The term hypersensitivity refers to a condition where a subject develops symptoms to a stimulus that would usually be tolerated amongst the general population. Allergy is a clinical condition causing hypersensitivity reactions in which an 'immunologic mechanism is proven or strongly implicated'. This immunologic mechanism is typically IgE-mediated, although non-IgE-mediated allergy can occur. 'Atopic' individuals have a general tendency to develop allergic symptoms, due to immune responses, upon exposure to a range of allergenic substances often at low doses. This condition is commonly inherited (Johansson et al., 2001).
The term 'food hypersensitivity' is used to describe an adverse reaction that occurs in response to a food. More specifically, an immunological reaction to food is termed 'food allergy', which can be IgE- or non-IgE-mediated; all other reactions are referred to as 'non-allergic food hypersensitivity' (Johansson et al., 2001).
Allergic disorders in the UK are common, with reported diagnosis in 30% of adults and almost 40% of children (Gupta et al., 2004). Furthermore, the incidence is thought to have increased over the past few decades. In particular, various generalised/systemic allergic diseases are increasing in prevalence, with hospital admissions for food allergy and anaphylaxis, and less so for urticaria and angio-oedema, rising significantly since 1990. The incidence of allergic conditions such as hay fever and eczema has risen since the 1960's but now seems to be levelling off (Gupta et al., 2007). This point could, however, reflect a reduction in the numbers of affected people seeking medical care, for example due to more widely available 'over the counter' treatments.
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Allergic disease in the UK affects people of all ages, nationalities and social groups. Not only does the condition put a strain on the individuals affected, both in terms of a restriction in lifestyle and fear of potential reactions, but, with the scale of the condition, it also imposes a burden on the NHS. It has been estimated that patients with allergic diseases account for 6% of general practice consultations and almost 1% of hospital admissions (Gupta et al., 2004).
There are various suggestions why the incidence of allergic disease is increasing, such as the 'western lifestyle'. For example, the increasing availability of exotic foods and development of food technology are both responsible for the introduction of novel food allergens. The 'hygiene hypothesis' proposes that a clean environment and widespread antibiotic use reduces stimulation of the immune system, particularly components that protect against allergy (Ewan and Durham, 2002).
Food allergy is rapidly becoming a cause for concern in the Western world due to its increasing prevalence and potentially serious outcomes. In a questionnaire-based study of over 33,000 members of the French population, the incidence of food allergy was found to be 3.24%, with the most commonly implicated foods including egg, milk, tree nut, peanut, crustaceans, fruit and vegetables (Kanny et al., 2001). In a similar study of French school children, almost 7% were reported to be food allergic. Of the affected children, there appeared to be a greater incidence of allergy in the age range of 6-10 years than in other age groups. The most commonly allergenic foods were peanut, cow's milk and egg. Shellfish, tree nut and exotic fruits were also implicated. In about a quarter of the children studied the condition was outgrown, particularly so with cows milk allergy. This trend was not seen in children with tree nut allergy, and was relatively rare in those with allergy to peanut (Rancé et al., 2005).
The spectrum of allergic symptoms can be very broad, both between individuals and between different reactions of the same individual; symptoms can affect varying organs, and can range from being very mild to life-threatening or fatal (table 1).
Table 1. Symptoms of Allergy (Brown et al., 2001; Sampson, 2003)
Oedema and pruritis of lips and tongue, metallic taste
Pruritis, urticaria, flushing, angioedema
Nausea, abdominal pain, vomiting, diarrhea
Dysphagia, dysphonia and hoarseness, dry cough, pruritis and 'tightness' in throat, shortness of breath, 'tightness' in chest, wheezing, congestion, sneezing
Hypotension, chest pain, syncope, dysrhythmia
In a questionnaire-based study of food-allergic members of The Anaphylaxis Campaign in the UK, the most common symptoms included oral and respiratory problems, abdominal pain, and cutaneous affects. The oral and respiratory symptoms were experienced more commonly in children than adults, and this may reflect the finding that asthmatic children tended to have more severe reactions than non-asthmatic children. Adults, on the other hand, suffered more frequently from collapse (Uguz et al., 2005).
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Anaphylaxis is a severe IgE-mediated systemic allergic reaction, often occurring as a result of exposure to a trigger allergen, and involving multiple body systems (Sampson et al., 2006). The condition was first described by Portier and Richet over a century ago after inducing a fatal allergic reaction in dogs (Portier and Richet, 1902; cited in Sampson, 2003). The onset and development of the reaction is very rapid, as can be recovery upon increased endogenous production of adrenaline and angiotensin II. In other cases, however, an individual does not recover and anaphylaxis is fatal (Simons, 2005).
