergic condition. Thus, preventive interventions, diagnostic pathways and therapeutic proposals may not be identical in different populations, nor in individuals in the same population. And in an age in which a spate of cutting-edge technology and clinical breakthroughs has substantially altered our vision and practice regarding allergy medicine, it is increasingly clear that all these improvements have their specific applications and, in plain language, may not be everybody's cup of tea.
Role of innate immunity in allergy inception
Innate immunity is one of the areas of the immunologic research that have substantially modified our understanding of allergic diseases. Cells of the innate immune system have been referred to as ‘the unsung heroes’ by Parham  as, with the accruing knowledge in this area, it has become clear that if ‘most people are not perpetually sick, this is tribute to innate immunity squelching most of the infections that we contract’.
The innate immunity cell types are specialized in different effector functions:
1. Macrophages are responsible for phagocytosis and activation of bactericidal mechanisms and are major producers of pro-inflammatory cytokines;
2. dendritic cells in addition to releasing abundant pro-inflammatory cytokines, are dedicated to the antigen uptake from peripheral sites (in their immature form) and its presentation to T cells in the lymph nodes (by mature dendritic cells);
3. neutrophils are able to phagocyte and to activate different bactericidal mechanisms;
4. eosinophils act for the killing of antibody-coated parasites;
5. mast cells are able to release granules containing histamine and other active agents;
6. natural killer (NK) cells are able to kill tumor and virus-infected cells and release cytokines, primarily IFN-γ, but also TNF-α and GM-CFS and various chemokines.
Thus, innate immunity is highly efficient in dealing with a series of immunologic stimuli and paves the way to the adaptive immunity. To achieve their functions, innate cells act with a series of mechanisms. As an example, NK cells, major players of innate defenses, exert the following functions:
1. Cytotoxicity, including tumor or leukemia cell killing, killing of virus-infected cells, dendritic cells editing, antibody-dependent cell-mediated cytotoxicity (ADCC);
2. Cytokine production, finalized to induce inflammatory responses, regulate adaptive immune responses, regulate hematopoiesis, induce dendritic cells maturation and, in decidual tissues during pregnancy, promote vessels/tissue remodeling and induce Treg;
3. Proliferative capacity.
NK cells are essential in tumor surveillance and their activating receptors are engaged by specific ligands frequently overexpressed or expressed de novo upon tumor transformation . However, other events such as cell stress, cell activation, or viral infection can lead to the expression of these ligands, thus resulting in NK cell activation (see Fig. 1). NK cell maturation, witnessed by the expression of particular surface markers as CD161, CD56, CD16, KIR and CD57 and by the downregulation of CD94 and in particular of CD56 [14,15], is accompanied by a progressive decrease in NK cell proliferative potential and an increase in their cytolytic activity .
Given their characteristics, NK have been successfully exploited in the haploidentical hemopoietic stem cell transplantation setting for the treatment of adult and paediatric patients with high-risk leukemias for several years [17,18].
Notably, NK cells are thought to be involved also in the development of allergic diseases. However, up to now the role of NK cells in respiratory allergy has been only marginally investigated. Some studies showed that different cytokines can bias NK cells towards ‘NK-1’ or ‘NK-2’ functional phenotypes, and it has been reported that these polarized NK cell subsets may be unbalanced in asthma . Although, we could not detect NK-1 and NK-2 cell subsets within circulating NK cells, in a recent study , we showed that the Th1-polarizing interactions between NK and dendritic cells was significantly altered in patients with respiratory allergy. Remarkably, NK-cell recruitment in tissue and their polarization may be consequent to the effect of infection with different pathogens. Pathogen-associated molecular products (PAMPS) may activate different cell types via Toll-like receptors (TLR), resulting in the release of polarizing cytokines acting on both T and NK cells and leading to their polarization towards Th1 (or NK1) or Th2 (or NK2) profiles. Thus, the particular cytokine microenvironment induced by infection may influence NK cell function, which, in turn, shapes downstream adaptive responses. Notably, NK cells themselves express an array of TLR including TLR3, TLR9, TLR2, TLR7 and TLR8 that allow them to directly sense different pathogens resulting in an increase in cytolytic responses and IFN-γ/TNF-α production  references. For example, NK cells display complex interactions with dendritic cells that are amplified by PAMPS interacting with TLR expressed by both cell types.
Dendritic cell-derived IL-12 promotes the differentiation of NK towards NK1, able to stimulate Th-1 responses; conversely, the basophil-produced or mast cell-produced IL-4 addresses NK cells towards NK-2, promoting immunologic tolerance or possibly Th-2 induction [22,23].
Regarding the possible role of TLR in allergy, it should be noted that in allergic children, neonatal monocytes are less responsive to lipopolysaccharides (LPSs), dsRNA and bacterial lipopeptides, the natural ligands of TLR2, TLR3 and TLR4 . This is of particular interest considering that:
1. neonatal responses must undergo environment-driven Th1 maturation in the postnatal period ;
2. postnatal suppression of Th2 propensity has been attributed to exposure to an increasingly diverse microbial environment during the early years of life;
3. there is now clear evidence that the failure of Th2 suppression in allergic children is related to an underlying disorder of innate microbial recognition pathways;
4. this failure can arise from deviations in innate TLR-mediated responses that have been shown to be preexisting in allergic children .
Thus, it appears today that TLR may at least in part be responsible for the differences in allergic predisposition, which appears more likely to emerge in an environment with low microbial burden. The idea of modulating dendritic cells, NK and ultimately Th1/Th2 differentiation through a TLR stimulation is today a seducing possibility for allergy prevention and treatment. In this context, we are aware of both the cellular distribution of different TLR as well as various natural TLR ligands. These include:
1. for TLR1, TLR2 and TLR6, peptidoglycan from gram-positive bacteria, lipoprotein GPI (from trypanosoma cruzi), zymosan (from yeast), mycobacterial lipopeptides, measles and cytomegalovirus proteins;
2. for TLR4, LPS of both gram-negative and gram-positive, lipoteichoic acids (gram-positive), fibronectin and RSV F-protein;
3. for TLR3, ds RNA;
4. for TLR7 and TLR8, ss RNA;
5. for TLR5, Flagellin;
6. for TLR9, unmethylated CpG DNA;
7. for TLR11, uropathogenic bacteria.
In conclusion, depending on the type of cytokine(s) released during the early stages of an inflammatory response to infection, NK cells can differently contribute to the quality and magnitude of innate and adaptive immune responses. As NK cells ultimately influence T cell polarization, acting on the deep mechanisms of allergy development in early life creating a ‘positive’ pattern of TLR-induced, dendritic cells and NK-secreted cytokines is more than a theoretical possibility today.
Early events in allergy development
The unprecedented rise in allergic disease reinforces a pressing need to define the early events responsible, and develop strategies to prevent this. The very fact that the allergic diseases have increased so rapidly and so dramatically in very young children is clear evidence that failure of immune tolerance is a very significant event, and that the developing immune system is vulnerable to modern environmental changes. Equally so, this indicates developmental plasticity of these systems and provides the hope that this can be harnessed for better allergy prevention strategies.
It is critically important to consider these issues in the wider context. Modern environmental and lifestyle changes are associated with an unparalleled rise in many chronic inflammatory NCDs. The specific vulnerability of the immune system to recent environmental changes is also reflected in the dramatic increase in virtually all immune diseases. Furthermore, clinical expression of immune disease within the first year of life together with detection of immune dysregulation at birth provides clear evidence of very early effects. Environmental risk factors for early immune dysregulation (dietary patterns, environmental pollutants, microbial patterns and stress) are also recognized risks for many NCDs, highlighting the need for interdisciplinary collaboration focusing on inflammation as a common element and target for NCD prevention. Common risk factors may logically mean that common interventions will have benefits in preventing many NCDs. It is therefore important to consider other health outcomes when designing cohort studies and intervention studies.
The growing burden of disease in early life
The burden of allergic disease is growing with each generation. The greatest burden of this new epidemic is in young children, who are experiencing the most dramatic increase in food allergy, earlier presentations  and increasing persistence  of disease. Most recently, food allergy has recently emerged as a substantial public health issue. In developed regions like Australia preschoolers have experienced a five-fold increase in food anaphylaxis over just 10 years . Currently, more than 20% of 1-year-old infants are sensitized to foods and more than 10% have clinical food allergy [6,28]. Many infants go on to develop respiratory allergic diseases . As these younger generations reach adulthood, the burden of allergic diseases is expected to increase even more.
Sensitization can be a very early event
Previous strategies to prevent food allergy through ‘allergen avoidance’ have not only failed, but have instead been associated with increased risk of disease . This together with other observations in humans and animals suggests that earlier introduction of allergenic foods may be a more logical preventive strategy. On the basis of this, there are several randomized controlled trials (RCTs) worldwide assessing the merits of early introduction of allergenic foods, such as egg and peanut. This includes our own study of healthy infants (n = 1512) at ‘high risk’ of allergic disease, randomized to receive egg powder (or a placebo) daily from when they first introduce ‘solid’ complimentary foods at between 4 and 6 months of age. We are also conducting a smaller pilot study using a similar design in children with eczema. In the course of both studies, we have already noted a significant proportion of clinical reactivity at 4–6 months (at randomization). Specifically, an interim analysis of the eczema study revealed that 22% had early clinical reactions (19/86 infants entering this pilot study), including one case of anaphylaxis requiring treatment with adrenaline (unpublished interim data). This indicates that sensitization has occurred much earlier than when complimentary foods are typically started (4–6 months) and suggests earlier strategies may be needed in some children. Importantly, in all cases there was no previous known history of direct ingestion of the food (egg) by the infant, indicating previous exposure through other routes potentially through breast milk or across the placenta. Although cutaneous sensitization has been proposed in children with eczema, this does not explain rates of reactivity (around 8%) in the study of infants with no eczema (unpublished interim data). Before designing earlier interventions to promote oral tolerance and prevent disease, it is critical to define the underlying immune events, particularly to address the continued uncertainty surrounding questions of allergen ‘dosing’ vs. ‘avoidance’ in pregnancy and lactation. This highlights the need to more fully understand the role and mechanism of early allergen encounter, and how variations in this context can influence the risk of sensitization. It is likely that allergen exposure is a key part of a series of inter-related events that, when functioning normally, lead to appropriate allergen-specific tolerance. Changing environmental conditions are promoting less favourable conditions for tolerance, with these effects beginning before birth and culminating in the early postnatal period.
Revisiting concepts of in-utero allergen exposure
The role and significance of allergen exposure in utero has been controversial, but needs to be revisited in light of increasing evidence of very early sensitization. There is good evidence that environmental allergens can be detected both in placental tissue  and in the fetal circulation  from where they could foreseeably interact with the developing immune system. In addition to systemic blood, allergens are also detected in amniotic fluid in animals  and in humans . These and other proteins are ingested and hydrolyzed through the gastrointestinal tract after fetal swallowing; another mechanism of potential processing of maternally ingested/derived proteins. Logically, these proteins are also in potential contact with the fetal skin and lungs.
Immune responses in utero
Human fetal T cells are now known to be in a dynamic balance between activation and quiescence, rather than in a passive state of immature inactivity . Functionally distinct from adult, fetal T cells are highly responsive to activation [36,37], but also more predisposed to Treg differentiation with higher proportions of circulating Treg in fetal life (reviewed in ). There is also evidence that the human fetus develops regulatory responses to exogenous antigens (alloantigens , microbial antigens  and allergens ) encountered through the placenta. Although fetal T cells are responsive to allergens  as early as 22 weeks’ gestation  these responses appear to reflect the promiscuous fetal reactivity of ‘recent thymic emigrants’  rather than ‘conventional adult’ T-cell memory. Utilizing a set of 28 overlapping peptides spanning the Ovalbumin (OVA) molecule, we have previously shown that more than 60% of neonates respond to multiple OVA epitopes, suggesting that these fetal immune responses involve recognition of multiple regions within the molecule and are thus likely to be directed against the native antigen, as opposed to a small number of epitopes in a cross-reacting antigen . Notably, our collaborators have previously shown this initial burst of short-lived OVA responsiveness is limited by parallel activation of suppressive CD4+CD25+regulatory T cells when fetal myeloid cells are cultured for 5 days in the presence of allergen , consistent with the recognized fetal predisposition for ‘active tolerance’ . Furthermore, using previously published methods , we have demonstrated the presence of OVA-specific CD4+ effector responses in cord blood which were suppressed by addition of fetal CD4+CD25+CD127lo suggesting OVA-specific Treg activity in this cord blood fraction (unpublished data). In-vivo studies also provide evidence that fetal Treg ‘memory’ to other exogenous antigens (alloantigens and microbial agents ) crossing the human placenta are long-lived and that these can persist after birth to influence postnatal antigen-specific responses [36,39].
Cord blood levels of food allergens, such as egg (OVA) range between 0.05 and 5.67 ng/ml and correlate directly with maternal levels (r = 0.754, P < 0.001) . Egg ingestion in pregnancy also correlates well with cord blood OVA-IgG levels, which are a better measure of allergen exposure than serum allergen levels probably because of a degree of allergen-trapping in the placenta . As allergen exposure at this age appears to favour regulatory T-cell responses , it is perhaps unsurprising that fetal lymphoproliferation to allergens do not correlate with maternal environmental allergen exposures [45,46]. Previous studies using maternal dietary elimination of egg in pregnancy in an attempt to reduce egg allergy have had paradoxical effects. Although egg-elimination achieved a significant reduction in OVA-IgG levels, this was not associated with allergy reduction . Rather a nonlinear (bell-shaped) relationship with subsequent allergy risk has been observed . Specifically, higher levels of OVA-IgG were actually protective against infant allergic disease (as were very low levels) whereas mid-range OVA exposure in pregnancy was associated with the highest risk . This is consistent with aeroallergen studies but still requires further investigation. Other studies suggest that the maternal allergen-specific IgG induced through allergen exposure may have direct allergy suppressive effects in the offspring .
We speculate that transplacental allergen encounter induces initial waves of allergen-reactive CD4+ T cells which are preferentially conditioned to differentiate into regulatory populations (as demonstrated in vitro), thereby ‘setting the scene’ for postnatal exposure.
The importance of tissue milieu
Variations in the local tissue milieu during antigen/allergen encounter play a critical role in determining the pattern of effector responses and the efficacy of regulatory mechanisms . This accounts for both tissue specific differences in immune responses, but also the changes in immune development under different environmental conditions that may alter the local milieu. Accordingly, there is evidence that maternal environmental exposures in pregnancy can specifically modify fetal immune function including dietary patterns , cigarette smoke exposure [51,52] and microbial exposure [53–55], through either epigenetic changes or other mechanisms . Variations in the cytokine profiles in cord blood, likely to reflect the milieu in the placenta and fetal tissues, have been associated with allergic predisposition . We have also recently shown that the neonatal thymic milieu also reflects the T helper type 2 (Th2) skewed cytokine milieu of cord blood, with the same age-related postnatal changes . Thymic stromal lymphopoietin (TSLP) produced by thymic epithelial cells in Hassall's corpuscles conditions dendritic cells  to induce conversion of reactive CD4+CD25− thymic T cells into CD4+CD25+FOXP3+ Treg . Maturation of thymic Treg cells also requires activation of the TSLPR/IL-7Rα receptor complex by TSLP or IL-7 [61,62]. Of note, we also demonstrated that variations in TSLP in newborn thymic tissue correlate with the capacity to generate Treg, suggesting that there are individual differences in the thymic microenvironment in utero . Moreover, we demonstrated that reduced TSLP and reduced Treg in the first weeks of life was associated with the risk of subsequent sensitization (to foods) at 1 year . It is therefore possible and likely that immune modifying environmental influences in utero could be exerting effects on the milieu in various tissues to promote or protect from allergic disease .
The allergen encounter in the early postnatal period
Although it is clear that ‘the scene is set’ to some extent in utero, it is also evident that responses are strongly shaped in the early postnatal period. During this period both colonization and breastfeeding provide critical tolerogenic signals in the developing gastrointestinal tract when food allergens are ‘first’ ingested as part of an infant diet. Food allergens secreted in breast milk provide an important early oral exposure [44,63]. However, we have shown in a double-blind RCT of lactating women fed 55 g/day of egg (vs. egg-free placebo) for 21 days that there are individual differences in OVA secretion and that some women (25%) do not appear to secrete OVA in their milk . Our collaborators have also shown that allergen-IgG complexes in breast milk induce antigen-specific Treg cells in the newborn animals . With similar complexes now detected in human milk , we speculate that observed differences in maternal milk content could contribute to the efficacy of local tolerance and susceptibility to inflammation. In this context, it is of enormous interest that thymus size is greater in breastfed than formula-fed infants  and that animal models show that this may be due to variations in breast milk IL-7 levels. Specifically, IL-7 in maternal milk has been shown to transfer across the neonatal intestine to increase T-cell production in the thymus . Because of the role of IL-7 in generation of thymic Treg this suggests a hitherto unrecognized link between local and systemic tolerance mechanisms, which needs to be investigated further.
Transcutaneous exposure to foods is also speculated to be another route for sensitization in the early postnatal period, particularly in children with eczema . Antigen transfer through a defective epidermal barrier is a key mechanism underlying IgE sensitization , and epithelial barrier dysfunction has even been proposed as a primary and initiating event in the allergic phenotype (reviewed in ). Eczema, frequently the first manifestation of allergic disease (and a recognized risk factor for food allergy) has been clearly associated with epithelial barrier dysfunction and mutations in Filaggrin, a key protein involved in skin barrier integrity . Moreover, children with early-onset (<3 months) and more severe eczema have the greatest risk of IgE sensitization . Clearly, in these children an atopic phenotype (eczema) has been established as a consequence of much earlier events and it is still not clear if food sensitization is a parallel or secondary event.
