Presently, allergy and allergic drug reactions are occuting more frequently in industrial countries, potentially leading to serious health problems. Around 10% of hospitalized patients have drug allergy and about 7 % of these patients require ambulatory care .The World Health Organization outline adverse drug reactions (ADRs) as a unhealthy and unintended response to a drug, that arise at a dose normally used in humans . The allergic drug reactions can cause different conditions such as angioedema, urticaria, rhinitis and more. The most common allergic symptoms are itchiness, swelling, breathing problems, headache, and a stuffy nose . These reactions can rapidly become life-threatening situations, especially if the event occurs during anaesthesia . Therefore, investigations are applied to understand the immunological mechanisms behind allergic drug reactions and to invent new detection methods.
Allergy is a common disease which can be caused by antigens such as food, drugs or venom. The mechanisms behind allergic reactions are well known. The two most common mechanisms are IgE mediated and non-IgE mediated allergy. IgE is a glycoprotein and its concentration in serum is the lowest of the five Ig subtypes. The immunoglobulins IgG, IgM, IgA, and IgD are also processed by B Lymphocytes . IgE has also the shortest half-life (around two hours), but it can be maintained on the surface of mast cells for several days . The level of IgE can be influence by genetic factors, race, immune status and environmental factors. There are two known receptors for IgE: low-affinity IgE receptor (FcεRII; CD23) which is expressed on the surface of B-cells and eosinophils, while the high affinity IgE receptor (FcεR1) is expressed on the surface of other hematopioietic cells and the high affinity IgE receptor (FcεR1). The density of human basophil FcεR1 expression correlates directly with serum IgE concentrations in parasitic infections, non-parasitic infections, and inflammatory diseases. The same was seen with the density of human mast cell FcεR1 concentration and the free IgE level in vitro [6, 7]. IgE level is involved in different types of disease such atopic diseases, atopic dermatitis, atopic asthma, and parasitic infections . The role of IgE in allergic inflammation is well known .
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Th2 lymphocytes secrete IL-4 which i induces antibody class-switching in B-cells to create antigen-specific IgE. FcεRI binding sites recognise high affinity IgE receptor on mast cell and basophil surfaces and bind to it . Allergen cross-links with the Fab region of IgE on the cell surface and induces degranulation. Mast cells and Basophils release inflammatory mediators including histamine and tryptase. In addition, they synthese and release new preformed lipid mediators and cytokines . B-cells require two signals for class-switching to occur; cytokines IL-4 and 13 send the first signal, followed by ligation of B cell CD 40 by T cell CD40L 
In an article by Averbeck et. al. allergic reactions are classified into four types; Immediate hypersensitivity reactions (type I), cytotoxic antibody reactions (type II), immune complex reactions (type III), and delayed-type hypersensitivity reactions (type IV) . Immediate hypersensitivity reactions are mostly caused by low-molecular weight allergen. It was shown that naïve CD4+ T cells play an important role in the development of type I reactions. type I reactions can occur in minutes (early response), but can also occur after 8 - 12 hours (delayed response). The key effector cells in early type I allergic reactions are mast cells due to its secretion of performed granules such as histamine and heparin . The Type I response is the most common in the general population . In some allergic reactions, the cell is destroyed after the antibody is bound on it. This type of reactions is called as antibody-dependant cell mediated cytotoxicity (ADCC) and belongs to category humoral cytotocix immune response . Drug-induced cytopenia or chronic urticaria belongs to the type II response. This reaction is mostly mediated by IgG and IgM and is a common reaction with drugs [1, 5]. Immune complex reactions are caused by antigen and antibodie interactions which form aggregates in vessels and tissues. These immulne complexes are recognised by Fcγ receptors on mast cells and other leukocytes and and induce activation of these cells . Type IV reactions are antiboidy-independant. Instead, antigen-specific effector T cells are activated and clinical symptoms appear with 24-48 hours. It is known that IL-1β and TNF α are important for type IV reactions .