Target organs involved, and therefore the signs and symptoms of anaphylaxis, vary not only between individuals, but also between episodes (Simons, 2005). Symptoms, which may include skin rash, gastrointestinal symptoms, reduced blood pressure, bronchoconstriction and cardiac arrest, can occur within minutes of allergen exposure (McEuen et al., 2001).
Although the actual incidence of anaphylaxis is not clear, it is thought to be increasing; in a study of hospital admissions, the number of patients with a primary diagnosis of anaphylaxis almost doubled between 1991 and 1995. The majority of reactions were to therapeutic drugs, foods and insect venom. Although giving an idea of the changes in incidence, the figures provided by this study are likely to be underestimated as they exclude any patients presenting at Accident and Emergency departments but not being admitted to hospital. Suggested explanations for the increasing number of anaphylactic cases include changes in diet and increasing therapeutic drug use (Sheikh and Alves, 2000).
The first cases of anaphylaxis due to food in humans were published in 1969 by Golbert et al. (cited in Sampson, 2003). In an American study of 266 anaphylaxis cases, 34% were thought to be due to foods. Shellfish or peanut were believed to be the implicated food in around half of these cases (Kemp et al., 1995). A study carried out in North England showed that food is to blame for a higher proportion of anaphylactic cases; of 172 patients who had suffered an anaphylactic reaction, food was thought to be the trigger in over half. In the vast majority of these cases, peanuts and tree nuts were believed to be involved (Pumphrey and Stanworth, 1996). Other foods commonly implicated in food allergy include milk, chicken egg, seeds and fruit (Sampson, 2003).
Interestingly, in an Italian study of children who have suffered from one or more anaphylactic reactions, although food was thought to be the trigger for almost 60% of cases, only 13% of these cases were caused by nuts. Furthermore only 1% of food-induced anaphylactic reactions were caused by peanut. The major implicated foods were seafood and cows milk (Novembre et al., 1998). This is actually consistent with studies using data from death certificates recorded at the Office of National Statistics (ONS) in the UK which show no incidence of fatal anaphylaxis due to nuts in children under the age of 13 (Pumphrey, 2000), suggesting that nuts are actually more of a problem in teenagers and adults, and that children are more at risk from other foods.
Some studies imply that anaphylactic reaction to food is a higher risk in children and adolescents than adults (Novembre et al., 1998; Sampson et al., 1992), whereas others claim that adolescents and young adults are at greater risk (Bock et al., 2001; Brown, 2001). According to Brown (2001), adults tend to experience cutaneous and cardiovascular symptoms more commonly during food-induced anaphylaxis, whereas children are more likely to suffer from respiratory complications. Consistent with this, Novembre et al. (1998) found that, in children suffering from anaphylaxis to foods, cardiovascular symptoms were relatively uncommon. In this study, gastrointestinal symptoms were found to occur most frequently in children. In a study by Pumphrey (2000), all cases of food-induced anaphylaxis, both in adults and children, led to respiratory problems.
In a significant number of cases, a biphasic reaction is seen. In these patients the symptoms of anaphylaxis can reappear after the initial reaction has resolved. Risk factors for this may include delayed administration or insufficient dosing of adrenaline (Lieberman, 2005).
Various other conditions and medications can affect the likelihood and control of food-induced anaphylaxis. Risk factors include persistant, particularly uncontrolled, asthma, as well as other comorbidities such as acute infection. Certain CNS-active medications and chemicals, including ethanol and HI antihistamines, may affect an individual's ability to recognise triggers and signs of a reaction. Furthermore, ethanol also acts to dilate, and antihistamines to increase the permeability of blood vessels, thus enhancing inflammation. Additionally, some medications may interfere with the effectiveness of treatment, for example angiotension-converting enzyme (ACE) inhibitors and β-blockers (Simons et al., 2005).
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A study in children and adolescents of fatal and near fatal anaphylactic reactions to food over a period of 14 months in the US revealed that over 92% of patients had asthma (Sampson et al., 1992). Consistent with this, a similar study carried out almost a decade later found that 96% of patients suffering an anaphylactic reaction to food were also asthmatics (Bock et al., 2001). Both studies found that the majority of patients were aware of allergies to foods, but were unaware of consuming the food at the time of the reaction. The initial study also found that over half the patients suffered a rapidly progressive and uniphasic reaction, whilst the remainder suffered either biphasic or protracted reactions. The later study indicated that both sexes are affected equally by anaphylaxis to foods; although there is conflicting data which shows that there is a higher rate of fatal anaphylaxis in men than women (Pumphrey, 2001).