Understanding the environmental factors driving this rising predisposition will provide the best hope for reducing the burden of allergic disease through early primary prevention strategies. Emergent differences in immune function of newborns destined to develop allergic disease [26,41] emphasize the need to examine the role of predisposing utero events, as the placenta and the fetus are both vulnerable to exogenous and endogenous maternal influences during this period . Specific maternal exposures, such as microbial exposure, maternal diet, cigarette smoke and other pollutants, can modify fetal immune function and contribute to an increased risk of subsequent allergic disease (reviewed in ). These initiating events then appear to be rapidly consolidated in the early postnatal period in the growing proportion of children clinically manifesting an allergic phenotype in the first months of life. The most logical strategies to prevent allergic disease are those that will safely re-establish the more tolerogenic conditions seen in traditional lifestyles, particularly during these critical periods of development. So far this has been focused on specific and isolated interventions such as restoring microbial balance (probiotics), dietary patterns (prebiotics, n − 3 polyunsaturated fatty acids and other supplements) and minimizing adverse exposures including pollutants and stress. However, this may ultimately require a more holistic approach. Likely benefits of lifestyle interventions for the many other chronic inflammatory disorders (NCDs) associated with modern living underscore the need for interdisciplinary collaborations to overcome the rising global burden of these numerous modern conditions.
Immune interventions in paediatric asthma: a new beginning
The modern day epidemic of asthma and allergy shows no signs of waning. In order to curb this trend and reduce the prevalence of allergic diseases, we urgently need to find ways of preventing the development of atopy for which, once established, there is no cure and no medications that can alter its natural course.
Early environmental exposure of the immature immune system of children is critical in determining allergen sensitization vs. tolerance. There is emerging evidence of an imbalance in both innate and adaptive immunity in those predisposed to atopic disease . In addition, there is evidence of delayed maturity at birth in both Th2 (allergy-favouring) and Th1 (counterbalancing) responses in atopic children . So how do we influence the immune system in children at high risk of atopy to redress this imbalance? A number of strategies could be tried (see Box 1).
Reduction in allergen exposure
A dose-dependent relationship between allergen exposure and sensitization [74,75] and between sensitization and the development of allergic disease  is well established. A number of studies attempted to reduce exposure to allergen in early life as a means of influencing immune development and consequently lowering the risk of asthma and other allergic conditions. The Isle of Wight allergy prevention study was the first to show that a combination of strict avoidance of food and house dust mite (HDM) allergens during infancy has a profound effect on the development of atopy in genetically at risk children. The study showed a significant reduction in the incidence of asthma, atopic dermatitis and atopy in early childhood , and a continued effect was observed until later childhood . However, other studies [79–81] using similar, although not the same, allergen reduction strategies met with less success.
There may be a nonlinear relationship between allergen exposure and sensitization so that very low and high exposure may lead to tolerance, while a moderate level may cause sensitization . Recent epidemiological observations support the notion of immune tolerance induction, rather than allergic sensitization following early high dose oral exposure to an allergen. Grass pollen immunotherapy in allergic rhinitis may prevent asthma . This led to the hypothesis that high dose orally administered HDM allergen will induce immune tolerance in early childhood.
Oral allergen immunotherapy
We hypothesized that oral HDM immunotherapy will prevent sensitization to this key environmental allergen and, therefore, prevents the subsequent development of asthma. To test this hypothesis, we are recruiting 120 infants at high risk of atopy in a RCT. Immunotherapy (or placebo) will be administered from 6 months of age for a period of 12 months. Regular assessments with validated questionnaires and allergy tests are carried out at 3 monthly intervals. The results of this trial will be available in 2014.
Use of hypoallergenic formulae
After a 20 years’ controversy, a systematic review of the National Institute of Allergy and Infectious Diseases (NIAID) Grading of Recommendation, Assessment, Development and Evaluation GRADE panel concluded in 2012 that:
1. patients at risk for developing food allergy are defined as those with a biological parent or sibling with existing, or history of, allergic rhinitis, asthma, atopic dermatitis, or food allergy;
2. the exclusive use of extensively or partially hydrolyzed infant formulas should be considered for infants who are not exclusively breastfed and are at risk for developing atopic disease;
3. cost and availability of extensively hydrolyzed infant formulas may be weighed as prohibitive factors;
4. there is no evidence to suggest that exclusive feeding with a hydrolyzed infant formula is more likely to prevent atopic disease than exclusive breastfeeding (NIAID).
Thus, according to previous meta-analysis [84–86], the use of such formulae may be considered in a preventive setting.
On the contrary, the results of the meta-analysis and of the German Infant Nutritional Intervention (GINI) study  suggest that hydrolysates (pHF-W and eHF-C) prevent atopic eczema, which is not associated with later asthma but this does not support the idea that asthma can be prevented by the use of hypoallergenic formulae.
Vitamin D supplementation
Recent data from a variety of different sources indicate that vitamin D is an important immune modulator and may influence the development of the child's immune system and therefore the development of allergy and asthma. Vitamin D receptor is expressed on many cells of the immune system, including T cells, activated B cells and dendritic cells. In human umbilical cord blood cells, vitamin D inhibits IFN-γ, IL-4 and IL-13 production possibly through its effect on T-regulatory cells .
Vitamin D is generated in the skin in a sunlight-dependent process. Countries furthest away from the equator tend to have a higher prevalence of allergy . However, observational studies provide somewhat conflicting results. In the Southampton study , children born to mothers with higher serum vitamin D had significantly higher eczema and asthma at 9 years. Other studies, however, suggested that maternal vitamin D intake in the third trimester is associated with less reported wheeze at 3 and 5 years [91,92]. What we need is a RCT to examine whether vitamin D supplements during pregnancy and early childhood prevent the development of allergy and asthma .
A number of RCT are ongoing aiming to assess if antenatal vitamin D supplementation prevents the development of childhood wheeze and asthma. Weiss and Litonjua  in the United States are examining the effect of a large daily dose of 4000 IU of vitamin D3 during the second and third trimesters of pregnancy in 870 women with a personal history of allergic disease. The aim is to prevent the development of asthma in the first 3 years, although diagnosis of asthma is difficult at this age and further follow-up will be required (ClinicalTrials.gov NCT00920621). The Copenhagen Studies on Asthma in Childhood are assessing the effect of a daily dose of 2400 IU of vitamin D3 during the second and third trimesters in an unselected population of 600 pregnant women (NCT00856947). Again, the aim is to prevent wheeze assessed up to the age of 3 years. Warner et al. are assessing a group of 180 children born to women who participated in a RCT of antenatal vitamin D supplementation (http://www.asthma.org.uk/how-we-help/research/john-warner-vitami.html). They aim to assess these children for the presence of asthma and allergies. In Southampton, women are being recruited to test the hypothesis that vitamin D supplementation during pregnancy (vitamin D3, 500 and 1000 IU daily) will result in improved neonatal bone mineral content . Taking advantage of this study, we plan to assess these children for asthma and allergy status at the age of 3–4 years. We have examined cord blood cells from these women for the immunological effects of vitamin D supplementation during pregnancy. Although the majority of cytokine responses of umbilical cord mononuclear cells were not correlated with neonatal vitamin D3, TNF-α and TNF-β, both pro-inflammatory cytokines, were inversely related to neonatal vitamin D3. A similar, statistically significant, negative correlation was evident with IFN-γ, after mitogen stimulation.
In summary, the vitamin D status during pregnancy may influence immune responses at birth. Definitive proof of its association with the development of asthma and allergy awaits outcome of the current RCTs.
Early treatment of viral infections
Asthma patients are at higher risk of repeated viral infections. Respiratory infections, especially those caused by rhinovirus, in early life increase the risk of asthma by several folds . In our Isle of Wight birth cohort, the adjusted risk of asthma at age 10 years was four-fold in children, who had recurrent chest infection before the age of 2 years . In the European Community Respiratory Health Survey, early life viral respiratory infections increased the risk of asthma in adult life .
The target for viruses and pollutants is airway epithelium. Asthmatic epithelial cells in vitro are more susceptible to the cytopathic effect of human rhinovirus . So, why is the airway epithelium of children having asthma susceptible to viruses? One plausible explanation is the lack of production of interferon-β (IFN-β) by infected cells following viral infection . Further, ex-vivo experiments have shown that addition of IFN-β to asthmatic epithelial cell layers restores the ability of these cells to eliminate rhinovirus and reduce cytotoxicity. A study in our department is currently assessing the role of inhaled IFN-β in reducing the risk of asthma exacerbation following viral infection. Theoretically, if proven well tolerated, these studies can be extended to those children at high risk of asthma, to protect them from the effect of viral infection by prophylactic treatment with IFN-β, thus reducing the risk of asthma development in wheezy infants.
Use of bacterial products
Lyophilized extract of bacterial fractions from common nasopharyngeal bacteria have been available for decades in many countries. With their use, significant reductions in wheezing episodes have been reported . How a lyophilized extract of bacterial fractions from common nasopharyngeal bacteria can prevent or alter the course of a cold virus is still under scrutiny. The preparation used contained components from Haemophilus influenzae, Streptococcus pneumoniae, Klebsiella pneumoniae, Klebsiella ozaenae, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus viridans, and Branhamella catarrhalis. Are all of those eight bacteria needed to induce the effect? Should each be studied individually? Is the maximal effect attained from the dose used? Is there a dose–response relationship? What is the duration of effect? All these questions remain open, warranting further investigations.
Primary prevention of allergy with bacterial products
Among the strategies aimed to reduce the burden of allergic disease in children, the use of bacterial products finds its rationale in the immunological considerations exposed in the section by Lorenzo Moretta. In that section, we alluded to the natural ligands for TLRs. Among them, a particular interest has been developed for bacterial endotoxins. These potential TLR ligands have been epidemiologically associated with a reduction of allergy risk [100,101]. Intervention studies in animals [55,102] confirm that gene environment interactions in the context of allergy protection may avail themselves of this kind of mechanism.
Based on epidemiological studies indicating the inverse association among farm milk consumption and allergic disease , animal studies have indicated arabinogalactan from cowshed dust as a potentially preventive compound of the farming environment . The possible mechanisms underlying this association in the pregnant mouse model are the following:
1. expression of LPS-binding protein, TLR and sCD14;
2. modulation of immune responses (Tbet increased);
3. suppression of IgE, inflammation and airway hyperreactivity .
From these observations, studies have been designed to verify the effect of oral administration of bacterial lysates on food allergy in animals, with positive results . Bacterial lysates containing bacteria often causing upper and lower respiratory tract infections have been found to suppress airway inflammation in mice through recruitment of regulatory T cells . In this study, bronchial hyper-reactivity, airways inflammation and mucous secretion have been found reduced after administration of a bacterial lysate (Bronchovaxom, BV). In humans, the administration of probiotics (in particular Lactobacillus GG) to influence the development of atopic dermatitis has been intensively studied, with a fairly inconsistent outcome .
Thus, given the theoretical background and the experiences on animals, it makes sense to attempt and mimic the protective effect of microbial exposure in an interventional approach. We report here the first experience of oral application of gram-negative and gram-positive bacteria lysates in order to prevent atopic eczema in a high-risk population living in a nonfarming environment . In a randomized, placebo-controlled trial, 606 newborns with at least single heredity for atopy were treated between week 5 and month 7 of life with an oral bacterial lysate (Pro-Symbioflor; SymbioPharm, Herborn, Germany) containing gram-negative (Escherichia coli) and gram-positive (Enterococcus faecalis) heat-inactivated bacteria. The main outcome did not show any statistical difference, as both groups presented atopic dermatitis in 30% of cases at 3 years. However, atopic dermatitis prevalence was significantly reduced at the end of the intervention phase (31 weeks of age) in the subgroup of infants with single heredity for atopy: 10% (15/154) of infants in the active group had atopic dermatitis compared with 19% (27/145, P < .030) in the placebo group. This was more pronounced in the group of infants with paternal heredity for atopy (11% vs. 32%, P < .004). This study – the first using bacterial lysates in the child – indicates that bacterial antigen exposure is another plausible way for allergy prevention. The magnitude of effect is nonnegligible, and assuming 700 000 children from 0–2 years of age in Germany the application of such treatment could render 13 800 as the predictable number of healthy children due to intervention.
PART 2 - GUIDELINES IN ALLERGY MANAGEMENT
The World Allergy Organization allergy white book
The World Allergy Organization (WAO) Allergy White Book, compiled for publication under the supervision of the WAO Education Council and the editors Pawankar R, Canonica GW, Holgate S and Lockey R, was published in 2011. It represents the result of a common effort of 79 international expert allergists and clinical immunologists and outlines the data, which indicate that allergy is a major global public health problem. By covering the various allergic diseases it provides for each disease entity a concise clinical overview on the prevalence and treatment, the burden of the diseases, impact on quality of life of patients, their socioeconomic consequences, unmet needs, and future therapies and a healthcare perspective. The White Book also includes a tabulated presentation of the status of allergic diseases in 62 countries provided by Member Societies of the WAO. It is intended to serve as a comprehensive resource in explaining allergic diseases and its importance as a major public health problem for governments, policy makers, healthcare providers, sports and food authorities, lay public and allergy patients and the need for adequate service provision .
It brings to the attention of the scientific and lay (political, economic, cultural) world community some important points, including:
1. the dramatically rising prevalence of allergic diseases in developed and developing countries;
2. the importance of this rise in children;
3. the fragmentation of the services for patients with allergic diseases even in the developed world;
4. the absolute absence of specialized services in many countries;
5. the general lack of allergy specialists;
6. the risk that inappropriate care leads to avoidable morbidity and mortality and to substantial increase and unnecessary cost to healthcare systems and national budgets;
7. the risk of opening the way to nonscientifically based alternative and complementary diagnostic and therapeutic remedies.
As the prevalence of allergy has increased to such an extent, allergy must be regarded as a major healthcare problem. The mission of the WAO is to be a global resource and to advocate in the field of allergy, asthma and clinical immunology, advancing excellence in clinical care through education, research and training as a worldwide alliance of allergy and clinical immunology societies. The Organization presently embraces over 84 regional and national allergy, asthma and clinical immunology society members and affiliated organizations (see home page at www.worldallergy.org).
WAO is greatly concerned about the increasing global burden of allergic diseases. A major focus of the Organization is to create global awareness of allergy and asthma as a major public health problem. The Organization published the first State of World Allergy Report (SOWAR) in 2008, and now presents the first-ever global White Book on Allergy. It also has developed several documents that define the specialty of allergy.
Given the problem list presented, the WAO White Book outlines the data indicating allergy as a major global public health issue, and provides ‘high level’ recommendations to:
1. create an integrated approach to the diagnosis and management of allergic diseases;
2. increase public awareness of allergic diseases and their prevention;
3. provide greater education at the primary healthcare level and to nonallergy-oriented secondary care specialists;
4. train medical students, nurses and pharmacists to an appropriate level to enable them to collaborate with different organ-based specialists and allergy specialists in providing integrated care for allergy patients;
5. institute environmental control measures by the lowering of indoor and outdoor air pollution, tobacco smoking, and allergen and drug exposures, as appropriate;
6. encourage a preventive approach to allergic diseases, emphasizing the importance of continued research both in disease causation and management;
7. use model projects, to disseminate good practice, promote prevention and immune tolerance, and decrease the allergy burden in future years.
The contribution of evidence-based guidelines to food allergy medicine
The level of knowledge of general practitioners (in particular, general paediatricians – GP) on allergic diseases is low (see Table 1):
1. the majority of practitioners are unaware that oral full challenges are necessary to confirm the diagnosis of food allergy;
2. ∼ 30% of practitioners believe that a positive skin prick test (SPT) is sufficient to diagnose food allergy;
3. ∼ 30% of practitioners do not believe that cow's milk allergy (CMA) can expose children to fatal anaphylactic reactions .
Clearly, as in many fields of medicine, it is impossible for the general paediatricians to be constantly updated for three reasons: too little time; too much to read; too complicated information. In this situation, evidence-based medicine can be of use to help practitioners to get out of the jungle of the literature and to take the correct decisions .
The last 2 years, 2010 and 2011, have witnessed a ‘guideline fever’  in food allergy: three international guidelines have appeared, followed by a number of national guidelines and other initiatives currently in development. The WAO's Diagnosis and Rationale for Action against Cow's Milk Allergy (DRACMA) guidelines were the first document to be published in April 2010 , followed by the NIAID guidelines on the Diagnosis and Management of Food Allergy in the United States, published in December 2010 , and by the UK National Institute for Health and Clinical Excellence (NICE) guidelines ‘Food allergy in children and young people; diagnosis and assessment of food allergy in children and young people in primary care and community settings’, published in February 2011 .
Brief history and context of EBM
The aim of evidence-based medicine (EBM) is to enable practitioners to a conscientious, explicit, and judicious use of current best evidence in making decisions about the care of individual patients. The practice of evidence-based medicine requires integration of individual clinical expertise and patient preferences with the best available external clinical evidence from systematic research. The discipline is indebted to some pioneers:
1. Alvan Feinstein, who helped define evidence in the field of clinical epidemiology;
2. David Sackett, who developed and mentored a new breed of applied clinician–scientists and worked with them to create and disseminate the practice of evidence-based medicine throughout the world;
3. Gordon Guyatt, who coined the term ‘evidence-based medicine’ and edited the ‘Users’ Guide to the Medical Literature’, a comprehensive set of journal articles and textbook for clinicians that wish to incorporate EBM into their practices;
4. Brian Haynes, who established the American College of Physicians (ACP) Journal Club and developed the science of Search strategies (Pubmed).
As seen, this relatively recent science, sprung from the tree of epidemiology, has rapidly progressed into a well recognized, distinct discipline. Nowadays, the question is, however: where is EBM going?
As health professionals and patients are moving towards shared models of decision-making, there is a growing need for integrated decision support tools that facilitate the uptake of best evidence in routine clinical practice in a patient-centered manner. Today, the correct decision in an individual clinical case arises from at least three factors:
1. the clinical state and circumstances of case presentation, such as ‘a 2-year-old suspected of CMA in tertiary care setting’;
2. the research evidence; as an example, ‘Diagnostic accuracy of SPT’ or ‘effectiveness of allergen avoidance’;
3. last but not least, the societal and personal values and preferences, in the case examined, for instance ‘Avoiding allergic reactions at home is important’.
Suggesting to the patient ‘do that test’ or ‘avoid milk’ should come from an equation of these three factors filtered through the doctors’ expertise. Even if many conditions coincide, the decision will not be the same in every individual case.
Decision support tools for medical practice can be mapped on two dimensions:
1. the target user and his or her level of decision-making;
2. the level of uncertainty: either supporting more directive decision-making (behavior support) in the case of strong recommendations with a single best option or supporting dialogue (deliberation support) on the pros and cons of different options in the case of conditional (or weak) recommendations .