Allergic drug reactions
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IgE antibodies and T cells drive most immune hypersensitivity reactions to drugs. IgE mediated reactions are the most elucidated of the reactions. In contrast, drug- induced allergy involving T-cells is still not well understood. These can be classified into two types: Type A and B. Type A reactions are defined as dose dependent and predictable. It also has the high ration on adverse drug reactions (ADRs) . Type B is independent and unpredictable. Hypersensitvity drug reactions are type B reactions and are marked as immunological mechanisms (IgE or non-IgE- mediated). Different clinical studies involving ADRs have been performed throughout the world. An 18-month American study of t731 identified reactions found that in 32.7% of cases an allergic reaction was involved. A French clinical study investigated anaphylaxis during general anaesthesia and found 789 cases, of which 518 had an immune response to neuromuscular blocking agents, antibiotics or other drugs. Thong et al. investigated the mortality of drug allergy and found out that 0.09 per 1000 patients hospitalised die due to an allergic reaction to drugs administered . A UK study by Pumphrey showed that from 1992-2001 the most people die on an anaphylactic shock triggered by medication. The group of Pen et al. shows that 8.4 per 100 000 people per year in the UK are affected by anaphylaxis which is triggered by oral drug administration. However, not only drug introduced anaphylaxis can lead to mortality. Stevens-Johnson syndrome (SJS) and Toxic epidermal necrolysis (TEN) also contribute to drug-related deaths .
The mechanisms of sensitization to drugs are divided into two steps; sensitization and secondary exposure. The sensitization step includes the first exposure to the drug and is mostly asymptomatic. Sensitization occurs when the drug specific IgE is produced. If the individual is then exposed a second time, an allergic reaction to the drug may occur. This can cause different syndromes including anaphylaxis, urticarial, and contact sensitivity. It was shown that eosinophilia is detected in blood or tissue at the time of a reaction. However, it is not known if eosinophilia alone is sufficient evidence for an allergic reaction. Drugs have mostly a low molecular weight and have to be bioactivated before they can lead to hypersensitivity reactions. There is currently a limited amount of diagnostic testing for drugs to determine their potential for inducing allergic reactions .
It has been shown that not all drugs bind to the major histocompatibility complex and induce an immune response. Some drugs bind directly to T-cell receptor which is expressed by T cells . This phenomenon is called (p-i) concept . Most drugs have to bind to carrier proteins to induce an allergic drug reaction; this is called the hapten-carrier effect. Only drugs with a molecular weight over 100 Dalton like beta- lactam can cross-link the IgE on the mast cell surface and cause a specific immune response . Studies showed that the female population are more affected by drug allergy than men . Penicilins, cephalosporins, and neuromuscular blocking agents can lead to an IgE-mediated drug hypersensitivity (Type I). The activation of mast cell then release different factors including histamine, leukotrienes, prostaglandins, and cytokines. These molecules elicit vasodilation, increase vascular permeability, enhance mucus production and bronchoconstriction, and contribute to eosinophil recruitment. Symptoms of this release are shock and severe cardiac complications . The detection of drug hypersensitivity is mostly done with by considering clinical histories, skin-prick test, patch test, and a few in vitro tests. The little information about drug hypersensitivity has to be careful investigated because the pathogenic mechanism or even the responsibility of the drug itself has not been demonstrated until now .
Anaphylaxis is described as an acute-onset and fatal systemic allergic reaction. The development of optimal risk reduction strategies and prevention of recurrence is becoming vital and understanding effector mechanisms is required for their development. The rate of anaphylaxis cases is unknown but it seems to be rising. Under-diagnosis, under-reporting and variation of case definitions complicate the recording of anaphylactic events. It was shown that in the 1980s, 21people per 100,000 were reported to have suffered from anaphylaxis. This was increased to 49.8 per 100,000 in the 1990s. 70 per 100,000 were under 19 years old [11, 12]. Studies show that under the age of 15 years old, anaphylactic events are more common in males than in females. In contrast, after the age of 15, more females have an anaphylactic reaction [11, 13]. Different trigger factors can cause anaphylaxis, and studies have shown that food induced anaphylaxis is more characteristic for young adults. In contrast to food, insect stings, diagnostic agents, and medications affect more middle-aged and older adults .