2. The Immunology of Allergy
An allergen is an antigenic, often protein, molecule which can trigger an immune response within an allergic individual. Allergen exposure causes a sequence of events that leads to the symptoms of a reaction. For a specific allergy to develop, an individual must have been exposed to the molecule in question previously to the first reactive episode (Lehrer et al., 2002).
In IgE-mediated allergy, initial exposure leads to digestion, processing and presentation, via MHC proteins, of the antigen on the cell surface of specialised antigen-presenting cells (APC) including B lymphocytes (B cells). The presented antigen is then recognised by mature T lymphocytes (T cells), which stimulate B cell multiplication and maturation into antibody producing plasma cells. The B cells are then able to produce and secrete specific antibodies against the antigen; signals from T cells specify the isotype, which in this case is IgE (Prussin and Metcalfe, 2006). B cells can also be directly stimulated by fragments of the antigen (Lehrer et al., 2002).
The antigen-specific IgE antibodies bind to mast cell and basophil membranes via the high affinity IgE receptor, FcεRI. Mast cells are inflammatory, bone marrow-derived cells that are found in body tissues. Basophils, on the other hand, are granulocytes which circulate in the blood. Like mast cells, basophils differentiate and mature in bone marrow. On both of these cells, FcεRI is found in its complete, fully active tetrameric form, αβγ2, and IgE binds to the α-chain of the receptor via its Fc fragment (Prussin and Metcalfe, 2006).
During a subsequent exposure, the allergen binds to the specific IgE antibodies presented on mast cells and basophils, causing cross-linking of the molecules and aggregation of FcεRI. This leads to granule exocytosis in mast cells and basophils. From mast cells various preformed mediators are released, such as histamine, tryptase and chymase. Additionally new mediators such as prostaglandins and leukotrienes are synthesised and also released. In basophils, granule exocytosis releases mediators such as histamine, leukotrienes and basogranulin. It is the actions of these mast cell and basophil products that cause the clinical symptoms of an allergic reaction (Lehrer et al., 2002).
In non-IgE-mediated allergy IgM and IgG, cytotoxic events, or activation of complement anaphylatoxin can be involved in the stimulation of mast cell and basophil degranulation and thus mediator release. These mechanisms, however, are less understood (Simons et al., 2007).
The majority of food allergy cases are thought to be caused by of one of eight food types/groups: peanuts, tree nuts, eggs, milk, fish, crustacean, soybeans or wheat. It is generally the major proteins within a food that can be allergenic, so it is suggested that a reaction may be more likely to occur with higher allergen doses. Consistent with this, most allergenic foods are high in protein. Of the exceptions to this, the majority have a large lipid concentration. The main allergenic proteins have been established for several foods, for example, ovalbumin is responsible for the majority of allergic reactions to egg, whereas tropomyosin causes most reactions to shrimp and other crustacea. Cross-reactivity, both between different food types and between foods and other substances, is a known occurrence. This is likely to happen when there are similarities in the allergenic proteins of the two substances (Lehrer et al., 2002).
Food allergens are different from many other allergen types, such as inhaled allergens, as they must be unaffected by processes such as cooking and digestion. This suggests that they are generally very stable molecules, although must be small enough to cross the mucosal membrane of the gut. The high fat levels in some allergenic foods, such as peanuts, may act to preserve the allergenic molecules during cooking and digestion (Lehrer et al., 2002). According to Sicherer (2002), the higher rate of food allergy in children compared with adults may be, at least in part, explained by increased absorption due to 'leakier' gut barriers.
Clearly food allergens must be recognised by the immune system as foreign and have the capacity to evoke an immune response. Furthermore they must be polyvalent with no less than two IgE binding sites in order for cross-linking of mast cell or basophil-bound IgE to occur (Lehrer et al., 2002).
3. Diagnosis of Allergy and Anaphylaxis
3.1 Diagnosis of allergy
The variability in the severity and duration of the allergic reaction, the target organs involved, the threshold doses of allergen required for a reaction, the responses to therapy and the reaction outcome, both between and within individuals, makes allergy and anaphylaxis a particularly difficult condition to understand and diagnose (Sampson et al., 2006). Currently, diagnosis of IgE-mediated allergy, particularly to food, is largely reliant on the history of a patient and the performance of a skin prick test (SPT). In vitro tests for detection of IgE in serum samples are additional tools that can sometimes aid diagnosis. As a final measure, avoidance trials or, conversely, provocation testing can be effective in confirming the diagnosis (Holgate et al., 2000).
If a case is presented of possible food allergy, the frequency and severity of symptoms are recorded, as well as the dominant symptoms and family history. Potential trigger factors are identified (Holgate et al., 2000). The history of a patient is believed by many to be the most important tool currently available in diagnosis of food allergy (Simons et al., 2007).