A good example would be the decision to perform – or not – a hyposensitization with milk for ‘that’ CMA patient: this largely depends on values and preferences of the family and the doctor . Thus, a prudent physician should draw evidence not only from the literature, but should also look for the evidence of baseline risk and context for a given population, and the evidence of that societal and personal values and preferences. The effects of such research need to be integrated in a decision-making and implementation way.
These three levels of research of the best evidence require:
1. for the evaluation of the baseline risk, detailed estimates for benefit–harm determination;
2. for the evaluation of the effects, the determination of all important outcomes;
3. for the determination of societal and personal values and preferences, new(er) type of research involving utilities.
In synthesis, the modern re-definition of an evidence-based healthcare requires a conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. The practice of evidence-based healthcare entails integration of evidence about the baseline risk and context of a problem; the patient's or society's values and preferences for the interventions and outcomes; and the estimates of related effects for outcomes that are critical or important for decision-making. Integrating these three factors requires a decision analysis that ranges from approaches used by a single practitioner and/or patient through intuition, and simple analytical reasoning, to fully fledged, mathematically based decision-analysis.
The implementation often requires, or would benefit from, decision support tools (e.g. decision aids) and is influenced by known or unknown factors related to the three basic pillars of decision-making.
Interventions include diagnostic testing and strategies as well as diagnostic-treatment strategies.
Healthcare recommendations, e.g. guidelines, when integrating on these considerations, are an ideal tool to support decision-making.
Evidence to action = recommendations
Evidence-based healthcare can largely benefit from the GRADE guidelines. These systematically developed syntheses, aimed to support practitioners and patients in decision-making about specific clinical circumstances, are based on a precise methodology. The key elements of each synthesis include the scope of the guidelines, the interventions and practices considered, the major recommendations and their strength of evidence, and the underlying values and preferences . The formulation of the recommendation is the result of an interplay among clinicians and EBM specialists, aimed to identify the most appropriate outcomes and the effects of the different interventions. Not all outcomes are equivalent. To remain in the field of food allergy:
1. avoiding rashes is important;
2. avoiding death (by allergic reaction) is also important!;
3. bone health is important.
But are those considerations equal (and independent)? Clearly not, and this is the reason why a scale of outcomes has been included and explicated in the GRADE guidelines. All DRACMA recommendations are constructed on these values and preferences, and can vary if the external conditions change. They are flexible, in order to be used in different circumstances .
Thus, at a time when evidence-based healthcare decision-making is changing, evidence-based guideline methods can help to:
1. identify magnitude of problem;
2. develop appropriate questions considering all outcomes;
3. synthesize evidence;
4. define research agendas.
Therefore, they remain an essential support tool in order to make the best possible decision in individual patients.
Paediatric considerations in allergic rhinitis and its impact on asthma 2010
Asthma and allergic rhinitis are two of the most common chronic respiratory inflammatory diseases. In children, the major risk factor is respiratory allergy. Asthma affects about 300 million people of all ages and allergic rhinitis affects 400 million people worldwide. These diseases rhinitis impair the quality of life. The International Study of Asthma and Allergies in Childhood (ISAAC III) indicates that the prevalence of allergic diseases is rising (asthma 0.8–29.1%; allergic rhinitis 5–40%) . It has been shown that comorbid asthma and allergic rhinitis substantially impact patient well-being and that the worsening of allergic rhinitis symptoms in patients with asthma can be associated with worsening asthma symptoms. In particular, allergic rhinitis impacts on asthma in child's quality of life. For these reasons, physicians who treat patients with asthma should evaluate treatment options for improving symptoms of both allergic rhinitis and asthma when present concomitantly . Studies have shown that:
1. treating allergic rhinitis decreases asthma-related resource utilization ;
2. failure to consider treatment of rhinitis as essential to asthma management might impair clinical control of asthma ;
3. pollen immunotherapy reduces the development of asthma in children with seasonal allergic rhinitis .
Despite this, allergic rhinitis is still largely ignored, underdiagnosed, misdiagnosed and mistreated. Given its characteristics – high prevalence, impact on QoL, significant comorbidities, high socioeconomic costs, poor control – allergic rhinitis is the ideal target for a large guideline policy .
The Allergic Rhinitis and its Impact on Asthma (ARIA) guideline was conceived during a WHO workshop in 1999 and was published in 2001 [126–128]. Among the progresses obtained with ARIA:
1. the concept of unified airways disease was introduced;
2. allergic rhinitis has been reclassified as mild/moderate–severe and intermittent/persistent, this classification closely reflects the impact of allergic rhinitis on patients;
3. in its 2010 revision, ARIA developed clinical practice guidelines for the management of allergic rhinitis and asthma comorbidities based on GRADE allergen avoidance has been indicated when possible;
4. patients’ education is indicated in all cases.
Regarding treatment, it's not everybody's cup of drugs: therapy should be individualized, keeping in mind efficacy, side effects and costs. As an example, ARIA recommends only second-generation antihistamines. The ideal antihistamine:
1. should have a rapid onset of action and longer duration of action, preferablly 24 h;
2. should have high receptor selectivity and additional antiallergic/anti-inflammatory effects;
3. should have minimal or no sedative effects;
4. should have no interactions with CYP450 isoenzymes, and no drug–drug interaction.
ARIA recommends newer generation intranasal corticosteroids (INCS), for their efficacy and lower systemic bioavailability. A meta-analysis done of INCS, looking at therapeutic index for INCS based on efficacy and adverse effects comparing the characteristics of Dexamethasone, Budesonide (BUD), Fluticasone propionate, Fluticasone furoate, Flunisolide, Mometasone furoate, Triamcinolone (TRIAM), and Beclomethasone dipropionate (BDP), indicated that:
1. of highest efficacy are BUD > Mometasone furoate >TRIAM;
2. with fewest side effects are Mometasone furoate and TRIAM followed by Fluticasone propionate .
Paediatric aspects of rhinitis are particularly focused on in the 2010 revision. Among the themes afforded, some deal with prevention of allergic rhinitis and are thus referred to as the whole ‘at risk’ population.
a. Should allergen avoidance methods be used by parents to prevent development of allergy in children? Recommendation 2 indicates that ‘For pregnant or breastfeeding women, we suggest no antigen avoidance diet to prevent development of allergy in children (conditional recommendation | very low-quality evidence)’. This recommendation places a relatively high value on adequate nourishment of mothers and children, and a relatively low value on very uncertain effects on the prevention of allergy and asthma in this setting.
b. Should passive tobacco smoke be avoided? Per recommendation 3, ‘In children and pregnant women, we recommend total avoidance of environmental tobacco smoke (i.e. passive smoking) (strong recommendation | very low-quality evidence)’. This is based on the fact that smoking and exposure to second-hand smoke are common health problems around the world causing a substantial burden of disease for children and adults. Although it is very rare to make a strong recommendation based on low or very low-quality evidence, the ARIA guideline panel felt that in the absence of important adverse effects associated with smoking cessation or reducing the exposure to second-hand smoke, the balance between the desirable and undesirable effects is clear.
c. Should exposure to dust mites be avoided? According to recommendation 4, ‘In infants and preschool children, we suggest multifaceted interventions to reduce early life exposure to house dust mite (conditional recommendation | low-quality evidence)’. This recommendation places a relatively low value on the burden and cost of using multiple preventive measures (e.g. encasings to parental and child's bed, washing and so on) and a relatively high value on an uncertain small reduction in the risk.
d. Should pets at home be encouraged/discouraged for children at risk of asthma? Children at high risk of developing asthma are those with at least one parent or sibling with asthma or other allergic disease. For such children, recommendation 5 suggests: ‘In infants and preschool children, we suggest no special avoidance of exposure to pets at home (conditional recommendation | low-quality evidence)’. This recommendation places a relatively high value on possible psychosocial downsides of not having a pet, and relatively low value on potential reduction in the uncertain risk of developing allergy and/or asthma.
Other recommendations refer to the treatment of established allergic rhinitis in children.
a. Should allergen exposure be reduced? According to recommendation 7, ‘In patients with allergic rhinitis and/or asthma sensitive to house dust mite allergens, we recommend that clinicians do not administer and patients do not use currently available single chemical or physical preventive methods aimed at reducing exposure to house dust mites (strong recommendation | low-quality evidence). We suggest multifaceted environmental control programmes be used in inner-city homes to improve symptoms of asthma in children (conditional recommendation | very low-quality evidence)’. This recommendation places a relatively high value on possible reduction in the symptoms of asthma in children, and relatively low value on the cost of such programmes.
b. Should intranasal antihistamines be used? Recommendation 14: ‘We suggest intranasal H1-antihistamines in children with seasonal allergic rhinitis (conditional recommendation| low-quality evidence). In children with perennial/persistent allergic rhinitis, we suggest that clinicians do not administer and patients do not use intranasal H1-antihistamines until more data on their relative efficacy and safety is available (conditional recommendation | very low-quality evidence)’. The recommendation to use intranasal H1-antihistamines in patients with seasonal allergic rhinitis places a relatively high value on reduction of symptoms, and a relatively low value on the risk of rare or mild side effects. The recommendation not to use intranasal H1-antihistamines in patients with perennial/persistent allergic rhinitis places a relatively high value on their uncertain efficacy and possible side effects, and a relatively low value on possible small reduction in symptoms.
c. Should new generation oral H1-antihistamines rather than intranasal H1-antihistamines be used in children? Per recommendation 15, ‘We suggest new generation oral H1-antihistamines rather than intranasal H1-antihistamines in adults with seasonal allergic rhinitis (conditional recommendation | moderate quality evidence) and in adults with perennial/persistent allergic rhinitis (conditional recommendation | very low-quality evidence). In children with intermittent or persistent allergic rhinitis, we also suggest new generation oral H1-antihistamines rather than intranasal H1-antihistamines (conditional recommendation | very low-quality evidence)’. These recommendations place a relatively high value on probable higher patient preference for oral vs. intranasal route of administration as well as avoiding bitter taste of some intranasal H1-antihistamines, and relatively low value on increased somnolence with some new generation oral H1-antihistamines. In those who experience adverse effects of new generation oral H1-antihistamines an alternative choice may be equally reasonable.
d. What about the possible use of leukotriene antagonists in allergic rhinitis in children? Recommendation 16: ‘We suggest oral leukotriene receptor antagonists in children with seasonal allergic rhinitis (conditional recommendation | high-quality evidence) and in preschool children with perennial allergic rhinitis (conditional recommendation | low-quality evidence)’. The recommendation to use oral leukotriene receptor antagonists in children with seasonal allergic rhinitis and in preschool children with perennial allergic rhinitis places a relatively high value on their safety and tolerability, and relatively low value on their limited efficacy and high cost.
e. Thus, leukotriene receptor antagonists are effective. Should they be used instead of antihistamines? Recommendation 17: ‘We suggest oral H1-antihistamines over oral leukotriene receptor antagonists in patients with seasonal allergic rhinitis (conditional recommendation | moderate quality evidence) and in preschool children with perennial allergic rhinitis (conditional recommendation | low-quality evidence)’. This recommendation places a relatively high value on avoiding resource expenditure. In other words, antihistamines are more convenient.
f. And what about INCS in children? Recommendation 18: ‘We suggest intranasal glucocorticosteroids in children with allergic rhinitis (conditional recommendation | moderate-quality evidence)’. This recommendation places a relatively high value on the efficacy of intranasal glucocorticosteroids, and a relatively low value on avoiding their possible adverse effects.
g. Should we use intranasal decongestants? According to recommendation 27: ‘We suggest that clinicians do not administer and parents do not use intranasal decongestants in preschool children (conditional recommendation | very low-quality evidence)’. The recommendation against the use of an intranasal decongestant in children places a relatively high value on avoiding the risk of serious adverse effects, and relatively low value on a possible benefit from a reduced nasal blockage.
h. Of course, ARIA provides suggestions for Specific Immunotherapy (SIT). According to recommendation 33, ‘In children with allergic rhinitis we suggest subcutaneous specific immunotherapy (conditional recommendation | low-quality evidence)’. This recommendation places a relatively high value on probable reduction in symptoms of allergic rhinitis and the potential prevention of the development of asthma, and relatively low value on avoiding adverse effects in children and resource expenditure. Regarding Sublingual Immunotherapy (SLIT), recommendation 35 suggests: ‘In children with allergic rhinitis due to pollens, we suggest sublingual allergen-specific immunotherapy (conditional recommendation | moderate-quality evidence). In children with allergic rhinitis due to house dust mites, we suggest that clinicians do not administer sublingual immunotherapy outside rigorously designed clinical trials (conditional recommendation | very low-quality evidence)’. The recommendation to use sublingual immunotherapy in children with seasonal allergic rhinitis places a relatively high value on small reduction in nasal symptoms, and relatively low value on avoiding adverse effects in children and resource expenditure. The recommendation to use sublingual immunotherapy in children with perennial allergic rhinitis only in the context of clinical research places a relatively high value on avoiding adverse effects and resource expenditure, and relatively low value on a possible small reduction in nasal symptoms.
Thus, ARIA guidelines (now disseminated and implemented in over 50 countries of the world) have also focused on paediatric aspects. As paediatricians probably see children with both rhinitis and asthma, they understand that allergic rhinitis and asthma are interlinked; all patients with asthma should be assessed for upper airways disease and proper treatment of allergic rhinitis in patients with comorbid asthma leads to improvement of asthma outcomes and better control.
The World Allergy Organization policy on guidelines
The mission of the WAO is to be a global resource and advocate in the field of allergy, asthma and clinical immunology, advancing excellence in clinical care through education, research and training as a world-wide alliance of allergy and clinical immunology societies. As part of these tasks, WAO is publishing informative reviews about topics of greater relevance in the field.
When a WAO article has to be developed, it starts with a proposal approved by the Executive Committee. The reasons for publishing an article are various, among them the need to disseminate new information, professional advancements and the need to inform practice decisions in allergy medicine. When doing a proposal, keep in mind the FINER rule: the article must be
F – Feasible
I – Interesting and important
N – Novel
E – Ethical, and
R – Relevant.
The Executive Committee nominates an Editorial Committee, often composed by members of the appropriate WAO Committee. There are basically five types of articles:
1. WAO guidelines [114,130–132];
2. WAO position articles [133–135];
3. WAO statements ;
4. member society statements;
5. journal article submissions (to the WAO Journal or to other journals) as WAO reviews [137,138] or reports of WAO surveys .
Position articles and WAO statements undergo a process of peer review and are approved by the WAO Executive Committee and the WAO Board. If the article is a position article, it is submitted for reconciliation to the member societies who are allowed a 60-day period for review, comment and expression of their views through an electronic vote.
A particularly important, comprehensive WAO article is the White Book of Allergy , with its Executive summary.
PART 3 – NOT EVERY FOOD ALLERGY IS SIMILARLY EXPRESSED
Is food allergy increasing? Evidence and reasons
The diagnosis of food allergy has been increasing in recent years. There is a debate as to whether such an increase reflects a true increase in prevalence or an increase in awareness among patients and/or better diagnosis by physicians.
Estimates of food allergy prevalence
Reported estimates of food allergy varied widely, being influenced by several factors such as study design, population demographics and diagnostic criteria. Variables that can lead to discrepancies among reported prevalence rates of food allergy are as follows:
1. Study design
a. cohort vs. cross-sectional,
b. point vs. interval (duration),
c. population demographics,
f. dietary habits,
a. self-diagnosed: medical history or questionnaire (by mail vs. interviewer-administered by phone or in person),
b. sensitization: skin testing vs. serum-specific IgE; number of foods tested,
c. challenge test: open vs. single-blind vs. double-blind.
According to reports during the past two decades (see Table 2) [140–148], food allergy affects 3% of the general population, 5–7.6% of children, 12% of adolescents, and 2.4–19% in adults. These figures are generally higher than expected. An important observation is that proven food allergy is much less frequent than being claimed by the population, even when a standardized questionnaire is used. Another issue is that a positive SPT or specific IgE (sIgE) merely reflects sensitization that may or may not be clinically relevant. For example, in British children 3–4 years of age, 3.3% had peanut sensitization but only 1.4% had symptoms . Another study on teenagers in the United Kingdom  showed that 12% gave a history of reactions to foods, yet skin test was positive in 5%, and only 1% had a positive open oral challenge.
In the European Community Respiratory Health Survey (1991–1994), a standardized methodology used an interviewer-administered questionnaire to 17 280 adults 20–44 years from 15 countries . Food allergy was reported by an average of 12.2%, with a very wide range from 4.6% in Spain to 19.1% in Australia.
Food allergy in specific allergic diseases
In children with atopic eczema, at least one-third had positive food challenge or a strong history of food allergy [148,149]. In anaphylaxis, foods were responsible for 17% of cases presenting to emergency departments in Australia  and for 36% of cases in the United States – exceeding drugs and insect stings . In patients with various degrees of asthma, food caused wheezing in 2–15% [152–154].
Allergy to specific foods
CMA was reported in 2.2% of Danish children below 3 years , in 2.8% of Dutch infants below 1 year , and in 4.9% of Norwegian infants below 1 year . Sensitization to hen's egg was noted in 1.3% of German infants below 1 year .
In a random telephone interview of a sample of the general population in the United States, reactions to fish and/or shellfish were reported by 2.8% of adults and 0.6% of children .
Peanut allergy was reported in 1.4% of British children 3–4 years of age  and in 1.2% of the American general population .
Interval comparison studies
Allergists in general have been observing a trend of increase in evaluating patients for food allergy, with increasing rates of positive results . However, more objective scientific evidence would be interval comparison studies on the same population, using the same or similar methodology.
In Sweden, during a 5-year period (1994–1998), peanut sIgE testing increased by 2.6-fold and positivity increased by 32% . During that period, there was no apparent concomitant increase in peanut consumption. In the Isle of Wight, United Kingdom, children 3–4 years of age in 1996 showed peanut sensitization in 1.1% and peanut allergy in 0.5% . Six years later, the figures were tripled, 3.3 and 1.4%, respectively . In the same region, a birth cohort of 969 showed SPT positivity to food in 2.2% at 1 year, 3.8% at 2 years, and 4.5% at 3 years of age, with a 5% cumulative clinical food allergy by challenge or a strong history .
In the United States, a general population survey by a random telephone interview in 1997 showed a prevalence of 0.6% of allergy to peanut or tree nut . A similar survey in 2002 showed a figure of 1.2% .