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The anaphylaxis mechanisms are divided in immunologic mechanisms or non-immunologic mechanisms. IgE and the high-affinity IgE receptor on mast cells or basophils drive the most anaphylactic reactions. It is known that IgE has an important role in anaphylaxis. Furthermore, IgE is involved in anaphylaxis intensity but also in sensitizing, activation and mediator release. IgE also enhances expression of FcεRI on mast cells and basophils . Peanuts, tree nuts, shellfish, fish, milk, and egg are the most common food antigens which lead to anaphylaxis [11, 13]. Different types of medication can cause anaphylaxis such as β-lactam, aspirin, ibuprofen, neuromuscular blocking agents [11, 13-16]. In addition, oversulfated chondroitin sulphate contaminated heparin and folic acid which are in vitamins and supplements can trigger anaphylaxis.
The tyrosine kinases and calcium influx in mast cells and basophils are activated and release immediately different granuleassociated preformed mediators including histamine, tryptase, carboxypeptidase A3, chymase, and proteoglycans . In both mechanisms (immunologic and nonimmunologic) mast cells and basophils were activated and induced an allergic reaction. The nonimmunologic mechanism is not yet fully understood. It includes exercise, cold air, coldwater exposure, radiation or ethanol . It seems that not only one mechanism is responsible for an anaphylactic shock. The balance of positive and negative intracellular molecular factors affect the mast cell response . It is known that the stem cell factor (SCF), especially the Kit receptor, is important in IgE/antigen-induced mast cell degranulation and cytokine production. In addition, it has been shown that the inhibitory sialic acid-binding immunoglobulin-like lectins are located on human mast cells . The involvement of genetic factors in human anaphylaxis should be considered, however, studies showed that are only a few known genes involved .
Simons et. al. has drawn attention to the fact that it also give other immunologic mechanisms which causes anaphylaxis but do not involve IgE. One of it is IgG antigen complexes which can also lead to an anaphylaxis shock. Tsujimura et. al. has expressed a similar view and showed that basophil play a role in IgG mediated anaphylaxis .
At the moment, the clinical diagnosis is made on patient history and physical examination. The clinical diagnosis can be supported by laboratory testing which measures plasma histamine and trytase concentrations in serum or plasma. The timing is important for the tests. Histamine needs to be tested in blood samples between 15 and 60 minutes form event, tryptase has to be tested in 15 to 180 minutes from event. However, not every time the tryptase or histamine levels are elevated. food-triggered anaphylaxis showed no rise in tryptase concentration. Therefore, other mast cell or basophil activation markers should be measured [4, 11, 13, 18]. After an anaphylactic event, additional tests should be done to minimize risks for future anaphylaxis. Injection of Epinephrine is used in anaphylaxis cases. Next to ephinephrine, H1-antihistamines and inhaled β2 -adrenergic can be also used in anaphylaxis events [11, 13].
The number of cases of anaphylaxis has increased over the last two decades, and the most common factors are foods, medications and insect stings. In some rare anaphylaxis cases, no factor exists (confirmation with skin testing or IgE test) and the possibility of mastocytosis or a clonal mast cell disorder must be considered. In emergencies, epinephrine autoinjectors must be available. In addition, the individual has to know when and how to use it. A personal emergency action plan and up-to date medical identification has to be available. Anaphylaxis was defined as a serious allergic reaction which happens rapidly and can lead to death . Different organ which are affected by anaphylaxis; skin (90% of episodes, respiratory tract (70%), gastrointestinal tract (30%-45%), cardiovascular system (10% to 45%), and central nervous system (CNS: 10%-15%) . Asthma, COPD, mastocytosis, nonselctive β-blockers, or stinging insect species increase the clinical risk of anaphylaxis . The heart is an organ which may be particularly affected. Mast cells are located in the myocardium and in the intima of the coronary arteries. Anaphylaxis can uncover subclinical coronary artery disease 
Mast cell and Basophil
Mast cells are mainly located in skin and mucosal tissues. In addition, they are also found in lungs and connective tissue of asthmatic individuals. Both share morphological and functional similarities such as same casophilic granules, expression of αβγ2 form of FcεRI . Both cell types play a central role in inflammatory and immediate allergic reactions .