Skin prick tests can be important for the verification or exclusion of atopy, however the clinical history is considered whilst interpreting the results. The test is performed by placing allergen extracts on the skin before piercing the top skin layers (Bohle and Vieths, 2004). Both positive and negative controls (histamine and diluent, respectively) must be used, and a positive result is detected by a skin wheal of at least 2 mm greater than the negative control. In many cases, but not all, a skin wheal of at least 6 mm is more likely to be indicative of clinical relevance. There are some circumstances in which the results of an SPT may be disrupted, for example if the patient is taking antihistamines or has a skin condition such as eczema. Furthermore, the test must be performed within a hospital as there is a small risk of systemic reaction (Holgate et al., 2000). It is thought that, although SPTs are effective in diagnosis of allergy to inhaled allergens, they are not as reliable in food allergy (Muñoz-López, 1998).
The main in vitro tests for allergy diagnosis are serum measurements of total IgE and allergen-specific IgE. Total IgE levels are elevated in the serum of many allergic patients, but not all. Therefore this test is not particularly reliable for predicting clinical reaction to a food. Allergen-specific IgE is the preferred in vitro test, and may be useful in the diagnosis of food allergy when predictive risk values are available. Immunoassays for detection of allergen-specific IgE in serum include radioimmunoassay (RIA) and enzyme-linked immunosorbant assay (ELISA). Chemiluminescence methods can also be used. All of these tests generally involve coating a plate/well with the allergen in question before adding a small amount of serum to allow any IgE specific to the allergen to bind. The antibody can then be detected in varying ways. The radioallergosorbent test (RAST) is a well known method in which the antibody is detected by adding radiolabelled anti-IgE and counting the radioactivity (Holgate et al., 2000).
SPT is thought to be the more sensitive test, whereas RAST is believed to be more specific. SPT has the additional advantages of being less expensive to carry out and yielding results quickly. However, unlike RAST, SPT is affected by various drugs and skin diseases, and carries a risk of systemic reaction. Furthermore, RAST allows for a greater range of allergens to be tested (Holgate et al., 2000).
The main problem with both of these tests is that they are not always reliable for predicting clinical reactivity (i.e. risk of an allergic reaction) to an allergen or the severity of a potential reaction. In a large proportion of cases a patient is able to eat a certain food type without a reaction even though a skin test has revealed sensitivity to that food type. It must therefore be considered that, even if a patient responds to an SPT, this is not evidence that the particular allergen tested is the trigger for the patient's symptoms. Individuals who are sensitised to a food allergen but do not react after consumption of that food type are known as clinically tolerant. Neither SPTs nor in vitro tests can reliably distinguish between clinically reactive and clinically tolerant patients (Simons, 2005). A potentially more dangerous problem, although rare, is that certain individuals may test negative to an allergen in an SPT but react to a food challenge (Simons et al., 2007).
A less common test in the diagnosis of food allergy is the measurement of blood basophil histamine release after exposure to allergen. This can be carried out on an ELISA plate: blood samples are added after coating the plate with allergen, and any released histamine detected by fluorescence or RIA. This is a sensitive and specific method, which takes only a few hours; however it does have limitations. In particular a small proportion of people possess basophils that do not release histamine in vitro. Thus a negative result must be confirmed with a positive result to an anti-IgE control (Holgate et al., 2000).
Provocation tests for the diagnosis of food allergy are generally only performed when other tools have not yielded a clear result, for example if the clinical history of a patient and an SPT are conflicting (Holgate et al., 2000). Food challenges involve exposing the patient to a particular food in an incremental fashion, and can be open or double blind. This test can confirm whether an individual is clinically reactive or tolerant to an allergen. There is a risk of systemic reaction with this test, and it must therefore be carried out in a hospital setting and supervised at all times (Simons, 2005). Additionally, the challenge is expensive and timely. Despite this, the double blind, placebo controlled food challenge (DBPCFC) is currently thought of as the 'gold standard' test in allergy diagnosis (Bohle and Vieths, 2004).
3.2 Diagnosis of Anaphylaxis
In the case of a patient being admitted to hospital showing signs of anaphylaxis, there is currently no available test that can reliably confirm whether an allergic reaction to food has taken place. On one hand this may put patients who have previously suffered a severe reaction to food at risk of a second if the trigger was not identified. On the other hand, patients may unnecessarily avoid foods that they incorrectly believe to put them at risk. This can lead to lifestyle problems and an unbalanced diet (McEuen et al., 2001).
Furthermore, death caused by anaphylaxis is often misdiagnosed at post mortem due to a lack of characteristic signs that distinguish from other causes of fatality. Research is currently being carried out to investigate the possibility that sudden infant death syndrome (SIDS) is a result of an anaphylactic reaction to cows milk (McEuen et al., 2001).