In a survey administered electronically to a large random representative sample of US households with children, food allergy in children was reported as 8% with almost one-third have multiple food allergy . A report in 2008 from the US Center for Disease Control based on the National Center Health Statistics Data  revealed that reported food or digestive allergy in children increased by 18% during 1997–2007. It also revealed that the annual hospital discharges with any diagnoses related to food allergy in children below 18 years increased by 365% in 2004/2006 compared with 1998/2000.
Why food allergy is increasing?
The above data indicate a definite increase in food allergy prevalence worldwide that cannot be totally attributed to an increased awareness and diagnosis. The factors driving such an increase may be classified into three groups: allergies in general are increasing, the causes of food allergy are increasing, and the contributory factors are increasing.
First, allergies and asthma have been on the rise, which may be explained by the ‘hygiene hypothesis’ [166,167]. Another factor is the high survivorship of patients with atopy, resulting in an increased perpetuation of the genetic trait. Also, there are data indicating that food sensitization during early childhood increases the risk for later development of atopic dermatitis, bronchial asthma and allergic rhinitis . The presence of food allergy in children below 18 years was reported to be associated with much higher frequency of respiratory and skin allergies .
Second, there is a remarkable increase in the exposure to food allergens, both in quantity and variety. Increasing causes of food allergy are as follows:
1. food consumption (obesity ‘pandemic’) including the highly allergenic foods (fish, shellfish, peanut, tree nuts, milk, egg);
2. fating out; ‘Buffet restaurants’; ‘All you can eat’.
3. food varieties all year-round;
4. cross-reactivities among foods and between foods and noningestant allergens (e.g. pollens, mite, cockroach, natural rubber latex);
5. commercial foods incorporate multiple nutrient and nonnutrient allergenic ingredients;
6. incorporation of food proteins in medical diagnostic and therapeutic agents, particularly dermatologic.
Also, exposure to food can be through noningestant routes, such as skin contact  or inhalation . Numerous medications, topical or systemic, incorporate food protein such as casein, whey, peanut oil, ovalbumin, ovomucoid, lecithein, wheat, and others.
Third, possible contributory factors that facilitate the development of food allergy are increasing [170,171]. One such factor is suppressing gastric acidity by the intake of antacid medications, which are commonly used, often without appropriate indication. Acidity is required for appropriate enzymatic digestion of food protein and reducing its allergenicity [172,173]. Experimental studies have shown that the administration of antacids to mice enhanced their developing allergy to fish protein . There were also increases in gastric mucosal mast cells and eosinophils, serum sIgE, and skin test reactivity. Similar findings were noted with hazelnuts . Human studies [175,176] confirmed the antacids’ promoting effect on IgE-mediated food sensitization.
Another suspected contributory factor is the increasing use of infant multivitamin supplementation. In-vitro studies [177,178] suggested that certain vitamins influence the development of T-helper cells into Th1 or into Th2. According to cohort data from the National Center for Health Statistics 1988 National Maternal–Infant Health Survey compared with the 1991 longitudinal follow-up of the same cohort, vitamin supplementation was associated with an increased risk for food allergy [179,180]. The indoor living and reduced exposure of skin to sunlight with consequent reduced vitamin D seems to be another factor [181▪]. More studies are very likely to reveal additional contributory factors behind the increasing prevalence of food allergy.
Many studies provided evidence indicating a true worldwide rise in food allergy prevalence. Some of the responsible factors are relevant to allergic diseases in general and some are specific for food allergy. More studies are needed to confirm such factors and might reveal additional ones. Physicians need to be cognizant of this trend, particularly with the wide variety of food allergy manifestations, some of which are associated with a significant impact on quality of life and even fatalities.
EuroPrevall: the epidemiology of food allergy in Europe
Self-reported intolerance to foods, including adverse reactions such as urticaria or other allergic symptoms after eating a particular food, has been described in up to 35% of children . The true frequency of food allergy is presumably much lower. Various diagnostic methods are used in epidemiologic studies:
2. symptom history, elimination diet;
3. sensitization (IgE, SPT);
4. food challenges (FC, open/nonstandardized);
5. double-blind placebo-controlled FCs (DBPCFC).
This variability is reflected by different frequencies of food allergy using different methods .
The EuroPrevall birth cohort was established to examine the incidence of food allergy in European infants and young children, the regional patterns of allergy, the role of parental, prenatal and early risk and protective factors using standardized questionnaires and clinical assessments including DBPCFC . The birth cohort is part of a European Union funded collaborative project (www.europrevall.org) which examined frequency, costs and basis of food allergy in children and adults across Europe, complemented by studies [184–186] in China, India and Russia using the same standardized protocols.
In the birth cohort, 12 049 children were recruited across nine European countries, 77% of whom were followed up for 30 months. The methodology was stringent, in order to arrive at a valid estimate of confirmed food allergy .
1. unselected consecutive newborns were recruited at centres;
2. questionnaires at baseline (birth) assessed pregnancy, family history of allergy, house environment and other details;
3. interviews (telephone or face-to-face) were administered at 1, 2, and 2.5 years of age;
4. if eligible, a DBPCFC was performed using a standardized methodology together with SPT and specific IgE determination.
Symptomatic children were also followed with repeated challenges to detect tolerance development.
The baseline results have been published . At the moment, statistical evaluation of food allergy frequency and the influence of allergies in family, sex, duration of breastfeeding, solid food introduction, food consumption and environmental factors is ongoing.
Despite the difficulties inherent in such ambitious, large-scale studies, EuroPrevall is the first large multicentre study on food allergy in the world based on the diagnostic gold standard (DBPCFC). It will be able to provide detailed data on early life predictors of food allergy, behavior and environmental exposures and nutritional influences.
Individualized management of children with allergic rhinitis: confidence, compliance and satisfaction
Are patients/parents confident in our treatment?
Let's first address the question, are patients/parents confident with our treatment for allergic rhinitis? One of the best reports that investigated this question was entitled Burden of allergic rhinitis; results from the Paediatric Allergies in America survey . In this survey, parents of 500 children with current allergies and 504 children without nasal allergies (aged 4–17 years) were interviewed by telephone about their children's condition and treatment between March and April 2007. The children with allergic rhinitis were symptomatic and/or had received treatment within the previous year. Interestingly, parents of children with allergies felt that their children's health was excellent and only 43% compared with the group without allergies in which over 59% answered that their children's general health was excellent. In looking at their emotional status, many of the children with nasal allergies complained of being tired and irritable and also miserable, either frequently or sometimes. This indicates that children's emotional status is affected by nasal allergy symptoms. Nasal allergies also had a detrimental effect in children's quality of life compared with those without allergies. This shows that children's general health is detrimentally affected when they have allergies.
Studies have shown that perennial allergic rhinitis increases frequency of sleep disorders in children . Other recent studies [190,191] have also confirmed that allergic rhinitis sufferers do tend to sleep poorly and that this poor sleep can lead to increased daytime somnolence, also, increased depression or anxiety, more difficulty with learning and reduced cognitive function and reduced quality of life. Nasal allergies have also been shown to detrimentally affect work and general activities in paediatric patients compared with those children without allergies. A case–control study  from the United Kingdom reported that a detrimental effect on examination performance in teenagers who suffered with seasonal allergic rhinitis.
Are patients/parents satisfied with allergy treatment options?
In 2006, an Allergies in America Survey reported that among 2500 allergic adults who completed telephone interviews only 56–58% were ‘very satisfied’ with management of their allergic disease . Interestingly, there was a disparity because when 400 healthcare providers were asked if the patients were satisfied with their management up to 81% of healthcare practitioners reported that all or most of their patients were ‘very satisfied’ with their management. This disparity suggests that the disease management could benefit from improved communication between patient and physician. One of the main reasons for patients’ dissatisfaction with their management was the effectiveness of their medication. The main complaints here were that the medication for treatment was not effective enough or that they needed a change in medication.
Why do patients stop their medications?
To understand why patients stop their medications one needs to understand the concept of compliance and adherence. Adherence is the preferred term for the description of patients’ behavior of correctly following medical advice, including taking medications correctly, which means the proper dose with the right frequency, and at the correct time. Nonadherence to prescription medications is considered one of biggest problems in healthcare here in the United States where it has been found that 15% of all new medications are never filled, and of those filled about half of patients stop their therapy in the first 6 months . Nonadherence is also a problem among children with median adherence rate of 58% being reported . In the Allergies in America Survey, the main reasons for medication nonadherence were loss of effectiveness over time, lack of symptom relief, and also concern about side effects.
What can physicians do to help improve adherence?
Various investigations have reported that physician communication when prescribing new medications is critical to help the patients’ understanding about why it is important to be compliant with their treatment recommendations. Physicians often fail to communicate critical elements of medication use, including name of drug, number of tablets to be used, frequency of dosing, and adverse effects; when these are not explained it contributes to increased noncompliance in patients . The measures that can be helpful to enhance medication adherence include simplifying the treatment regimen, using printed instruction materials, counseling the patient about the recommended treatment regimen, using reminders, and reinforcements, and also involving family members .
Individualized management strategies
In conclusion, it is important to individualize patients’ management recommendations for all chronic conditions, particularly allergic rhinitis. When there is poor confidence it is important to address the parents’/patients’ concerns and to encourage patients to become more self-reliant and more confident in the management of their illness. In terms of compliance and adherence, it is important for the patient to understand the treatment recommendations and to educate the parent/patient about the treatment rationale. When the parent/patient is not happy with their current management regimen this can result in poor satisfaction, and when this occurs it reduces confidence and also reduces compliance, and can result in a vicious cycle that can be detrimental to a patients care.
Allergic rhinitis is a chronic condition that detrimentally impacts children with this diagnosis. Using appropriate management strategies not only improves the patients’ quality of life, but it can also increase the families’ confidence about management, increase their compliance with the prescribed treatment, and ultimately result in satisfaction with our recommendations.
It is increasingly clear that asthma is a syndrome, not a single condition. Lumping all asthmatics together is neither useful in planning treatment nor helpful in determining mechanisms of disease and genetic associations. This review highlights recent advances both in methods and results of attempts to separate out homogeneous groups of asthmatics (phenotypes). This is still very much a work in progress, but essential if understanding of the different manifestations of asthma is to be achieved.
A phenotype is here defined as a feature or cluster of features, which allows the separation of a subgroup from the general population. It should be noted that these need not be stable over time (and indeed frequently are not, although there must be sufficient stability for clinical utility). Importantly, some useful action must result from phenotyping, such as a change in treatment, a new understanding of pathophysiology or insights into prognosis. Phenotyping for its own sake cannot be justified.
In theory, phenotyping can be investigator driven or objective. Investigator driven can be criticized on the basis that the prejudices of the investigator are imposed on the data. An example is sputum cytology, which may be eosinophilic, neutrophilic, mixed type or pauci-inflammatory. So-called objective techniques rely on mathematical interrogation of the data, using techniques such as principal component analysis, cluster analysis or the sophistication of systems biology . However, even these are vulnerable to investigator prejudice; only data that are entered can be analyzed. So for example only recently has the importance of bacterial infection and the airway microbiome been appreciated [199,200], and until this happened, these data could not be included in analyses.
Before going on to sophisticated analyses, it is essential to get the basics right; allocating phenotypes to a child having asthma who is not taking basic medications is a waste of time. We first ensure the diagnosis is correct, and address comorbidities, and then use a detailed, nurse led protocol to address the basic management, specifically adherence, environmental allergen and tobacco smoke exposure, and psychosocial issues [201,202]. This process divides the children into difficult asthmatics (>50% of those studied, just need to get the basics right) and true, therapy-resistant asthma patients (STRA). Although STRA had lower spirometry, more bronchodilator reversibility and a higher exhaled nitric oxide (FeNO) at baseline, the overlap between the groups was too great for these differences to be clinically useful. A recent follow-up study [203▪] shows that the difficult asthma patients are able to reduce their level of prescribed treatment, while having improved spirometry and fewer asthma attacks.
Preschool wheeze phenotypes
Among the earliest phenotypes was the epidemiological classification proposed by the Tucson group, namely ‘transient’, ‘persistent’ and ‘late-onset’ wheeze . However these can only be defined retrospectively, and do not guide treatment, so their clinical utility is limited. Likewise, although atopy is important, the presence of atopy does not correlate with treatment with inhaled corticosteroids (ICS) . In any case, it is becoming increasingly clear that atopy is not ‘all-or-none’ but can be graded in severity by the size of SPTs or the titres of specific IgE . Thus, the European Respiratory Society Task Force proposed that preschool wheeze phenotypes should be divided into ‘episodic viral wheeze’ (EVW, wheezing just at the time of a usually clinically diagnosed viral infection) and ‘multiple trigger wheeze’ (MTW, wheezing with viral colds and also between colds with typical asthmatic stimuli such as exercise and allergen exposure) . The justifications for these phenotypes are that (a) MTW, but not EVW have eosinophilic airway inflammation and thickening of the reticular basement membrane [208▪▪]; (b) MTW have worse airflow obstruction, worse gas mixing and elevation in FeNO than EVW ; and in the teenage years, those who had pure preschool EVW were no more likely to be wheezing or atopic than never-wheezers in the preschool years . These phenotypes have implications for treatment; EVW can be treated with intermittent therapy, either inhaled corticosteroids or leukotriene receptor antagonists [211▪,212], whereas MTW should be at least considered for a prophylactic approach with low-dose ICS.
Determining phenotypes in really severe asthma
Elsewhere we have described in detail our protocol to phenotype STRA , with noninvasive measurements of lung function and inflammation; bronchoscopy, bronchoalveolar lavage and endobronchial biopsy; and a single triamcinolone injection. Four weeks later the noninvasive measurements are repeated. We aim to answer four questions:
1. What is the pattern of any airway inflammation?
2. Is there phenotypic discordance (defined as lack of concordance between symptoms and inflammation )?
3. Is the child steroid responsive?;
4. Does the child have persistent airflow limitation (PAL)?
Unfortunately, paediatric definitions of PAL and corticosteroid responsiveness are lacking. Our preliminary data suggest that more than one dose of triamcinolone may be needed to define target lung function, and reliance on a single dose of triamcinolone will lead to over-diagnosis of PAL .
Severe asthma phenotypes in children
It is very clear that there are marked differences between adults and children with STRA [215,216▪▪,217▪]. These include:
1. no sex differences in children;
2. STRA children are markedly atopic, and nonobese;
3. spirometry may be normal;
4. airway eosinophilia to a hugely variable degree, but not neutrophilia as in adults;
5. no evidence of signature Th2 cytokines despite airway eosinophilia in the vast majority.
However both children and adults have marked morbidity, including steroid bursts, and admissions to hospital and intensive care.
The Severe Asthma Research Program (SARP) study  has identified four clusters of mild/moderate and severe paediatric asthma: cluster 1, late-onset symptomatic with normal lung function; cluster 2, early-onset atopic asthma with normal lung function; cluster 3, early-onset atopic asthma with mild airflow limitation; and cluster 4, early-onset atopic asthma with advanced airflow limitation. Cluster 1 was more common in mild/moderate asthma, and cluster 4 in severe, although no cluster was specific to a particular severity. Unfortunately, as with all such studies, there was no second validation cohort. The SARP group also failed to find evidence of Th2 cytokines in paediatric severe asthma .
The absence of signature Th2 cytokines may have therapeutic implications; whereas adults with eosinophilic, exacerbating asthma do well if treated with the anti-IL-5 monoclonal antibody mepolizumab [220,221▪], this may not be appropriate for children. Further work is needed to clarify this. Finally, a recent study  showed that paediatric phenotypes are much less stable than adults’; 42% underwent at least one sputum phenotype change over a 12-month period.
Lessons from childhood: does late-onset adult asthma exist?
Adult asthma specialists have described a group of late-onset asthma patients, who are female predominant, less atopic, and have relatively severe impairment of lung function for a short period of disease . However, the Tucson prospective study  has shown that these patients in fact at age 6 had wheeze, low lung function and bad airway hyper-reactivity. This underscores that retrospective recall in adult life is notoriously unreliable. In one prosepctive study , retrospective re-call in adult life of even major respiratory illnesses like pneumonia and pertussis was litttle better than flipping a coin, with false positives and false negatives common.
The exacerbating phenotype
Asthma exacerbations can be reduced by appropriate management, but not abolished. Typically, they are triggered by viral infection, although massive allergen exposure, for instance in the thunderstorm asthma epidemics , may also cause acute asthma attacks which are clinically indistinguishable from viral exacerbations. There is no doubt that the combination of viral infection, allergen sensitization and exposure to that allergen leads to a high risk of asthma attacks , suggesting that strategies to reduce environmental allergen burden may be helpful. Exacerbations are by no means a universal feature of asthma [228▪▪], and there may be genetic factors which predispose to asthma attacks [229,230]. Measures that should be taken [231,232▪▪] include prescribing low dose ICS (but there is no evidence that escalating to higher doses is beneficial); ensuring that baseline control and baseline lung function have been optimized; and determining the allergic triggers and reducing environmental exposure to these allergens. Unfortunately, a study [233▪] which tried to use a sputum eosinophil strategy to control exacerbations did not replicate the success of an adult study.
Summary and conclusion
The science of phenotyping paediatric infancy is still at an early stage. The first need is obviously to get the basic management steps right. It is important that phenotypes are robust (validated in more than one cohort) and are helpful in clincial management or the understanding of mechanisms. Given the rarity of STRA, it is likely that international collaboration, with the adoption of common protocols [234,235▪,236▪], will be needed to achieve this.
PART 4 – INDIVIDUALIZED DIAGNOSIS-TREATMENT OF ALLERGIC DISEASE IN THE ERA OF MOLECULAR TOOLS
Allergen extracts: will we solve the standardization puzzle?
As discussed by Paolo Matricardi, a time has come when our research progress allows us to:
1. know the details of allergic sensitization to pollens protein by protein using the molecular approach in serological assays;
2. discuss even the finer details of the allergen sensitization;
3. conceive the hope to produce very selective, individualized products for therapy.
At this time, allergen extracts, still in use for in-vivo diagnostic and therapeutic use, remain the protagonists of allergy diagnosis and treatment. They should be standardized, of course, but still they are not . Thus, a comparison of extracts in different countries reveals huge discordance with potencies from various sources for Dermatophagoides pteronyssinus, cat dander and Bermuda grass [238–240]. The total protein content of extracts was found to be lower than the reference standard, as was relative potency measured by enzyme-linked immunosorbent assay inhibition for Bermuda and Fel d 1 content for the cat extracts. The exception was one European cat dander extract containing almost twice the Fel d 1 content compared with the US reference. Generally, the European extracts contained four-fold to six-fold less potency than the US standard, and the local Mexican extracts contained 10-fold less potency. In this situation how can we determine the ‘proper’ potency for testing? The answer depends on primary use of extract: diagnosis vs. therapy, epidemiologic studies vs. clinical management, standardization vs. potency.