Mast cells are based in blood vessels and at epithelial surfaces and play a major role in diseases of immediate hypersensitivity and of mastocytosis but also in responses to pathogens, autoimmune diseases, fibrosis and wound healing . They are produced in the bone marrow. The maturation is influenced by stem cell factor binding to the receptor c-kit and by other cytokines such as interleukin. SCF receptor which belongs to the c-kit is an important factor in the embryonic development. SCF is also responsible for the mast cell adhesion, migration, proliferation, survival and release of histamine and tryptase. Mast cells contain the majority of the body's histamine. Mast cells are long-lived, surviving for months or even years in the tissue .
The extracellular release of mast cells mediator and exocytosis occur by organelles which are located in the mast cells cytoplasm. In an article by Amin et. al. three possible reasons of exocytosis and extracellular release are possible. Chemical substances, such as toxins, venoms, and proteases are one of the possible reasons for release. Secondly, endogenous mediators, including tissue proteases, cationic proteins derived from eosinophils and neutrophils can induce release . Immune mechanisms can potentially lead to exocytosis and extracellular release that may be IgE-dependent or IgE-independent. IgE-dependent degranulation is a consequence of an antigen binding. During allergic response, the IgE is secreted from B-cells and becomes attached to mast cells though binding with the high-affinity IgE receptor on the surface of mast cells. A subsequent exposure to the same allergen cross-links the cell-bound IgE and triggers the release of preformed prostaglandins, histamines and cytokines. The Ca2+ influx is increased and promotes the mast cells degranulation; ionophores that increase cytoplasmic Ca2+ also promote degranulation, whereas agents who deplete cytoplasmic Ca2+ suppress degranulation. IgE-independent degranulation is also caused by antigen. However, activation of mast cells occurs directly without pre-sensitisation .
Two types of mast cells are known; mucosal and connective tissue mast cells (MCTC). The MCTC mast cells express tryptase and chymase . MCT cells are present inside of the mucosa of the respiratory and gastrointestinal tracts and induce mucosal inflammation. MCTC cells are present in connective tissues such as the dermis, submucosa of the gastrointestinal tract, heart, conjunctivae, and perivascular tissues .
Mast cell produced mediators are divided into preformed mediators, newly synthesized lipid mediators and cytokines/chemokines. TNF-α is a preformed mediator and newly synthesized molecule but is also a major cytokine. In addition to TNF-α,histamine, serine proteases (tryptase and chymase), carboxypeptidase A, and proteoglycans (stored in cytoplasmic granules) belongs also to the group of preformed mediators . MCT cells contain tryptase as neutral proteases and tryptase, chymase, cathepsin G, and carboxypeptidase in MCTC cells. Human cells have α-andβ-tryptase . Human mast cells also generate IL-4,IL-5 and IL-6 and also several neutral protease including tryptase and chymase. Thus, mast cells are key players in host defence, with a role in immune surveillance, phagocytosis, and immune activation .
Figure : Products of Mast cell after cell activation .
Basophils are involved in artificial cantharidin blisters or spontaneous bullous lesions in atopic dermatitis (AD) and other eczematous diseases . Basophils are produced in bone marrow. They differentiate and mature there and develop from CD34+ progenitors . The half-life of basophil is a few days and they express cytokine receptor such as IL-3R, IL-5R, and GM-CSFR and chemokine receptors including CCR2 and CCR3. Basophils are also able to infiltrate inflamed tissues, especially in the skin disease atopic dermatitis and the airway with respiratory allergies . Basophil expresses the FcεR1, secrete Th2 cytokines, metachromatic staining and release of histamine after activation . A major characteristic of basophils is their quick and strong expression of IL-4 and IL-13 and they are also involved in allergic responses and immune regulation . As well as producing effector cytokines, basophils secrete pre-stored and newly sensitized pro-inflammatory mediators such as histamine and VEGF . It was also shown that basophils have a small proliferative capacity and express a variety of cytokine receptors (e.g. IL-3R,IL-5R, and GM-CSFR), chemokine receptors ( CCR2 and CCR3), complement receptors (CD11b, CD11c, CD35, and CD88), prostaglandin receptors ( CRTH2), immunoglobulin Fc receptors (FcεRI and FCγRIIB) and TLRs .