Tests that are available to diagnose anaphylaxis include measurement of plasma histamine levels, which typically increase in an anaphylactic reaction. However plasma histamine levels peak very quickly after the onset of symptoms and usually decline to normal levels within an hour. This test is therefore often impractical as the onset of anaphylaxis does not commonly occur in a clinical setting. This problem may be overcome by instead taking urine histamine or histamine metabolite measurements for 24 hours following the onset of anaphylaxis (Simons et al., 2007).
Measurement of total serum or plasma tryptase levels can be helpful if the sample is collected within three hours of the onset of symptoms. Ideally the measurement would be compared with samples taken over 24hrs after the symptoms of anaphylaxis have subsided. If the tryptase level is increased compared with baseline levels whilst symptoms are present this is indicative of anaphylaxis. Although this test is useful in anaphylaxis induced by insect sting, it has not proved to be particularly effective in diagnosing food-induced anaphylaxis as serum tryptase levels are generally not significantly increased (Simons et al., 2007).
4. Aim of Project
Anaphylaxis is believed to be underreported, and this may be due to several reasons. For example, symptoms can be hard to determine in young patients or in those suffering from shock, and symptoms may appear and disappear very quickly. Many symptoms are not specific to anaphylaxis, such as collapse, nausea and vomiting, and there is a lack of consensus on specific diagnostic criteria. Also, an allergic condition may not be recognised when an individual suffers their first reaction (Simons et al., 2005). At post mortem, anaphylaxis as a cause of death is often rejected, despite implicative clinical records, due to a lack of specific evidence (Pumphrey and Roberts, 2000).
There is no reliable test currently available which can confirm a severe allergic reaction to food, thus making anaphylaxis very difficult to diagnose. There are two main implications of this. Firstly, individuals who have suffered from an allergic reaction to a food may be at risk of another if the reaction was not confirmed and the allergen therefore not identified. On the other hand, an individual may unnecessarily avoid certain food types if it is incorrectly thought that a reaction to a particular food allergen has occurred. This may lead to lifestyle restrictions and have negative affects on the diet of the individual (McEuen et al., 2001).
Ideally a test should be developed for the diagnosis of anaphylaxis which would detect a marker specific to mast calls and/or basophils that is secreted upon activation of these cell types. Furthermore, the marker in question should be relatively stable in biological fluid. Also of benefit would be a test for predicting the severity of future reactions of an individual. This would likely involve measuring baseline levels of a mast cell or basophil product.
4.1 Potential Markers of Anaphylaxis
Histamine is stored in and released from mast cells and basophils, and has long been known to have a role in allergic reactions. It has thus been considered as a potentially useful marker of anaphylaxis. In a study of bee sting-induced anaphylactic reactions, plasma histamine levels increased, reaching a peak level about 5-10 minutes after the challenge; levels then declined quickly, returning to baseline levels between 15 and 60 minutes (Schwartz et al., 1989). Thus it seems that serum histamine levels may be a reliable marker of anaphylaxis, however is impractical in the majority of cases as must be measured very early on in the reaction, and thus levels may have returned to normal by the time the patient reaches medical care.
In an emergency department-based study approximately half of adults with acute allergic reactions were found to have elevated plasma histamine (>10 nmol/L) levels. The majority of these patients did not have severe anaphylactic reactions, suggesting that histamine levels are a marker of non-anaphylactic as well as anaphylactic reactions. However levels did correlate with initial heart rate, and were higher in subjects with more severe symptoms such as wheezing and angioedema (Lin et al., 2000). In food allergic patients, intraluminal provocation with food antigens resulted in an increase in intestinal histamine release. This result was not seen in control subjects (Santos et al., 1999).
Tryptase is a serine protease believed to play a key role in events mediated by mast cells. It is found in mast cell secretory granules and, upon cell activation, released along with numerous other mast cell products. Tryptase may therefore be useful as a marker of mast cell activation. Tryptase release can be local for example in asthma, or systemic for example in anaphylaxis (Caughey, 2007).
In 1990, tryptase was isolated from human lung tissue, and polyclonal and monoclonal antibodies were produced which bind to the enzyme. Use of the antibodies within the study showed that tryptase was specific to mast cells; and thus it was suggested that they can be used as a tool to identify human mast cells. In addition to this, tryptase detection by specific antibodies may be useful in differentiating these cells from basophils (Walls et al., 1990).