If we use allergen extracts for intradermal diagnostic tests, we should expect a wide range of false positive tests. This was shown 70 years ago for ragweed, timothy, wheat, horse serum, cat hair, feathers, silk, orris powder , 50 years ago for dust and grass , and more recently for insect venoms . In SPTs, a standardized extract may prove to be able to give reproducible results , but again, which is the optimal level of standardization? In a preliminary attempt to establish it, the available information on the European allergen extract units and potency was reviewed . Here again huge differences in strength were found. They were dependent in part on the source material, the extraction process, and the decisions on reconstitution based on arbitrary in-house reference standards. All of these vary from one manufacturer to another. International standardization guidelines would answer some of these issues.
In the past, some studies reported optimal cut-off levels for SPT. As an example, SPT cut-off wheal was established at 5.7 mm mean diameter in adult patients with seasonal allergies and at 5.6 mm mean diameter in patients with HDM allergy (using D. pteronyssinus extract) . In a nasal challenge-based study  on cat dander rhinoconjunctivitis, a 6 mm diameter distinguishes cat allergic from nonallergic patients.
These excellent studies suffer from inherent methodological bias, not only due to the known lack of generalizability of data from specific caseloads, but also due to the reference standard itself: defining ‘true positives’ and ‘true negatives’ implies that it is possible to determine which is which, and implies that there is a ‘gold standard’ test or assessment which establishes the truth. This is not the case with respiratory allergy, for which clinicians’ judgment – based on patients’ reports – is necessarily taken as standard. The case of food allergy is different. In food allergy, we have a gold standard, the oral provocation challenge. Against it, a revision of existing data was able to pose the following statements about the SPT wheals:
1. egg allergy less than 2 years old, 3–5 mm wheal translates into a 93–94% positive predictive value (PPV) or 100% specificity;
2. egg allergy more than 2 years old, 6–14 mm determines a 95–100% PPV or 100% specificity;
3. peanut allergy less than 2 years old, 4 mm is associated with 100% specificity;
4. peanut allergy more than 2 years old, 7–16 mm return a 93–100% PPV or 100% specificity .
In conclusion, not only every patient is a single case in allergy, but also extracts are not the same. Standardization is a difficult task to achieve for many reasons:
1. using extractive products, differences will be unavoidable in obtaining source material;
2. differences in extraction procedures are difficult to standardize;
3. this will result in differences in declaring a standard measure;
4. and even when it would be declared, it will be difficult to maintain the ‘reference’.
At the moment, the allergist should clearly know the characteristics of the diagnostic products she/he has in her/his tray, and bear in mind that her/his allergens should be used differently for different purposes (establishing the prevalence of a disease, clinical management or risk assessment). In other words, this tray is similar to a painter's color palette: the allergist's art is to use, patient-by-patient, situation-by-situation, the appropriate test. It's not everybody's cup of allergens.
The advent of molecular diagnosis in food allergy
Diagnosis of food allergies in general starts with the anamnesis followed by skin tests using native food or commercial extracts. Food challenge tests and elimination diets are used to confirm a suspected food allergy. Skin test and determination of specific IgE demonstrate sensitization to food. Sensitization is an important condition, but no reliable proof of a clinically relevant food allergy. According the German guideline on ‘In-vitro diagnosis and molecular basic of IgE-mediated food allergies’  indications for in-vitro IgE-diagnosis in paediatric patients are the risk of severe reactions to foods (e.g. peanut, soy, milk, fish and crustaceae), unclear history and positive skin test obtained with food extracts not suitable for skin testing (e.g. tomato, spices), certain skin diseases making skin testing impossible and medication hampering skin testing or it interpretation. Moreover, in-vitro IgE testing is recommended if a food allergy is suspected despite an uncertain history and unclear skin tests results.
In-vitro IgE-diagnosis can be performed by using either crude allergen extracts or purified allergen molecules. The basic idea of component resolved diagnosis (CRD) is the molecular analysis of allergen sensitization pattern, to replace extracts with panels of purified recombinant or natural allergens, and to use assay systems allowing quantification of allergen-specific IgE. For targeted IgE testing by CRD allergens from one or few sources should be selected according to the case history of the respective patients. Furthermore, allergens applied for CRD need to be well characterized in terms of the purity, physicochemical properties, and IgE-reactivity . In line with this, performance of allergen-specific IgE tests should be validated using sera from well characterized patients [preferable with positive double-blind placebo-controlled food challenge (DBPCFC) to the respective food] and from carefully selected control groups (e.g. sensitized but asymptomatic patients). Moreover, it is recommended to confirm serological data by controlled oral provocation. In case recombinant allergens are used for CRD, is should be considered that recombinant allergens represent a selected isoform or variant of the natural counterpart. Moreover, potential artifacts should be assessed or misfolding of proteins, which might result from heterologous expression systems need to be excluded. In contrast, purified natural allergens potentially comprise impurities including other allergens from the same source. Reported benefits of CRD are:
1. increased sensitivity compared with extracts;
2. the correlation of certain allergens with clinically relevant food allergy and severe reactions;
3. the possibility to predict persistent vs. outgrowing food allergy if suitable markers (potentially sequential IgE-epitopes) are available;
4. to define cross-reactivity patterns;
5. to detect geographic differences in sensitization patterns.
For example, detection of IgE to soybean can be enhanced by using the Bet v 1-homologous soy allergen Gly m 4 instead of soy extract, but Gly m 4 alone still is a poor prognostic marker for soybean allergy . Moreover, sensitization to soy allergens Gly m 5 and Gly m 6 has been shown to correlate with severe allergy to soybean . The data are in line with recent reports from Ito et al. who showed even clinical relevance in children: the majority of soybean allergic children were sensitized to Gly m 5 (67%) and Gly m 6 (58%), and IgE levels to soybean and Gly m 5 were significantly higher in patients with severe reactions in comparison to asymptomatic patients. Diagnosis of hazelnut allergy is likewise complex. Using six hazelnut allergens subjected to CRD across Europe, due to low specificity of the serological assay it was not possible to conclude any clinical relevance from positive IgE-results to Cor a 1 and Cor a 8 in patients with unclear history. But, CRD provided some evidence for the lipid transfer protein (LTP) Cor a 8 as a biomarker for potentially severe reactions in adults . Likewise, sensitization the storage protein Cor a 9 correlated with severe reaction in paediatric patients .
In summary, CRD has a high potential to increase diagnostic sensitivity and to improve the clinical interpretation of in-vitro test results. There is a relevant evidence for certain allergens (e.g. LTPs, storage protein from soybean and peanut) to serve as potential biomarkers for severe reactions. In general, the interpretation of serological data should only be performed in combination with carefully recorded case history and clinical observations.
Immunotherapy and respiratory allergy in children: the American experience
Noon's original article on grass immunotherapy was published over 100 years ago ; the patients were adults. It became clear very early that children should be treated with this new therapy. This was based on the prevailing concept of preventing bacterial diseases through vaccination. It became important to define populations at risk for allergies. In Cook and Vander Veer's seminal article on Human sensitization  he described the inheritance of allergic illness. He further defined it in another series of patients in Spain . They reported that 75% of all children with bilateral antecedent allergy would develop allergy, about 50% of all children with unilateral antecedent allergy would develop allergy and about 40% with negative antecedent allergy would develop allergy.
Tuft  in his textbook of Clinical allergy (1937) was recommending early treatment for children with allergies. In Cook's Textbook of Allergy, 1947 , Albert Vander Veer answered the question of when to begin treatment. His answer: ‘In general as soon as definite clinical hay fever is diagnosed treatment should be instituted.’ He also added that for children under 6 years of age, one could temporize by sending the child to a pollen free area. A more recent review summarizes the issues of beginning immunotherapy in children .
Another important group of allergists developed in Rochester, New York where the program was directed by Jerome Glaser. In 1968 Douglas Johnstone and others published some very significant studies. They were able to show that children undergoing high-dose immunotherapy had better results and also developed fewer allergies than those in the placebo or low-dose groups. They also showed that children treated with immunotherapy had decreased asthma . They studied 210 children with perennial bronchial asthma in a prospective study in which one-half of the children received placebo injections whereas the other half received conventional hyposensitization therapy, beginning the study in 1953. Of the 130 children still under observation at the time of their 16th birthday, 22% of the placebo-treated children were free of asthma compared with 72% of the treated children. In the treated group, the rate of loss of asthma may be related to the dose of antigen received in hyposensitization therapy. Whereas 66% of the ‘1/5000’ group were free of asthma at the end of the study, 78% of the ‘highest-tolerated dose’ group were symptom free in their 16th year. The findings of this study suggest although do not prove that even young children with mild asthma, especially if they also had hay fever should not be denied hyposensitization therapy in the hope that the child will outgrow his illness. The likelihood of a child outgrowing asthma was not significantly influenced by his sex, age of onset, or severity. These studies were subsequently validated approximately 30 years later by the PAT study and the work of Möller et al..
Although almost all the US studies have shown efficacy of immunotherapy in children there was one study that seemingly did not show efficacy with a multiple allergen study in children with asthma. This was the Adkinson study . This study was a double-blind, placebo-controlled trial of multiple-allergen immunotherapy. The patients were 121 allergic children, equally divided between inner and outer city populations with moderate-to-severe, perennial asthma. Injections of up to seven allergens were increased weekly to reach a target maintenance dose of 0.7 ml of concentrate; if a dose produced systemic reactions on two attempts, the next lower dose was used for maintenance therapy. Maintenance therapy was given every 2 weeks for 24 months and then every 3 weeks until the completion of the study. The results were that the median medication score declined from 5.4 to 4.9 in the immunotherapy group (P < 0.001) and from 5.2 to 5.0 in the placebo group (P < 0.001), but there was no significant difference between the groups (P > 0.6).
The number of days on which oral corticosteroids were used was similar in the two groups.
Partial or complete remission of asthma occurred in 31% of the immunotherapy group and in 28% of the placebo group (P > 0.5).
There was no difference between the groups in the use of medical care, symptoms, peak flow rates, methacholine PC20. The authors concluded that immunotherapy with injections of allergens for over 2 years was of no discernible benefit in allergic children with perennial asthma who were receiving appropriate medical treatment. A major flaw was lack of cockroach immunization in treating an inner city population of asthma patients, especially, as there is a significant body of information indicating the importance of this allergen in that population .
Why the difference in results between Johnstone and Adkinson? Methods were different in that Adkinson excluded animal dander patients and did not test or treat with cockroach in a group that would have had contact with both directly or indirectly. Also ‘usual’ medications had significantly changed with the advent of inhaled corticosteroids. Length of treatment and follow-up was also different. Also it seems that Adkinson et al. used a single extract mixture and it is conceivable that the molds with enzymatic activity inactivated wholly or partially the mixture they were using.
Many allergen extracts contain mixtures of proteins and glycoproteins. Proteolytic enzymes can degrade other allergenic proteins. There have been reports of interactions between extracts when mixed together. Extracts such as Alternaria species have been shown to reduce the IgE-binding activity of timothy grass extract when mixed together. Studies designed to investigate the effect of combining mold/fungi extracts with pollen extracts have demonstrated a significant loss of potency of grass pollen, cat, birch, white oak, box elder, dog, and some weeds .
Thus, the 20th Century American Allergists/Immunologists established allergen immunotherapy was effective in children and adults. Further, therapeutic efficacy was demonstrated as well as preventive aspects. Also during this period mechanisms were elucidated.
So far the 21st century US achievements include the third revision of the immunotherapy practice parameters [266,267], which standardizes much of the practice of immunotherapy, improved safety data and studies proving the cost efficacy of immunotherapy. In the practice parameters for allergen immunotherapy , immunotherapy for children is addressed in Summary Statement 17: ‘Immunotherapy for children is effective and well tolerated. It has been shown to prevent the new onset of allergen sensitivities in monosensitized patients, as well as progression from allergic rhinitis to asthma. Therefore immunotherapy should be considered along with pharmacotherapy and allergen avoidance in the management of children with allergic rhinitis/rhinoconjunctivitis, allergic asthma, and stinging insect hypersensitivity.’
Another 21st century US achievement deals with establishing the real value of specific allergen immunotherapy in a new medical economic era. Excellent studies have been produced by Hankin et al.. Their objective: to examine characteristics associated with receiving immunotherapy, patterns of immunotherapy care, and healthcare use and costs incurred in the 6 months before vs. after immunotherapy. This was a retrospective analysis of Florida Medicaid claims data (1997–2004) of children (<18 years of age) with new diagnoses of allergic rhinitis. Results: of 102 390 patients with new diagnoses of allergic rhinitis, 3048 (3.0%) received immunotherapy. Male patients, Hispanic patients, and those with concomitant asthma were significantly more likely to receive immunotherapy. Approximately 53% completed less than 1 year and 84% completed less than 3 years of immunotherapy. When they compared the costs before immunotherapy to the 6 months after immunotherapy they found that the patients who received immunotherapy used significantly less pharmacy (12.1 vs. 8.9 claims, P < 0.0001) resources ($330 vs. $60, P < 0.0001),
There were fewer outpatient visits (30.7 vs. 22.9 visits, P < 0.0001), ($735 vs. $270, P < 0.0001) and less inpatient (1.2 vs. 0.4) admissions, P < 0.02) ($2441 vs. $1, P < 0.0001).
Hankins conclusions: ‘There is substantial, compelling information to support the cost effectiveness of subcutaneous immunotherapy. However, third party payers remain unaware of this. This is a public health policy issue and has preventive care implications. Poor subcutaneous immunotherapy adherence leads to treatment failure.’ They pointed out that until subcutaneous immunotherapy adherence is improved, sublingual immunotherapy is likely to face similar treatment failure. A more recent article further supports the value of immunotherapy .
Another important achievement of the 21st century deals with improved safety of immunotherapy. Adverse events have been studied with regard to immunotherapy. A presentation by the Food and Drug Administration deals with allergy related adverse events . An adverse event is any undesirable experience associated with the use of a medical product; ‘associated with the use’ refers to a temporal relationship, which may or may not be causally related to the product. A serious adverse event is death, hospitalization, life-threatening, disability, congenital anomaly and/or other serious outcome. The adverse event reporting system (AERS) collects reports of adverse events from drugs, therapeutic biologics, allergenic extracts, blood and blood products, and does not include preventive vaccines. Data are collected through MedWatch (Form 3500 or 3500A). Since 1969, there have been more than four million reports in all these categories. Criticisms of this system are limitations of a passive surveillance system, failure to calculate incidence rates, under-reporting, absence of denominators, reporting bias and publicity or litigation may stimulate reporting. Newer products are more likely to be reported than older ones. There is no control group and there may be missing and inaccurate data. Also, reported diagnoses not verified and there is a lack of consistent diagnostic criteria and no reports of concomitant medications. There is a low likelihood of detection of long latency events; often the investigators are unable to assess causation. A summary of analysis from AERS database search shows that serious adverse events to allergen extracts are relatively rare, consistent with the medical literature. Reported adverse events are not associated with any one product or class of extracts (standardized or nonstandardized). Therefore, these adverse events are associated with the procedure of immunotherapy which, when performed in accordance with generally accepted principles, is well tolerated. Serious adverse events to allergen extracts are relatively rare, consistent with the medical literature. Reported adverse events are not associated with any one product or class of extracts (standardized or nonstandardized). Therefore, these adverse events are associated with the procedure of immunotherapy which, when performed in accordance with generally accepted principles, is well tolerated. There are, however, 16 products with potential safety issues that are not associated with the allergenic activity of those products.
Similar results have been obtained from AAAAI and ACAAI Committees; they found that 76 (∼1 per 2.5–3.0 × 106 injection visits) fatal reactions occurred between 1973 and 2001 and there were 273 near-fatal reactions (∼1 per 1.0 × 106 injection visits) , but from 2007 to 2011, there was none .
In summary, the American experience in the 20th century showed efficacy and mechanisms indicating efficacy of immunotherapy for respiratory illness. In the 21st century, its value for treatment has been established as well as improved safety.
Sublingual immunotherapy and respiratory allergy in children: a European perspective
Sublingual immunotherapy (SLIT) was first described in a DBPC trial in 1986 . Since then, more than 60 trials have been published, all reviewed in a number of meta-analysis, guidelines and Position articles. What's new from the last 2 years in SLIT from our European vision?
In 2010, a Cochrane Review pointed out the state of the art for injection immunotherapy for asthma . Injection SIT was confirmed effective for mites and pollens, but it was pointed out that it is difficult to compare its efficacy to that of drugs. One trial found that the size of the benefit is possibly comparable to inhaled steroids. Adverse effects exist and the authors stated that – given its greater safety – patients should be allowed an informed choice between injection SIT and SLIT. At that time, SLIT was known to be effective for allergic rhinitis  and asthma  in paediatric patients 3–18 years of age. Its efficacy has subsequently been supported by a further Cochrane Review on allergic rhinitis  and by the WAO Allergy White Book . In synthesis, what is known about SLIT is that the large majority of the studies (using mites, grass, ragweed, wall pellitory) reported a significant effect on symptoms. There were enough studies to draft specific meta-analyses for different aspects [133,279–284], and a series of so-called ‘big trials’ with grass pollen extracts confirmed an effect of SLIT vs. placebo ranging from 25% to more than 50% [285,286].
What's new in 2011–2012? The scientific production is increasing, and in 2011, more than 250 studies were published on SLIT. Among them, many deal with practical everyday questions.
A couple of trials and a QoL study addressed the issue of precoseasonal vs. continuous grass sublingual immunotherapy in children. From their results, we know that:
1. both protocols are effective, determining similar decreases for symptoms and medication score, with a reduction of nasal symptoms in the precoseasonal group ;
2. the continuous regimen performs better than the coseasonal in the first season, whereas in the subsequent years the two regimens are nearly equivalent ;
3. the health-related quality of life reaches scores already close to the normal in the first pollen season during treatment with SLIT .