Figure : Products of Basophil after cell activation .
Resarch by Tsujimura et. al. indicates that basophils are involved in IgG1-mediated systemic anaphylaxis. On base of this result it was suggest that basophils are infiltrate into the skin tissue when the allergic responses of mast cells are not sufficient for the antigen elimination . If this is the case other inflammatory cells such as eosinophils and neutrophils are addicted. This finding leads to the suggestion that it might give to major pathways which causes systemic anaphylaxis. One involves basophils, IgG, IgG receptor and PAF and another way includes mast cells, IgE, FcεRI and histamine .
In addition, basophils play a critical role in the development of IgE-mediated chronic allergic reactions by functioning as initiator cells. It was also shown that the depletion of basophils leads to an almost complete abrogation of skin inflammation IGE-CAI . Basophils are responsible for the differentiation of CD4 T cells to a Th2 phenotype. They are also involved in giant papillae disease. It is also shown that a high number of basophils are located in patients with atopic asthma .
Histamine is the best investigated inflammatory mediator and between 3-5 pg is present in each human mast cells . Histamine has a half-life of approximately 1 minute. Histamine N-methyltransferase degraded histamine to tele-methyl histamine and also diamine ocidase degraded it to immidazole acetaldehyde. Sodium ions effect the dissociation of granule with histamine from the proteoglycans in the extracellular fluid. Histamine affects different areas such as smooth muscle, endothelial cells, nerve endings, and mucous secretion . Histamine is released when the body detects toxic substances and it will be also released when the mast cells detect injury. It causes nearby blood vessels to dilate allowing more blood to reach the site of the injury or infection . It has been shown that injection of histamine induces an increase in heart rate, increased skin temperature, flushing, itching, bronchospasm, headache and fall of blood pressure. Histamine is related to the urticaria degree. Additionally, histamine levels were increased in humans with angioedema, and tachycardia. Studies suggested that a lower histamine level correlated to milder symptoms after an allergic drug reaction .
Tryptase is related to histamine and might be a stimulator for histamine release. It was found that tryptase stimulates histamine release in tonsil tissue .In addition, intracellular cyclic AMP level can control histamine release .
In addition to histamine, tryptase is set free after degranulation of mast cells . The tryptase gene placed on the chromosome 16 and has four subunits with active enzyme sites . Tryptase belongs ia a serine peptidase and has enzymatic mechanisms . In human mast cells there exist four tryptase genes (TPSG1, TBSB2, TPSAB1, and TPSD1) which are divided in two general groups: membrane anchored and soluble. It is known that the membrane-anchored γ tryptase has inflammatory potential. In contrast, soluble human β trypase has an extracellular effect. It also may be involved in pathogenic inflammatory episodes and be present in basophils . In addition, β trypase plays an important role in asthma [26, 27]. α-tryptase is not stored in mast cells like β trypase and it is suggest that α tryptase is not involved in the pathophysiology of anaphylaxis. Next to α tryptase, δ tryptase showed also catalytic domain defects which leads to reduce of activation. Tryptase can lead to inflammation, matric destruction and tissue remodelling. Therefore, different mechanisms are applied such as damaging pocagulant, matrix, growth and more .
It is known that tryptase digests fibrinogen, fibronectin, pro-urokinase, pro-matrix metalloprotease-3, protease activated receptor-2 and complement component C3 in vitro. However, the tryptase function in vivo is unknown. Tryptase can also mobilise fibroblast, promote accumulations of inflammatory cells, and potentiate histamine-induced airway bronchoconstriction . Studies showed that Tryptase also activates protease activated receptors (PAR). PAR receptors are responsible for the nitric oxide- mediated vascular relaxation in the cardiovascular system. In addition, PAR -2 receptors are located on sensory neurones and are important for the inflammatory process and induced HUVEC proliferation [27, 30].