There are four forms of human tryptase: α-, β- and δ-tryptase are all soluble, where as γ-tryptase is membrane-bound. β-tryptase is further subdivided into subtypes I, II and III. Of the soluble proteins only β-tryptase is active and believed to play a role outside the mast cell; α- and δ-tryptase are generally inactive. For this reason, β-tryptase is thought to be the main form of the enzyme that is involved in the inflammatory events that occur upon mast cell activation (Caughey, 2007).
Because of its release upon mast cell activation and its specificity to mast cells, tryptase has been identified as a candidate for a marker of anaphylaxis. Levels of the enzyme in serum from venous blood have been found to increase from around 30 minutes and peak 1-2 hours after experimentally-induced anaphylaxis to bee sting. After this time levels reduce, with a half life for the enzyme of approximately 2 hours (Schwartz et al., 1989). The Resuscitation Council promotes the measurement of tryptase in anaphylaxis diagnosis (Project Team of The Resuscitation Council (UK), 1999), however studies that have investigated the enzyme's potential in this role have yielded conflicting results.
In one study, β-tryptase measurements determined in blood at post mortem were increased in more subjects with fatal anaphylaxis than most other causes of death. Exceptions to this included deaths due to heroin injection and car crash organ injuries. Interestingly, of the anaphylactic deaths, the lowest tryptase level was found in a subject who reacted to food allergens; the measurement obtained for this case was not considered elevated. Increased tryptase levels were also found in cases of sudden infant death syndrome (SIDS) (Edston et al, 1998), however it has been suggested that anaphylaxis may play a role in this cause of fatality. In a study by Buckley et al., increased serum β-tryptase was found to be significantly higher in infants with SIDS than those who died of known causes, and this was considered to be indicative of anaphylaxis (Buckley et al., 2001).
In an emergency department-based study of adults with acute allergic symptoms, elevated serum tryptase levels were observed in approximately one fifth of patients and this was most commonly in subjects with urticaria and tachycardia. High β-tryptase levels were not common, however were found almost ten times more commonly in subjects with hypotension, tachycardia or wheezing than in patients without these symptoms. All subjects with high β-tryptase measurements also had elevated total tryptase levels. In this study very few cases were considered as anaphylactic, so it may be that increased tryptase levels are more characteristic of allergic disease with greater severity (Lin et al., 2000).
In a study by Vila et al. serial serum and saliva tryptase measurements were taken before and following food challenges in patients who had previously suffered systemic reactions to food. Only 25 % of patients with a positive food challenge had elevated serum tryptase levels, and no significant differences in levels were observed between the serial measurements in any of the patients. There was no increase in tryptase levels in subjects who did not react to the food challenge. Tryptase was detected in the saliva both before and after the food challenge in one positive subject; in all other patients, including controls, saliva tryptase was undetectable (Vila et al., 2001). In another study however, administration of food antigens intraluminally resulted in increased intestinal tryptase release, as well as histamine, peroxidase and prostaglandin D2, in food allergic but not control patients. Plasma tryptase levels, consistent with other studies, were similar in both food allergic and control subjects, and were not increased in either group after the antigen challenge (Santos et al., 1999).
Available data generally suggests that, although high tryptase levels are an indicator of anaphylaxis, low levels cannot exclude a diagnosis of this condition. Conversely, elevated tryptase can be found in other conditions, and thus additional factors such as the patient's history should be considered. It is widely agreed that further research into the use of tryptase measurements in diagnosing anaphylaxis, particularly as a result of food, is needed.
Like tryptase, chymase is a serine protease. It is synthesised in mast cells where it is stored within the secretory granules. From here the enzyme is released, along with other mast cell products such as heparin, histamine and tryptase, in mast cell degranulation following exposure to an allergen. Chymase is thought to be involved in a range of events implicated in inflammation (McEuen et al., 1998)). Furthermore, the enzyme has the ability to generate angiotensin II from angiotensin I (Urata et al., 1990).
The mast cell granule in which chymase is stored is reported to have a pH of 5.5. Chymase was found to be relatively inactive at this pH; however appears to be immediately active when released. Thus perhaps chymase is synthesised in an inactive pre-protein form and then converted within the mast cell to its mature form. This mature protein may be stored in the mast cell granule, and when released, become instantly active due to the removal of the pH suppressing effect. The formation of mature chymase from its inactive form, pro-chymase, is catalysed by the enzyme dipeptidyl peptidase I (DPPI) via the removal of a dipeptide (McEuen et al., 1998).
Chymase is found mainly in the subpopulation of mast cells that localise in the dermis of the skin (MCTC); little or no chymase is expressed in the MCT cells of mucosal tissue (Caughey, 2007). In a study by McEuen et al. (1998), affinity chromatography revealed several forms of human chymase. These showed differences in tissue distribution, and thus it was suggested that the possibility of variations in chymase action at different inflammatory sites should be investigated.