It is known that SIT displays a long-lasting effect, but what if this effect vanishes? A trial published in 2011 affords this issue in patients allergic to HDM . If after 10–15 years the clinical benefit is attenuating, a new course of SLIT is able to reduce nasal eosinophilia, bronchial hyper-reactivity and the rate of new allergic sensitizations. Interestingly, the second course of vaccination induced benefits more rapidly than the first one. The behavior of bronchial hyper reactivity and nasal eosinophils paralleled the clinical score. Thus, it is possible that SLIT acts more or less as a regular immunization, which can require a booster after some time.
Another unresolved question is the effectiveness of SCIT vs. SLIT. A study in HDM-sensitized children with asthma/rhinitis treated with SCIT or SLIT and concomitant rescue medications demonstrated an important clinical improvement vs. untreated children, in both modalities. This happened for total rhinitis and asthma symptom score, total medication score, VAS and skin reactivity to HDM (P < 0.05) for both treatments when compared with pharmacotherapy. A significant reduction of serum-specific HDM-IgE in SCIT and SLIT was observed. Moreover, titrated nasal provocative dose significantly increased in both immunotherapy groups when compared with the pharmacotherapy group. No adverse effects were reported in SLIT, while two patients demonstrated serious adverse events in SCIT. After 1 year of treatment, Der p 1-driven IL-10 significantly increased in SLIT compared with pharmacotherapy, whereas Bet v 1-driven TGF-β (negative control) increased significantly in SLIT only. No changes were observed for Th1-Th2 cytokines . Given its safety profile, SLIT was considered by the authors more advisable in allergic children. On the contrary, a combination of sublingual and subcutaneous has been evaluated and appeared promising in that it successfully combined the advantages of the two alternatives: rapid onset and potency in SCIT together with safety and avoidance of injections in SLIT .
Of importance, the European regulatory authorities are more and more interested in the field, and according to the EU Directives 2001/83–2003/94 and the following D.L. 219, allergens are gaining the status of medicines . As a consequence, they should undergo registration as do all other drugs.
Thus, from a scientific point of view SIT is living a golden age. The same cannot be said from the practical point of view: despite the huge scientific acquisitions and despite big efforts for disseminating the information – including many scientific meetings in 2011 and 2012 – still a minority of allergic children profit from the benefits of this treatment. This is probably due to some barriers hampering an appropriate use of SLIT:
1. underappreciation of the science supporting SIT;
2. complexity and time consumption in performing appropriate SIT studies, as compared with clinical trials with drugs;
3. absence of reimbursement of SIT in most countries;
4. limitation of resources available for SIT promotion, due to the small size of the manufacturers’ companies.
These barriers mark the limit of the SLIT use in Europe. Strategies to break them down are urgently needed if we aim to put an important piece of new scientific information at the service of allergy sufferers.
IgE response to grass pollen allergens in children: a molecular approach and implications for immunotherapy (not everybody's cup of pollens)
Allergen-specific immunotherapy (SIT) and in particular subcutaneous immunotherapy (SCIT) of seasonal allergic rhinitis (SAR), is usually considered a ‘second-line’, slow-acting, disease-modifying treatment. According to the majority of guidelines, many doctors would agree with the statement that SCIT should be proposed only when antisymptomatic treatment with drugs is not effective, accepted, and/or tolerated. As an example, the American guidelines state ‘Candidates for immunotherapy are patients whose symptoms are not controlled adequately by medications and avoidance measures or those experiencing unacceptable adverse effects of medications or those who wish to reduce the long-term use of medications’ . This opinion reflects the fact that in RCTs, the effects of SCIT and sublingual immunotherapy (SLIT) measured in terms of total symptom score (TSS) and total nasal symptom score (TNSS) in SAR do not exceed a 30–35% reduction during the first season after treatment initiation . Thus, the question arises: can we obtain more from SIT in terms of prevention and cure?
Many ways have been explored to improve the efficiency of specific immunotherapy with grass pollens. Among them, the idea to use a molecular approach is at the moment on the floor. For some years, we have had a clearer idea of the allergenic molecules of grass and their role in grass sensitization. This has been made possible by the availability of specific IgE assays using smaller and smaller amounts of blood and giving more and more information. If in the 1970s, we were able to measure specific sensitization using 50–150 μl (RAST) per allergen; ImmunoCAP is using molecules produced from rRNA 40 μl per allergen, and at the moment we are able to test the IgE response to more than 100 molecules from a sample of only 20 μl using the component-resolved diagnosis in a microarrayed dispositive. Using such information, it has become clear that not all patients allergic to grass pollens are the same. We are able to measure at least eight different sensitizing molecules from grass: Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11, Phl p 12. A sample of 176 grass-allergic children living in Rome returned a highly heterogeneous situation, with 39 profiles (15 of which represented 80% of the population) and many profiles of cosensitization to other pollens . Thus, it seems that ‘grass allergy’ is not a unique, homogeneous entity, and that most pollen allergic patients display a ‘unique’ sensitization profile.
The current SIT preparations may be either underpowered (they may contain too few molecules for patient ‘A’; type I mismatch) or overpowered (they may contain too many molecules for patient ‘B’; type II mismatch). They may also be over-underpowered (type III mismatch), or even completely unrelated to the individual sensitization profile (type IV mismatch). Hence, a tailored component-resolved therapy (CRT), as proposed many years ago by Valenta et al., would be obtained by exactly matching the SIT preparation with the sensitization profile of the patient. This approach may improve the efficacy of SIT but, given the registration rules regulating SIT, is unfeasible. Indeed, too many different grass pollen SITs should be registered. According to the data obtained by Tripodi et al., 15 different preparations should be registered to immunize according to the CRT approach 80% of the population of allergic children living in Rome, and an additional 24 preparations would be necessary to immunize the remaining 20%.
So far, we have a single clinical experience of recombinant SIT for grass pollens. In 64 grass-pollen allergic adults with rhinitis, 29 of whom were actively treated and 28 placebo-treated, a mixture containing 10 μg of Phl p 1, 5 μg of Phl p 2, 10mcg of Phl p 5a, 10 μg of Phl p 5b, and 5 μg of Phl p 6 was administered. The reduction of the symptom score did not reach statistical significance, but the combined symptom and medication score showed a reduction of 39% (P = 0.44) in disease severity; moreover, the serum levels of specific IgE to Phleum pratense decreased . The authors registered an excellent improvement in quality of life of treated patients, but recorded systemic side effects in 24% of them. Should we apply that particular product to the population examined in Rome, we would treat, according to the CRT approach, 4% of the patients, namely those sensitized to Phl p 1, Phl p 2, Phl p 5a, and Phl p 6. In 28% of them we would have administered an underpowered product (type I mismatch), in 32% an overpowered product (type II mismatch), in 30% we would register a type III mismatch and in 5% a type IV mismatch. It is clear that the mismatch in the molecular profile can expose patients to the following theoretical situation :
1. Type I mismatch – lower efficacy or efficacy through the so-called ‘linked suppression’ phenomenon;
2. Type II mismatch – beneficial prevention of new sensitizations or risk of new sensitizations (both possibilities should be experimentally tested);
3. Type III mismatch – a combination of the consequences shown in type I and type II mismatch;
4. Type IV mismatch – lowest chance of efficacy.
Thus, grass-SIT should and could be individualized; grass SIT is not everybody's cup of pollens.
When a child comes to the clinical observation with a grass pollen-induced rhinitis, his/her molecular profile of IgE sensitization is relatively complex and highly heterogeneous. However, recent data from birth cohorts have shown that the sensitization process is a progressive one starting with sensitization to one molecule and growing towards complex sensitizations. In particular, it has been recently shown that a weak IgE response against grass pollen starts in most patients-years before the perception of the first symptoms, when only one or a few molecules are recognized .
During the child's growth, this weak IgE response becomes progressively stronger and molecularly more and more complex. Moreover, testing IgE sensitization at a preclinical stage allows predicting seasonal allergic rhinitis at its molecular monosensitization or oligosensitization stage. If so, we may intervene when the wrong immune response is still weak and therefore more susceptible to be corrected. This could be defined as specific immuno-prophylaxis (SIP) and adopted in the early stages of the disease. If realized with the sensitizing molecules, SIP could be also defined as ‘Component Resolved Prophylaxis’ (CRP) or, at disease onset, ‘Early Simplified Component-Resolved Therapy’. This interesting approach should be tested through properly designed trials.
Candidates for novel vaccine formulations in allergy
Meta-analysis on several double-blinded studies demonstrate that allergen immunotherapy is highly effective in carefully selected patients with IgE-mediated respiratory diseases and bee venom allergies. It represents the only routinely administered antigen-specific immunomodulatory treatment given for immunologic diseases of any kind . Regarding its mechanisms, the role of the T helper type 2 (Th2) cell-mediated immune response against allergens is well recognized. In allergic individuals priming of allergen-specific CD4+ Th2 cells by antigen-presenting cells (APC) results in the production of type 2 cytokines, such as interleukin (IL)-4, IL-5, IL-9 and IL-13, which are responsible for the initiation, maintenance and amplification of human allergic inflammation. IgE production, eosinophil and basophil activation, mast cell degranulation, airway hyper-reactivity and mucus hypersecretion are the ultimate consequences of this process.
In this chronic Th2-mediated systemic disorder, Th2 are not the only immune effector cells capable of inflammation maintenance, chronic evolution and tissue remodeling. Learning from our knowledge on the role not only of Th1, but also Th17 in chronic inflammatory or autoimmune disorders [300–302], we have a wider idea about the pathogenetic mechanisms of allergic disease. Several data provide evidence that the Th17 expressed IL-17 in the lung plays a pathogenetic role in promoting neutrophil influx, the production of pro-fibrotic cytokines by bronchial fibroblasts and the release of eosinophil chemoattractants by the airway smooth muscle cells. IL-17 mRNA has been found increased in the lung, sputum and bronchial alveolar lavage (BAL) fluids or sera of asthma patients, with levels correlated to the severity of airway hypersensitivity . IL-17 expression increases upon allergen stimulation in in-vivo and in-vitro systems and decreases after glucocorticoids treatment . Its levels in sputum of asthma patients are inversely related to PC20 of the methacholine challenge test, and an increased expression of IL-17-induced cytokines in airways correlates with the severity of asthma . With these characteristics, it is not surprising that Th2 and Th17 development and function have been found influenced by a common environmental milieu, sensitive to interactions among allergens, epithelial cells and dendritic cells . The epithelial and dendritic cells play a central role in conditioning flexibility of different T effector (Th2, Th17, Th9 etc) cells in inflamed tissues.
For a long time, it has been known that SCIT is able to skew allergen-specific responses from Th2 to a more protective Th1 phenotype – a mechanism known as immunodeviation [307,308]. In addition, both SCIT and SLIT produce alterations of T cell proliferation, cytokine production and serum IgG1/IgG4, resulting ultimately in increased specific IgG4 seen with high-dose protocols and prolonged treatment. These immunoregulating actions depend in part on Th17, are dose-dependent, and may correlate with the efficacy of SIT.
In the last decade, the mechanisms of immunodeviation and immunoregulation induced by SCIT or SLIT have usually been presented as alternative, mutually exclusive events. However, more recent data indicate that such immunological changes include the involvement of both allergen-specific Th1 and Tr1 cells and that, possibly, the supposed dualism may be considered as two sides of the same coin. Thus, immunodeviation and immunoregulation are complementary mechanisms, essential to induce a long-term tolerance . The ideal SIT should therefore be able to redirect the allergen-specific Th2/Th17 cell responses to a more protective and balanced Th1/Tr1 phenotype.
How it is possible to improve immunotherapy through these acquisitions? As a matter of fact, today different ways for effective forms of immunotherapy are being experimented with:
1. recombinant allergens;
2. hypo-allergenic allergens;
3. T-cell peptide vaccines;
4. antisense ODNs for cytokines or chemokines receptors;
5. Th1-immunostimulants (TLR agonists – TLRa);
6. allergen-immunostimulant complexes .
It is of interest to observe that among these possibilities researchers are focusing on the use of TLRa as Th1 immunostimulants. TLR are pattern recognition molecules expressed by many types of cells and represent crucial triggers for adaptive immune response . TLRa are the exogenous stimuli able to condition tissue environmental milieu due to their ability to stimulate PPR/TLR on epithelial cells or subepithelial mucosal dendritic cells. Due to their high degree of plasticity, innate (innate lymphoid cells, ILC) and adaptive (T-helper cells) effectors are highly sensitive to the environmental conditions. TLRa are able to trigger epithelial cells or dendritic cells selectively, modifying the environmental milieu and redirecting the allergen-specific Th responses to a more protective IFN-γ/IL-10 cytokine pattern .
The natural ligands of endosomal TLR are oligodeoxi/ribonucleoside analogues, mainly of bacterial origin. We described a synthetic purine derivative, chemically related to adenine (9-benzyl-2-butoxy-8-hydroxy adenine), referred to as SA-2, which triggers TLR7 in both human and murine cells [313–315]. Its action in mouse model includes:
1. redirection of allergen-specific Th2 responses through TLR7 interaction and IL-12/IFNα/γ release;
2. downregulation of IL-17-related response through IL-10 induction.
These findings suggest that such TLR7 agonist, downregulating Th17 (as well as Th2) response, can be considered a candidate for novel vaccine formulations in allergy. Conjugated with an allergen, it can:
1. promote antigen uptake and dendritic cells but not B-cell activation;
2. potentiate the differentiation and activation of IFN-γ-producing T cells;
3. enhance the immunogenicity and reduce the allergenicity of the preparation.
At the moment, the conjugation procedure of modified adenine to allergenic protein has been achieved and chemically tested, and studies on its effects are in progress. These studies aim to verify the effects on the expression of Th2-related molecules, IFN-γ and IL-10 production, eosinophils, and immune inflammation.
In synthesis, we know today that immunoregulation or immunodeviation of allergen-specific T-effector cells is a complementary mechanism operating in SIT, able to induce and maintain a protective Th1/Tr1 phenotype. They can become targets of immunostimulating adjuvants (TLRa) able to modify the environmental milieu and to condition the T-effector cell profiles. Some of these agonists, as TLR7 agonists, are the best candidate adjuvants since they induce a balanced proinflammatory (IL-12, IL-27) and regulatory (IL-10) cytokine pattern. Thus, these molecules seem the best candidates for novel vaccine formulations in allergy today.
PART 5 – NOT EVERYBODY'S CUP OF MILK
The management of gastrointestinal cow's milk allergy
CMA, similarly to all other food allergies, has multisystem manifestations that range from a skin rash, to gastrointestinal symptoms to anaphylaxis. IgE-mediated allergy occurs within minutes to an hour after allergen exposure and is often referred to as ‘immediate hypersensitivity’ . It can affect several target organs: the skin (urticaria, angioedema), respiratory tract (rhinitis, asthma), gastrointestinal tract (oral allergy syndrome, nausea, vomiting, pain, flatulence, emesis, and diarrhea), and/or the cardiovascular system (anaphylactic shock). A significant proportion of infants and the majority of adults with CMA do not have circulating milk protein-specific IgE and show negative results in SPTs and RAST. These non-IgE-mediated reactions tend to be delayed, with the onset of symptoms occurring from 1 h to several days after ingestion of milk. Again here, a wide range of symptoms can occur, but are most commonly gastrointestinal or dermatological. The management thus starts with the recognition of the cow's milk role in the symptoms or syndrome, strategies for avoidance of cow's milk and then timing the reintroduction of this important ingredient of diet for all ages.
IgE-mediated CMA generally starts in infancy. Gastrointestinal symptoms, vomiting and diarrhea are the most common symptoms leading to a diagnosis of CMA in infants fed cow's milk-based formula. There is a wide differential diagnosis for the infant who has persistent vomiting, including gastrointestinal reflux, pyloric stenosis, intestinal malrotation, increased intracranial pressure, inborn errors of metabolism and congenital immunodeficiencies. Unless other symptoms may point to one of the diagnoses listed, CMA is usually the initial diagnosis and management is started. Testing may or may not be indicated in different circumstances as presented in the Diagnosis and Rationale for Action against Cow's Milk Allergy (DRACMA) guidelines . Testing for CMA in infants is usually not resorted to due to misconceptions that young infants cannot be tested, and due to the vast array of formulae available to replace cow's milk in an infant's diet. However research data suggest that in various countries, for example, the United Kingdom and South Africa, there is an average of 2 months’ delay before an infant with gastrointestinal symptoms, ascribed to CMA, gets to be switched to an alternate formula. There is also a large cost to the management of CMA [318,319]. The formulae now available to replace cow's milk include soy-based, partially and extensively hydrolyzed cow's milk, amino acid formulae and more recently, rice-based formulae. The utilization of each depends on availability, cost and the recommendations of the guiding organizations. Soy-based formulae had been the most commonly used formula to replace cow's milk-based formulae, but are no longer first choice . The timing of re-introduction of cow's milk into the diet of a child who has been diagnosed with CMA is commonly thought to be at 1 year of age. However, clinical experience shows that CMA does not always resolve at 1 year of age and can extend into the adolescent and adult years . The consensus is that when cow's milk-specific serum IgE decreases, preferably below 5 KU/l is the time to perform an oral food challenge in the office to ascertain that introduction of cow's milk will be well tolerated . The reduction in IgE, if accompanied by an increase in milk-specific IgG4 may even be more indicative of the resolution of the allergy .
The less well known and diagnosed cow's milk reaction with severe gastrointestinal manifestations is the food protein-induced enterocolitis syndrome (FPIES) . Infants with this condition present with severe gastrointestinal symptoms of protracted vomiting to the point of dehydration and prostration. The syndrome occurs in infants who are breast-fed and are supplemented with cow's milk-based formula. Commonly, the scenario occurs a few times before the diagnosis is thought of. Tests for serum cow's milk-specific IgE are negative. There is no diagnostic test other than a food challenge, which needs to be done in an inpatient setting, with intravenous access placed and fluid available for fluid resuscitation. The food challenge is not performed in the graded fashion that is recommended for the diagnosis of IgE-mediated CMA, but rather the whole amount of cow's milk is given in one feeding. If symptoms occur then fluid resuscitation is administered. Because the procedure of diagnosis poses a risk, it is recommended to make the diagnosis on the basis of clinical suspicion alone . This creates a problem in identifying the time when the syndrome would have resolved in any particular patient. In general, it is recommended to maintain complete cow's milk avoidance until the age of 2 years, and that cow's milk would only be introduced in the diet after an inpatient food challenge results in no symptoms. Recent data suggest that patch test may be of value in the diagnosis of FPIES . However, there are no data for the value of patch test in determining the natural course of the syndrome. In other words, it is not clear if a previously positive patch test to cow's milk turning negative would herald the onset of resolution. In FPIES, similarly to the IgE-mediated CMA described in the first paragraph, the management is by replacement of cow's milk with another infant or toddler amino-acid-based formula. In this syndrome, soy may need to be avoided as the syndrome has been described after the ingestion of soy-based formulae and after other grains and cereals including rice. Therefore, rice-based formulae may not be an appropriate substitute [325,326].