The findings that chymase is released from mast cells after allergen exposure, and that it acts to promote inflammatory events, suggests that the enzyme may play key a role in allergic reactions and anaphylaxis. In a post mortem study, chymase was detected from cardiac blood-derived serum in all deaths due to anaphylaxis but in less then 2 % of deaths by other causes. Furthermore, serum chymase levels correlated positively with tryptase levels in all anaphylactic deaths. In one case of anaphylaxis serum chymase was only just detectable; however the subject had died at the onset of the reaction suggesting that levels may not have reached a peak. The majority of the anaphylactic cases in the study were triggered by drugs, including antibiotics and anaesthetics (Nishio et al., 2005).
In 1989 activity resembling that of carboxypeptidase was identified in mast cells derived from both lung and skin. The enzyme responsible was purified, and suggested to be a secretory granule zinc-metalloexopeptidase. It was found to have functional similarity to CPA, for example in that it is able to hydrolyse synthetic dipeptides and angiotensin, but structural similarity to CPB. Thus this novel member of the zinc-containing carboxypeptidase family is referred to as mast cell carboxypeptidase (MC CP). MC CP release from mast cells is induced by immunologic challenge, and it may therefore have a role in inflammatory and allergic mechanisms. Furthermore, it may be useful as a biochemical marker of skin mast cells (Goldstein et al., 1989).
Levels of carboxypeptidase A, along with tryptase and chymase, were found to be higher in blood from asthmatic, allergic and drug reactive patients compared to healthy controls. Interestingly, in co-incidence with this, peripheral blood basophilic cells were found in higher numbers, and expressed these mast cell proteases in asthmatic, allergic and drug-reactive patients, but not in healthy individuals. Thus basophil, as well and mast cell, carboypeptidase may be involved in inflammatory and allergic disease (Li et al., 1998).
Dipeptidyl Peptidase I
Dipeptidyl peptidase I (DPPI) is a cysteine protease expressed in the cytoplasmic secretory granules of bone marrow-derived leukocytes, such as myelomonocytic, cytotoxic T and mast cells. DPPI is released from these cells, and was therefore suggested that the enzyme acts extracellularly. Roles of DPPI, as found in vitro, include degradation and turnover of proteins, and activation of enzymes including chymase. DPPI may also have a role in the growth and differentiation of mast cells (Wolters et al., 2000).
DPPI is potentially involved in allergic reactions. It has been suggested that activation of mast cells, for example by IgE-bound antigen, may trigger the release of the enzyme from the secretory granules of these cells. The finding that DPPI interacts with tryptase and chymase perhaps supports its role in allergy (Wolters et al., 2000).
DPPI may also be involved in other inflammatory states such as asthma. Within the airways of dogs, DPPI is located mostly in mast cells, whereas in alveoli it is mostly in macrophages. DPPI was found to cleave various extracellular matrix proteins, and it was thus suggested that DPPI may be involved in matrix protein turnover and remodelling of the airways in asthma (Wolters et al., 2000).
In 1999 a basophil-specific antibody was produced. The antigen to which this antibody binds was found mostly intracellularly, and localised to the secretory granules. Furthermore, within the granules it is associated with the matrix rather than membrane. Because of these properties, the antigen was named Basogranulin (McEuen et al., 2001).
In later studies, in which basophils were stimulated with both FcεRI-related and unrelated stimuli, basogranulin was released alongside histamine with a bell-shaped response curve. Peak levels of basogranulin occurred at 15 minutes, before that of histamine (Mochizuki et al., 2003). Basogranulin was found to be present in all granules within the basophil, likely meaning that, upon basophil activation and degranulation, basogranulin is always secreted. It was therefore suggested that this granulocyte product may be a novel mediator of allergic inflammation, and perhaps a useful marker of basophil involvement (McEuen et al., 2001).
Basogranulin was found to have a very large size (5 x 106 d), and it is predicted that this will restrict the rate of diffusion of the protein. Tissue accumulated protein, i.e. protein that has left the circulation, is expected to be highly localised (McEuen et al., 2001).
Various other potential markers of anaphylaxis have been identified. Furthermore, some studies have focused on markers that could potentially predict the risk or severity of allergic reactions in different individuals.
Granzyme B (GzmB), derived from basophils, was suggested to be a novel mediator of allergic inflammation. The cytokine IL-3, located within basophil granules, was found to induce expression of GzmB, resulting in changes in basophil granule constituent composition. 18 hours following lung provocation in patients with allergic asthma, an increase in BAL fluid GzmB levels was identified. Furthermore, this correlated with rising levels of IL-13, another basophil granule cytokine. Thus it was suggested that GzmB may be a mediator of asthma, and levels may reflect the late phase reaction intensity (Tschopp et al., 2006).