Cow's milk has been associated with gastroesophageal reflux and Eosinophilic Esophagitis disease in the infant, and it was noted that the reflux is associated with accumulation of eosinophils in the esophagus. An early study reported that the substitution of cow's milk formula with an amino acid-based formula, neocate, not only relieved the reflux symptoms, but resolved the esophageal eosinophilic accumulation. The study  noted that the reintroduction of cow's milk brought back both the symptoms and the eosinophil pathology. As the diagnosis of eosinophilic esophagitis started to be coined, attention was directed to the cause of the disorder. In infants who present with fussiness and persistent vomiting, a correlation between the symptoms and cow's milk formula was made. Large case series were published in which the diagnosis of eosinophilic esophagitis was made based on endoscopic and pathology criteria, removal of cow's milk from the diet and feeding infants, toddlers and even adolescents with elemental formula resulted in more than 90% resolution of the disorder [328,329]. There is however difficulty in consistently finding evidence of cow's milk sensitization, either by skin tests, by serum cow's milk-specific IgE or by patch tests with cow's milk. Even in the absence of this evidence, cow's milk elimination and substitution with elemental formula for prolonged periods of time, months to years, remains one of the mainstays of management of eosinophilic esophagitis. The cow's milk avoidance is usually prescribed to be complete and strict. When patients with eosinophilic esophagitis are managed with multiple food eliminations, the course of action is to maintain the elimination until the symptoms completely resolve and the esophago-gastro-duodenoscopy shows complete resolution of the eosinophilic inflammation before foods are introduced back in the diet. During the food re-introductions, it has been advised that cow's milk be the last food to be re-introduced in the diet. This is a management strategy that highlights the extent to which cow's milk is thought to be associated with the disorder, which is a chronic disorder with remissions and recurrences. On the contrary, this disorder is the only cow's milk-precipitated syndrome that has therapies other than dietary modifications. These therapies are all still experimental, and include puffed and swallowed inhaled steroids, swallowed steroid slurry and the anti-IL5, mepolizumab and reslizumab [330–334].
In summary, gastrointestinal CMA and the syndromes associated with it are common, need to be recognized and mostly require cow's milk elimination for their management. The elimination in infants and toddlers requires replacement with alternate formulae that include soy-based, rice-based or hydrolyzed or elemental formulae for various periods of time.
Phenotypes in cow's milk allergy. Seeing or not seeing the allergens?
Not every CMA is the same: some phenotypes prove more persistent than others, and it is possible that such natural history is modified by the exposure to cow's milk proteins. The general rule of ‘avoidance’ applies to CMA , whose standard treatment is the dietary elimination of cow's milk proteins. During breastfeeding, and in children 2 years of age or older, a substitute formula may not be necessary. However, in nonbreastfed infants and in children less than 2 years, replacement with a substitute formula is mandatory as milk is a staple food and the treatment of CMA entails a nutritional risk in particular in this category of children . In the recent past, it was generally supposed that milk avoidance was useful to gain milk tolerance. This belief has been recently questioned, and a systematic review of the literature concluded that there is no evidence that strict food avoidance, compared with less strict avoidance, has any effect on the rate of natural remission to a specific food allergen . This begs the question of what to do if small doses are tolerated. We know from challenge-confirmed series that a substantial number of milk-allergic children tolerate little doses. For instance, in the Milan Cow's Milk Allergy Cohort (MiCMAC) 60.7% children tolerate at diagnostic challenge at least 4.4 ml of milk . Can we recommend to these children a nonabsolute avoidance? Can we avail the use of little doses? If so, the change from a milk-avoidance diet to a milk-limited diet could provide a significant improvement to the quality of life of milk-allergic individuals .
The first study addressing this issue was the ‘baked milk’ study . One hundred children with milk allergy were challenged with heated milk products; heated milk-tolerant patients were subsequently challenged with unheated milk. Sixty-eight patients tolerated extensively heated milk only (heated milk-tolerant), 23 reacted to heated milk (heated milk-reactive), and nine tolerated both heated and unheated milk (milk tolerant). Heated milk-tolerant children displayed smaller SPT and lower sIgE to milk: they were essentially ‘less allergic’ to cow's milk. As such, they were probably intrinsically prone to the development of tolerance. This observation can find its basis on many studies, which suggest such associations. In the MiCMAC cohort study for instance, a larger weal diameter at SPT with fresh milk was significantly correlated with the failure to achieve tolerance, although this has not been seen in all studies. All patients with CMA and a negative SPT at 1 year of life had developed tolerance by their third year of life. However, 25% of 1-year-old infants with a positive SPT were still allergic at the same time. High levels of cow's milk IgE antibodies identified at diagnosis and during the course of disease were also predictive of longer duration. It has been reported that a reduction in milk-specific IgE levels correlates with the development of tolerance  and that a 99% reduction in milk-specific IgE antibody concentrations of more than 12 months translates into a 94% likelihood of achieving tolerance to cow's milk protein within that time span . In the ‘baked milk’ study, heated milk-tolerant children ingested heated milk products for 3 months and were then re-evaluated at 3, 6, 12 months. During this period, IgG4 significantly increased, milk-specific IgE remained unchanged and milk SPT was recorded reduced. Children receiving extensively heated milk essentially reported no acute milk-induced allergic reactions as a result of this diet. In this study, it was suggested that there are at least two different phenotypes of IgE-mediated milk allergy in children:
1. Type I individuals, baked milk-tolerant, are eventually able to terminate Th2 responsiveness and clinical reactivity, resulting in ‘transient’ CMA;
2. Type II individuals are not able to downregulate Th2 responsiveness and may have persistent CMA.
Still, after this study, the same researchers were of the opinion that there is no consensus for permitting patients who tolerate some small amounts of allergen (e.g. small amounts of peanut) to ingest up to their threshold dose because doing so is risky and the immune consequences are unknown . Thus, the answer to our question (can we avail the use of little doses?) is ‘no’. However, this study (and the similar ‘baked egg’ study ) suggested that the exposure to little doses of modified food allergenic proteins is not prejudicial in terms of natural history. This paved the way to the hope that this type of diet can augment the development of tolerance, thus reducing the frequency of prolonged or permanent milk allergy. In other words, the exposure to allergen could be beneficial in terms of recovery. This idea found supporters in may research groups, especially those dealing with cow's milk oral tolerance induction. These groups argued, from anecdotal cases in which milk avoidance seemed to trigger increased allergic reactivity , that avoidance does not make for tolerance, but for a longer duration of CMA. One of these case series concluded: ‘there is a considerable chance of developing acute allergic reactions to cow's milk after elimination in children without previous problems after cow's milk intake’ . However, when observing the survival curves describing the tolerance acquisition pattern of children with CMA on milk elimination diet , it is clear that the opposite is true: ‘There is a considerable chance of developing tolerance to cow's milk after elimination in children with previous problems after cow's milk intake.’
Thus, in the absence of prospective studies, it seems inappropriate and potentially dangerous to advocate deliberate exposure to foods involved in serious reactions against current recommendations and particularly so among food allergic children, at least until more basic and clinical research become available .
At the moment, we have two prospective studies dealing with milk exposure among children with CMA. One of them concludes that exposure to cow's milk by dietary baked milk accelerates the resolution of CMA in children . Briefly, children tolerating baked milk (muffin) underwent sequential food challenges to baked cheese (pizza) followed by unheated milk. Immunologic parameters were measured at challenge visits. Although the original protocol was designed to have a prospective control group, through a random assignment of baked milk-tolerant patients to dietary baked milk or strict avoidance arms, the recruitment was reported unsuccessful. Thus, in this study the comparison was done using a retrospective group, matched to active patients for age, sex, and baseline milk-specific IgE levels. This clearly limits its conclusions on the natural history of development of tolerance.
The second study  uses the exposure to cow's milk hydrolysate formulae as measure of intervention. Children with IgE-mediated CMA were randomly switched to one of three treatment groups: rice hydrolysate formula, extensively hydrolyzed cow's milk formula and soy-based formula. Patients not exposed to cow's milk protein residue achieved cow's milk tolerance earlier than patients who followed an extensively hydrolyzed cow's milk diet. This may be due to residual antigenicity in hydrolyzed milks. Using soy cosensitization as a marker of polysensitization, this effect was not retrieved in the subgroup of polysensitized children, suggesting that the association of cosensitization with a longer duration of disease could not be modified by allergen exposure. As the effect of dietary intervention is stronger in patients not sensitized to soy, we inferred that when atopic disease has progressed to multiple sensitizations, the elimination of allergenic exposure may not be sufficient to reduce the duration of CMA.
Even though these studies demonstrate conflicting results, they indicate that the effect of allergen exposure on CMA duration is dependent on the clinical phenotype. This echoes the findings of two earlier cohort studies [349,350], in which a sub-population of patients was described expressing an early poly-sensitization pattern associated with earlier, severe and persistent symptoms. The same pattern appears to be mirrored in the MiCMAC cohort, in which longer duration of CMA is akin to the persistent wheezing found by these two studies. Looking at these data, it is possible to identify a specific CMA phenotype characterized by the involvement of two or more organ systems, intensity of atopic expression (wheal diameter at SPT with cow's milk, low-threshold dose response at DBPCFC with milk, high total IgE level, cosensitization to both inhalant and ingestant allergens) associated with longer duration of CMA.
These data buttress the emerging concept that severity and course of atopic disease may be phenotype-dependent, warranting epidemiological, clinical and gene expression profile studies to document whether a hypothetical tolerance-prone (or persistence-skewed) phenotypical patient profile is observable at cellular and molecular pathology levels. The effects of allergen exposure in these different populations can be different: hypothetically, milk exposure could decrease tolerance in more severe phenotypes, increase tolerance in less severe, and could be neutral in mixed caseloads. If so, it's not everybody's cup of milk.
Tolerance to milk and egg after technological treatments
There are two widely discussed points in the field of food allergy:
1. the threshold of clinical reactivity;
2. the tolerance of allergenic ingredients after their technological treatment by food industry.
Although apparently separate, these topics are strictly correlated and according to the most recent scientific data they should be considered together in any clinical approach directed to face the problem of tolerance.
The importance of technological modifications is illustrated by recent studies.
Nowak-Wegrzyn  performed oral challenges with muffin and waffle containing 1.3 mg milk protein; the first was baked at 350°F (177°C) for 30 min in an oven, and the second (<0.625 inches thick to ensure thorough heating) was cooked in a waffle maker at approximately 500°F (260°C) for 3 min. The conclusion of the study was that the majority (75%) of children with milk allergy tolerate heated milk.
Similarly, oral challenges with a muffin and a waffle containing one third of an egg (approximately 2.2 g of egg protein) each were performed . The muffin was baked at 350°F for 30 min in an oven, and the waffle (<0.625 inches thick to ensure thorough heating) was cooked in a waffle maker at approximately 500°F for 3 min. The conclusions were that the majority of patients with egg allergy were tolerant of heated egg.
These clinical studies are very well performed, but some clarification is necessary. It is important to underline that heating as such cannot be considered the main responsible for the observed tolerance. In fact, the specific technological treatments, which are capable of modifying chemically the binding capacity of allergen to antibodies, must be considered involved in that phenomenon. The same authors underlined this concept in a later article, as discussed below.
The heating process can produce important chemical modifications in food allergens, but high temperatures are not always sufficient to eliminate the binding properties of allergens with the specific antibodies [352,353]. On the contrary, it is rare that milk or egg allergy is due to raw milk and egg since they are normally consumed after industrial thermal processes (pasteurization and sterilization of milk) or after cooking (egg).
As an example, Fig. 2 shows the effects of some technological treatments on the binding capacity of human circulating IgE antibodies. As shown, the binding capacity by the specific IgE antibodies was totally conserved after the four treatments used by the food industry. The temperature close to 200°C (used to bake muffin) cannot be used for milk sterilization, as the nutritional value of milk would be reduced and undesired by-products formed.
Similar results were obtained by using egg after usual cooking procedures and sera from allergic children.
As cited above, an article in which the specific technological treatment (baking) is stressed in its role in improving tolerance appeared in 2011 .
The clinical data described in that article aroused new scientific interest. In previous studies performed to develop analytical methods sensitive enough to detect traces close to the clinical threshold, we obtained incomprehensible results. On the contrary, these results, taken in parallel to the quoted clinical studies, seem to justify, from the molecular point of view, the oral tolerance of baked products. We thought to evaluate the effects of milk and egg baking preparing experimental cakes with known amounts of proteins. A first cake was prepared adding skimmed milk to obtain final concentrations of 770 and 385 ppm of total milk proteins. Cakes were cooked at 220°C for 30 min. After quantification using an ELISA kit for the detection of total milk proteins, the results clearly demonstrated that the quantity of allergen (Table 3) measured in the uncooked dough was close to the actual content. The differences are probably due to the unsuitability of the used method, which was not developed to detect a very high quantity of allergen.
On the contrary, the measured concentration of milk proteins in the final cake was highly below the expected value (below 3%), and in the cooked sample added with the highest amount comparable to that of milk-free cake.
Similar results were obtained by analyzing the cake prepared with known amounts (707 and 354 ppm) of egg proteins, as shown in Table 4.
The results obtained by analyzing the experimental cakes allow some considerations:
1. allergens used to prepare experimental cakes lost their capability to bind antibodies after baking;
2. apart from the heating process, different chemical reactions could be responsible for allergen modification; it is well known that during the heating process of foods containing proteins and sugars, several derivatives develop: among others the Maillard compounds;
3. allergens during baking could be covalently bound to the matrix (cereal ingredients) and be unavailable for antibodies or alternatively be masked in the upper part of gastrointestinal tract allowing a more efficient activity of gastrointestinal proteases;
4. finally, coming back to the first lines of this section, the presence of traces of milk and egg in baked products could be free of risk for allergic patients or alternatively be useful to induce oral tolerance at least in most allergic children.
Milk-specific immunotherapy comes of age
Cow's milk is one of the most common food allergies among children . Patients usually outgrow CMA but spontaneous resolution of IgE-mediated CMA is less common than that of non-IgE-mediated [155,354,355]. The only efficacious prevention of symptoms is a strict elimination of cow's milk from a diet and accidental ingestions are frequent . Observational studies [357–360] suggested that oral immunotherapy (also known as specific oral tolerance induction – SOTI) may be a promising treatment of CMA. As we did not identify any systematic review of oral immunotherapy in the management of IgE-mediated CMA, we performed one for the development of the WAO guidelines .
We recently systematically reviewed studies assessing the benefits and downsides of oral immunotherapy in CMA . We found six RCTs [362–367] and three small observational studies [368–370] that addressed that question. We found that the probability of reaching full tolerance was higher in children receiving oral immunotherapy compared with elimination diet only (RR: 9.98; 95% CI: 4.11–24.24). The probability of being able to tolerate small amounts of cow's milk (5–150 ml), thus most likely being protected against accidental ingestion, was also higher in those receiving oral immunotherapy (RR: 5.31; 95% CI: 1.16–24.45). The effects were similar in the observational studies.
Adverse effects were more frequent in children receiving immunotherapy. The most frequent were local symptoms that occurred with 16% of immunotherapy doses with most events presenting during the initial phase of rapid increase of milk dosage: lip and mouth pruritus (RR: 34.4; 95% CI: 4.8–244.7) and perioral urticaria (RR: 8.16; 95% CI: 1.21–55.04). Generalized adverse reactions also occurred more often in children receiving immunotherapy: generalized erythema or urticaria (RR: 12.05; 95% CI: 2.96–49.07), gastrointestinal symptoms (RR: 16.60; 95% CI: 4.12–66.93), mild laryngospasm (RR: 12.88; 95% CI: 1.68–98.59), mild bronchospasm (RR: 10.0; 95% CI: 2.41–41.43), increased need for oral glucocorticosteroids (RR: 11.29; 95% CI: 2.74–46.47) and need for administration of intramuscular epinephrine (RR: 6.14; 95% CI: 1.12–33.54).
Unfortunately, most of the effects were estimated imprecisely, as illustrated by very wide confidence intervals. Publication bias is also very likely because there are only few small studies showing large benefits and inconsistently reporting adverse effects.
The reactions during the initial milk challenge tests as well as the history of prior reactions have not been sufficiently reported; thus, it is difficult to estimate the severity of symptoms and determine whether the children included in the studies were similar to those seen in daily clinical practice. As the estimated benefits of oral immunotherapy seem very large, it is possible that the included children were particularly likely to benefit from immunotherapy.
There are several issues that remain unanswered. The immunologic mechanism of immunotherapy for CMA is not known. It has not yet been established whether the disappearance of symptoms when ingesting cow's milk represents a true immunological tolerance or rather a temporary desensitization achieved with oral immunotherapy. Related and clinically most important would be to determine whether children gain long-term benefit or a temporary improvement. All studies included only children, hence it is even less clear what would be the balance of benefits and harms in adult patients with allergy to cow's milk. Answering those questions would require performing additional well designed studies among representative patients. Those studies would require a sufficiently long follow-up that would measure long-term effects of oral immunotherapy on outcomes that are important for patients and their families.
Given the overall low confidence in the estimated desirable and undesirable effects the true clinical value of oral immunotherapy in patients with CMA remains uncertain. Decision to use oral immunotherapy seems to depend on a value that patients, parents and clinicians place on achieving at least partial tolerance of cow's milk compared with avoiding adverse effects of oral immunotherapy itself. Although oral immunotherapy of allergy to cow's milk (as well as other foods) proves to be really beneficial, its successful implementation might require additional adjustments in the healthcare system to provide reliable continuous availability of medical emergency support and resources needed to immediately react to serious adverse effects when they develop. Also, as is the case with any immunotherapy, success will largely depend on a long-term compliance with treatment as well as a significant commitment of the child's family.
Hygiene hypothesis and probiotics (indications, patients, and bugs)
In 1976, a Canadian survey on the prevalence of asthma, eczema and urticaria in white and metis (Cree Indian) communities found that the white community, living for a long time outside the reserves, displays a higher incidence of allergic diseases than the metis community. These individuals, living in the reserves, displayed lower allergies, but increased prevalence of helminth infestation as well as of other untreated viral and bacterial diseases. The authors concluded: ‘It is suggested that atopic disease is the price paid by some members of the white community for their relative freedom from diseases due to viruses, bacteria and helminths’ . More than 10 years later, epidemiologic studies confirmed the finding and the ‘hygiene hypothesis’ was formulated. This hypothesis proposed that, as a result of modern public health practices, individuals experiencing a relative deficiency in immune stimulation by microbes become vulnerable to the development of allergic hypersensitivities and their associated diseases [372,373]. The immunological bases of the hypothesis became clear when it was discovered that the intrauterine environment is strongly oriented towards a Th2 immune response, while the Th1/th2 balance at 2 years becomes inverted . The social evolution for thousands years modeled the immune human evolution as in Fig. 3.
The hypothesis was expanded when it became clear from studies of transgenic germ-free animals that the disruption or absence of gut microbiota accounts for the development of allergic airway responses in the absence of prior systemic priming. As an extension of the hygiene hypothesis, the ‘microflora hypothesis of allergic disease’ was postulated to highlight the role of the gut in modulating host immunity in early life  and possibly in later life . Cross-sectional and cohort studies in young children with allergic diseases have shown an association between microbial patterns of colonization and allergic disease not displayed by healthy controls . This finding is not universal, and some studies were unable to establish association of early gut ‘probiotic’ colonization with later atopy . In any case, these findings raise the idea of interfering through microflora manipulation in the immune response, orienting it at allergy prevention and treatment.
These attempts can be based in principle on three types of instruments :
1. probiotic: an oral supplement or a food product that contains a sufficient number of viable microorganisms to alter the microflora of the host and has the potential for beneficial health effects;
2. prebiotic: a nondigestible food ingredient that benefits the host by selectively stimulating the favourable growth and/or activity of one or more indigenous probiotic bacteria;
3. synbiotic: a product that contains both probiotics and prebiotics. Synbiotics may be separate supplements or may exist in functional foods as food additives.
At the moment, many studies have been published but no consensus exists on the effects of such interventions. A systematic review concluded that there is not enough evidence to support the use of probiotics, prebiotics or synbiotics for prevention or treatment of atopic dermatitis in children in clinical practice .
Scientific societies worldwide formulated recommendations for their use. ESPGHAN, in a systematic review of RCT comparing formula with added probiotics and /or prebiotics with standard formula, did not recommend the routine use of probiotic-supplemented formula in infants . A WAO position statement, Clinical Use of Probiotics for Paediatric Allergy (CUPPA), is ready to be published . It concludes that probiotics do not have an established role in the treatment of allergy. No single probiotic supplement or class of supplements has been demonstrated to efficiently influence the course of any allergic manifestation or long-term disease or to be sufficient to do so. The document calls for epidemiological, immunological, microbiological, genetic, and clinical studies to determine whether probiotic supplements will be useful in allergy medicine, in collaboration between allergo-immunologists and microbiologists.
Allergy prevention using probiotic supplementation
Allergic diseases, the most common chronic diseases in childhood, continue to increase in prevalence and this has been linked to the relative lack of microbial stimulation  especially in early childhood when the permeability of the gut is higher  and the gut immune system not fully developed . The development of oral tolerance requires contacts with microbes . Mice reared in germ-free environments do not develop tolerance, but this can be reconstituted with the administration of bifidobacteria . Further, less lactobacilli and bifidobacteria have been shown in the gastrointestinal tract of infants later developing allergy [388,389]. This led to the probiotic concept.
Supplementing microbes using probiotics, health-promoting nonpathogeneic bacteria is a well tolerated alternative [390–392]. There are at least 15 published double-blind, placebo-controlled intervention studies regarding the clinical effect of probiotic supplementation on development of allergy. About 60% of the studies show a favourable effect decreasing the risk of eczema during the first years of life, whereas the remaining studies fail to show an effect. Most investigators have chosen high-risk for allergy cohorts to study the probiotic preventive capacity. This review highlights recent work on prevention of allergic diseases using probiotics. Since the publication of earlier reviews on prevention and treatment of allergic diseases [393,394] several large prevention studies have been published which are the focus of this review.
Clinical probiotic studies on prevention of allergic diseases
In the first study (2001) , administration of Lactobacillus rhamnosus GG (LGG) to 145 pregnant mothers and thereafter to the breastfeeding mother or when not breastfeeding to the infant halved the development of atopic eczema by age 2 years from 46 to 23%. In 2007, we published the largest cohort reported (n = 1223) with 925 infants followed until age 2 years after supplementing pregnant women from week 36 of gestation with a mixture of four probiotics (LGG, L. rhamnosus LC705, Bifidobacterium lactis Bb12 and propionibacterium) and infants receiving the same probiotics and a prebiotic oligosaccharide from birth to 6 months showing a 20% reduction of eczema (32.3–26.0%) and a 30% reduction of atopic eczema (17.7–12.4%) compared with the placebo group . We had a good retention of children since at the 2 and 5-year follow-up visits 91 and 88% were attending. The second largest cohort from New Zealand is unique with a comparison of two different probiotic strains. Pregnant women (n = 474) were treated with L. rhamnosus HN001, bifidobacterium animalis subsp lactis HN019 or placebo 1 month prenatally and until 6 months to the breastfeeding mother and directly to infants from birth until 2 years. A 50% reduction of eczema in the lactobacillus group, 26.8% vs. 14.8%, HR 0.51 (0.30–0.85) but no change in the bifidobacteria group was found . The study highlights the importance of the bacterial strain chosen and not any probiotic strain is efficient. The probiotic intervention was prenatal combined with postnatal both to the lactating mother and directly to the infant and with a longer intervention than in most studies. Choosing the most beneficial probiotic and length of intervention needs to be settled.
These two largest cohorts show an eczema preventive effect from L. rhamnosus, but not all studies do so. A small German study (n = 105) replicated the initial LGG study  and gave LGG or placebo 1 month during pregnancy and 3 months to the breastfeeding mother and thereafter from age 3 to 6 months directly to the infant and found no difference in eczema frequencies at the age of 2 years . However, direct supplementation to infants started only at 3 months, which might be one explanation for the failed effect.
A Swedish study using L. reuteri 1 month prenatally to mothers and 12 months to infants found no difference in eczema until age 2 years, but showed a decrease in IgE-associated eczema during the second year. IgE-sensitization measured with SPT was also found less often in the active group compared with the placebo group (14 vs. 31%) while most studies do not show effects on sensitization.
Two studies used only postnatal probiotic supplementations. In a study from Australia, L. acidophilus or placebo was administered after birth to 231 newborns during 6 months and fails to show an effect on development of eczema at age 1 year. There was an increase in SPT positivity and SPT-positive atopic eczema cases in the probiotic group . In the same cohort followed for 2.5 years, no differences in any allergic disease or sensitization outcomes were evident . In another postnatal-only study in Asian infants, Soh et al. supplemented infants with Bifidobacterium longum and L. rhamnosus for 6 months but in two logs lower concentrations than other studies, and found no allergy-preventive effects. According to these studies [403,404,405▪,406], prenatal maternal supplementation appears important for allergy preventive effects of probiotics, leading to faster infant colonization and changes in the breast-milk composition can be important for the preventive effects.
Two bifidobacteria dominant studies show favourable effects in high-risk populations. A mixture of B. bifidum, B. lactis and L. lactis was given prenatally to mothers and to their infants for the first year . Cumulative incidence of parent reported eczema at 1 and 2 years appeared significantly less often in the probiotic group than in the placebo group already at 3 months, but thereafter the incidence of eczema was similar until 2 years and the thus relative risk reduction was strongest during the first months after birth. The study strength is hampered by a high drop-out rate. A small Korean study  using two bifidobacteria plus L. acidophilus 1–2 months prenatally and 3 months to breastfeeding mother and thereafter 3 months directly to the infant showed a strong reduction in eczema, OR 0.24 (0.07, 0.79) at 1 year.
All previously mentioned studies have assessed allergy preventive capacity of probiotics in high risk for allergy cohorts, but three studies used unselected cohorts. An interesting and easy to implement approach was used in a Swedish study , in which supplementation with Lactobacillus F19 during weaning from 4 to 13 months resulted in halved eczema frequencies at age 13 months. In a Norwegian study , a probiotic mixture (LGG, L acidophilus La-5 and Bifidobacterium animalis) given 1 month prenatally and 3 months to the breast-feeding mother showed atopic eczema to be less prevalent at 2 years in the actively treated group [OR 0.51 (0.30, 0.87)]. In a Finnish study LGG and Bifidobacterium Bb12 were given prenatally and postnatally to the breastfeeding mother resulting in less IgE-sensitization but with no effect on eczema frequencies until age 1 year. Supplementing the mother pre and postnatally without infant supplementation can possibly work and would be an easy way of supplementation.
The studies in which supplementation commenced during pregnancy and continued directly to the infants after birth seem to result in the strongest effect as seen in the two largest studies published [396,397]. Eczema development decreased significantly OR 0.74 (0.55, 0.98) and 0.51 (0. 30, 0.85) during the first 2 years of life using LGG (with three other probiotic strains) and L. rhamnosus HN001, respectively. The best evidence of an effect has been shown from use of L. rhamnosus, but even if the majority of these studies show a beneficial effect on allergy prevention, there is inconsistency in the results.
Long-term follow-up studies
Long-term follow-up of cohorts for allergy development is mandatory for a comprehensive evaluation of effects of probiotics. Respiratory allergies can reliably be assessed only from 4 to 5 years of age. Only three cohorts have published follow-ups until 4 years and beyond. Kalliomäki showed a clear decrease in eczema until 4 and 7 years, with no effects on respiratory allergies [412,413]. Our large cohort failed to show any effect on allergy prevalence until age 5 years. However, in the subgroup of caesarean delivered children, 17% of the cohort, a sustained effect on reduced incidence of allergic disease was shown. This was due to less eczema in the probiotic-treated group, with nonsignificant reductions in asthma and rhinoconjunctivitis. These children showed a delayed colonization with bifidobacteria compared with vaginally delivered children, which could be corrected with probiotic supplementation . Very recently the large Wickens study  reports their 4-year follow-up . They show a sustained eczema-reducing effect from 2 to 4 years with L. rhamnosus supplementation. Very interestingly this is the first study to show a reduction in respiratory allergies as they also show less rhinoconjunctivitis indicating that by preventing eczema stopping the atopic march might be possible.
Systematic reviews on probiotics for allergy prevention
Several systematic reviews addressing probiotics for allergy prevention have been published and a more analytical approach was used in two meta-analyses. The Cochrane Review included 12 RCT studies of which five were analyzed in the meta-analysis with allergy outcomes reported in 1477 infants with a reduction in infant eczema RR 0.82 (0.70–0.95), but studies were heterogeneous. All studies reporting significant results used L. rhamnosus in high-risk families. No benefits for other allergy outcomes were observed . A recent review summarized the publications on probiotics and prebiotics for preventing allergic disease including six randomized controlled studies. It concluded that an effect on development of eczema could be supported, with the strongest effect when supplementation started prenatally, in which seven of 10 studies showed a decrease in eczema frequency until 2 years, OR for eczema 0.76 (0.64–0.91) and atopic (IgE-associated) eczema 0.70 (0.56–0.88) . However, due to the heterogeneity of study design using different populations in diverse environments, various strains, study designs, especially time and length of intervention, it is difficult to perform stringent meta-analyses and hence conclusions should be treated cautiously.
In summary, results from studies on primary prevention of allergies using probiotics should be limited to used strain(s) and time (prenatal and/or postnatal) and length of intervention as well as populations (high-risk, normal risk) and setting (hygienic conditions in study environment). The use of prebiotics should also be taken into account.
Mechanisms of action in allergy prevention
The prenatal exposure to a farming environment has also pointed to the importance of prenatal exposure for allergy preventive effect . By colonizing the mother prenatally, the transfer of favourable bacteria to the infant starts during birth. Also, immunomodulation of the mother and changes in her breast-milk composition could benefit the infant with regard to allergy development [404,406]. Boyle et al.[405▪] assessed whether prenatal administration without supplementing infants would suffice for allergy prevention. Using LGG from 36 weeks of gestation until delivery had no effect on eczema prevalence by age 1 year. However, it appears that the prenatal immunomodulation is insufficient for a change in the child's immunologic response to the nonallergic phenotype and needs to be followed by stimulation of the infant's gut immune system, preferably directly to the infant.
The preventive mechanism by which probiotics operate might be the modification of the gut microbiota, strengthening of the mucosal epithelial barrier and immunomodulation. In-vitro and in-vivo immunologic effects have been shown from probiotic administration [419,420]. Probiotic administration has resulted in demonstrable changes in the levels of the given strains in faecal samples in reported studies and supplementation has been successful in all studies [108,396,398,400,407,414] in which colonization has been reported. However, there is no good evidence for a permanent colonization of the supplemented probiotic in infants or children and in adults given probiotics, they can be found in faeces only for short period of weeks .
No proven effective way of primary prevention of allergic disease has been found despite intensive research on environmental and dietary factors assessed including breastfeeding, maternal diets and dietary restrictions during pregnancy and lactation, short-chained fatty acid supplementation. The most promising allergy preventive alternative is administration of probiotic bacteria, preventing eczema until age 2 years showing, a good result already. The effect is clearly strain-specific, with L. rhamnosus showing the most promising effects, time-specific with prenatal combined with postnatal supplementation demonstrating most consistent effects. Choosing the most effective strain and the time and length of the supplementation needs to be settled. More long-term follow-up to assess the impact of probiotics on development of respiratory allergies is highly awaited.
PART 7 - HEREDITARY ANGIOEDEMA IN CHILDREN
Hereditary angioedema in children
Hereditary angioedema (HAE) was identified as an independent clinical entity in the 19th century and in 1963 C1 inhibitor (C1-INH) deficiency was discovered to be the genetic defect underlying this disease . In recent years, other forms of HAE without C1-INH deficiency have been defined affecting a very small number of patients usually not symptomatic in childhood . Here, we will just refer to HAE caused by genetic C1-INH deficiency.
Patients with HAE experience intermittent cutaneous or mucosal swellings as a result of failure to control local bradykinin production. Swellings evolve, typically, over a few hours and persist for a few days. As well as orofacial angioedema, painless swellings affect peripheries, causing disfigurement or interfering with school and other activities of daily living. Angioedema affecting the gastrointestinal tract or abdominal viscera causes severe pain, often with vomiting as a result of edematous bowel obstruction. About 2% of swellings involve the larynx and may be fatal, if left untreated . HAE affects around 1 in 50–100 000 of any ethnic group, with many of those affected being unaware of their diagnosis. Although, the deficiency is life-long, swellings only rarely occur before the age of 2 years and are less frequent before adolescence. Mean age at onset of symptoms is around 8–12 years. Incidence of swellings varies from more than one per week to less than one per year. Hence, HAE in childhood can cause significant disability and represent a medical emergency.
Diagnosis of HAE is based on the above-mentioned clinical symptoms and evidence of C1-INH levels below 50% of normal. In about 20% of HAE patients, C1-INH deficiency can only be assessed by measurement of C1-INH function because the mutation allows the production of a dysfunctional protein that results in normal C1-INH plasma levels. As a consequence of C1-INH deficiency there is consumption of the fourth component of human complement (C4) that is almost invariably below 10 mg per 100 mL: such a reduction of C4 supports the biochemical diagnosis.
C1-INH deficiency causes instability of the contact-kinin system that, through activation of plasma kallikrein, eventually leads to the release of bradykinin, a potent proinflammatory vasoactive peptide . Therapeutic interventions in HAE are aimed to avoid fatalities and reduce disability. It can be done by replacing the deficient protein (both plasma-derived and recombinant C1-INHs are available), specifically inhibiting plasma kallikrein or antagonizing bradykinin . Other therapeutic approaches, not directly derived from the pathophysiology of the disease, involve the use of attenuated androgens and of antifibrinolytic agents.
Only plasma-derived C1-INH and antifibrinolytic agents have been used in prepuberal children. Limited experience with the use of attenuated androgens has also been provided , but we do not recommend the use of these drugs during growth for the risk related to residual hormonal effect. Management of HAE in children should first be based on providing each patient carrying hereditary C1-INH deficiency with an amount of plasma-derived C1-INH sufficient to treat two angioedema attacks at the dose of 20 U/kg. This should be done also in still asymptomatic children because laryngeal edema can be the first clinical manifestation of the disease. Plasma-derived C1-INH is given intravenously and is generally recommended for all attacks, since they are potentially disabling. It is mandatory when larynx or larynx surrounding area is involved. Location of angioedema to the gastrointestinal mucosa can easily cause abdominal symptoms of bowel obstruction that are difficult to differentiate from a surgical emergency: in these situations the response to C1-INH treatment is an important tool for differential diagnosis.
In children with frequent symptoms of angioedema the need for a prophylactic intervention should be considered, but continuous administration of a therapeutic agent calls for careful consideration of the risk benefit balance. Reasonably, vast experience indicates that the antifibrinolytic agent tranexamic acid can safely be used in children at doses of 15 mg TID orally. This treatment may not be effective in all patients, but it is worth trying in children experiencing several days of inability per months despite optimized on-demand administration of C1-INH. When tranexamic acid is not effective and children remain severely sick programmed C1-INH administration can be introduced to prevent attacks. Prophylactic C1-INH has been performed as regular infusion of 500/1000 U twice a week as well as by so-called ‘individual replacement therapy’, which consists on administration of 500 U of C1-INH based on very early subjective signs of an upcoming attack [428,429].
In the near future, the therapeutic armamentarium for treating children with HAE will probably grow due to the ongoing paediatric studies with recombinant C1-INH and with the subcutaneously administrable kallikrein inhibitor ecallantide and bradyknin receptor antagonist icatibant.
In conclusion, HAE is present in children as a disabling and potentially life-threatening disease. Precise diagnosis is mandatory because this condition requires a specific therapeutic approach that has to be provided to the patients in advance in order to avoid fatalities. Appropriate use of therapeutic tools can also prevent and/or reduce the disability related to the disease.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 000–000).
Conflicts of interest
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