In a study investigating factors that may predict patients at risk of anaphylaxis to peanuts and tree nuts, subjects with low serum angiotensin-converting enzyme (ACE) concentrations (<37.0 mmol/L) were almost 10 times more likely to develop severe pharyngeal oedema compared with other study subjects (Summers et al., 2008). ACE plays a role in the breakdown of bradykinin, a protein that may be involved in anaphylaxis. In a previous study, surges in bradykinin were found to occur in patients with hereditary and acquired angio-oedema during an attack. Furthermore, in a hypertensive patient receiving an ACE-inhibitor, high levels of bradykinin accompanied an attack of angio-oedema; levels were normal when the drug was withdrawn (Nussberger et al., 1998).
In another study, serum platelet activating factor (PAF) levels were found to be increased in patients with anaphylaxis compared to controls. Furthermore, levels appeared to correlate with the severity of anaphylaxis. PAF is a proinflammatory phospholipid. It is synthesised in mast cells, as well as several other cell types, and is degraded by the enzyme PAF acetyl hydrolase (PAF AH). Serum PAF AH activity was also measured; and this was found to be inversely correlated with PAF levels. Patients with anaphylaxis had lower PAF AH activity than controls (not significant), and the numbers of patients with low PAF AH activity was proportional to the severity of the reaction. Patients with fatal anaphylaxis to peanuts had significantly lower PAF AH activity than a range of control groups, which included children with mild peanut allergy, patients with non-fatal anaphylaxis, deaths from non-anaphylactic causes, and children with life-threatening and non life-threatening asthma. From these findings it was suggested that the severity of anaphylaxis may depend on the inactivation of PAF by PAF AH; less inactivation may result in more severe reactions (Vadas et al., 2008).
4.2 Study design
Assays to determine circulating levels of many of these mast cell and basophil products, including tryptase, chymase, carboxypeptidase, and basogranulin, have been or are being developed within the laboratory. This study will involve further development of these assays where necessary, as well as development of new assays, such as for the enzyme DPPI. These developed assays will then be assessed for their use in measuring products in human serum and saliva.
Samples have been and will be collected from well established cases of allergy and anaphylaxis, including cases that occurred in response to food. Additionally, blood, urine and saliva samples will be taken at various time points during controlled food challenges in food allergic patients. Measurements in samples from allergic patients will be compared with those from control subjects. The extent to which the concentrations of the measured products are associated with the risk and severity of reactions will be investigated. Ultimately it is hoped that a multiplex assay can be developed for those markers that are useful in diagnosing or predicting a severe allergic reaction.
4.3 Experimental work carried out so far
The first part of the project has involved the development of a sandwich ELISA that can be used for the detection of human DPPI. Monoclonal antibodies of the IgM isotype have previously been produced from mice against human DPPI, and these are being used to detect the enzyme in the assay. Currently, commercially available rabbit polyclonal anti-mouse antibodies are used as secondary antibodies. Human recombinant DPPI is being used for assay development purposes.
The assay was developed using culture supernatant containing the anti-DPPI antibodies, and conditions that produced a good dose-response curve (figure 1.), with little background, were determined. The protocol for this assay can be found in the appendix. The mouse antibodies were then purified using an immobilized protein L column. A protein assay was used to determine which elution fractions contained the purified antibodies, and thus at what time point they are eluted. The assay also provided information on the amount of antibody collected (figure 2.). The antibodies were tested for purity in a western blot (see appendix for protocols). The results showed one thick band at approximately 20 kDa and one faint band at approximately 50 kDa (figure 3.), fairly consistent with other western blots of IgM antibodies.
The purified antibodies were tested using the developed sandwich ELISA protocol; however did not produce a good dose-response curve, as had been seen previously. Furthermore, the background was relatively high. When tested using a direct ELISA, the purified antibodies showed more promising results. This suggested that they still had the potential for use in an assay, and thus conditions for a sandwich ELISA are being re-optimised.
Figure 2. Purified DPPI Antibody Protein Concentration Curve
PAF AH activity assay
A commercially available assay kit (Cayman Chemical) has been used to measure activity of the PAF-degrading enzyme PAF AH in serum samples from kiwi-allergic patients (see appendix for protocol). Prior to the assay, samples were concentrated to approximately ½ to ¼ of their original volumes using Amicon centrifuge concentrators, as recommended in the assay protocol. Serum samples were divided into three groups: those from patients with mild allergy; those from patients with oral allergy syndrome; and those from patients with severe allergy. The results were compared with those of control subjects.