Tryptase is increased in an anaphylactic event and can be detected one hour after the event. However, the total tryptase concentration is not always corresponding to the measured histamine concentration. It was shown that Histamine was released from mast cells after incubation with tryptase [24, 30]. To support the diagnosis of anaphylaxis, mature β trypase level should be measured next to total tryptase . Anaphylaxis to parenteral agents (drug and insect venom) is associated with increased tryptase level, whereas anaphylaxis to oral agents, particularly food, is often not accompanied by elevated tryptase levels in the serum. APC366 is known as tryptase inhibitor and reduced histamine release [24, 28]. It also showed to reduce late bronchoconstriction response after antigen exposure .
McEuen et al. discovered the antigen basogranulin. The group used a basophil-specific antibody BB1 to detect the antigen basogranulin. It also suggests that basogranualin is a novel basophil-specific protein and involved in allergic inflammation . Basogranualin showed to be released with histamine and react to FcεR I- related and unrelated stimulation .Mochizuki et notes that basogranulin after release is not disperses as freely as histamine and this could be explanation of the lower level of basogranualin release . Therefore, it could be a marker for basophil activation, cell activation and quantification of basophils in chronic myeloid leukemia [31, 33]. The work of McEuen et al. indicates that basogranualin are more linked to the matrix than to the membrane .
Dipeptidyl Peptidase 1
Dipeptidyl peptidase 1 (DPP1) belongs to the papain family of protease and is a lysosomal cysteine dipeptidyl aminoppeptidase. DPP1 is also known as cathepsin C and has four identical subunits. It cuts of the dipeptide moieties from the N-terminal of target proteins. Increased DPP1 expression was found in lung, kidney, liver and spleen . It was found that DPP1 is responsible for the process of mast cell tryptase . In mice, DPP1 is involved in activation of chymase but not in tryptase [28, 35]. In addition, it is also important for other neutrophil-derived serine proteases which can decrease various extracellular matrix components. This causes tissue damage and chronic inflammation .
Studies have shown the involvement of DPP1 in different diseases such as sepsis, arthritis and other inflammatory disorders. In addition, knockout of DPP1 leads to the Papillion-Lefevre syndrome which is a gene mutation . Different DPP1 inhibitors are known. All inhibitors are small molecules and have an electrophilic 'warhead'. Inhibition of DPP1 showed a reduce in the level of neutrophil elastase, cathepsin G and proteinase-3 .
Carboxypeptidase is in the mast cells located and belongs to mast cell specific proteases. Human mast cell carboxypeptidase (HMC-CP) is a member of the protein family of zinx-containing carboxypeptidases . It is not known if basophils have a carboxypeptidase gene until now only in mast cells and mast cell like cell lines have the carboxypeptisase gene expression . It was shown that HMC-CP is located in MCTC type and expressed in granules which have tryptase and chymase [28, 38]. In addition, studies suggest that carboxypetpidase are related to chymase .Next to chymase, heparin plays also important role for carboxypetpidase. It was shown that heparin is necessary for mast cell carboxypeptidase A (MC-CPA) storage. MC-CPA cannot store if the serglycin core protein is missing in mast cells. It is shown that MC-CPA are involved in host defence against parasites .
MC-CPA is also involved in the processing of angiotensin I like chymase. It cleaves the carboyterminal His-Leu bond of antiogtensin I  and it could be that MC-CPA process the substrate protein or peptide after processing by chymase . Studies showed that carboxypeptidase A also hydrolysed des-Arg bradykinin . In vitro experiments showed that MC-CPA cut several proteins and peptides on the C-terminal . It was also shown that MC-CPA is involved in theproliferation of fibroblasts. In addition, ET-1 induced damage is avoided by MC-CPA and this could lead to the implication that MC-CPA has a role in regulating sepsis . Potato carboxypeptidase inhibitor (PCI) is the most used inhibitor for MC-CPA. PCI is used in studies which investigate different activities of MC proteases. SR48692, ethylenediaminetetraacetic acid, 1,10-phenanthroline, and 8-hydroxyquinoline are also possible MC-CPA inhibitors .
Chymase belong also to the family serine peptidase like tryptase and is stored in mast cells but not in basophils [26, 39]. One gene is located in humans. Chymase digest their targets mostly after aromatic residues (Phe and Tyr) . It is also known that chymase can change the bioavailability of cytokines such as activation of interleukin-1β precursor, digestion of IL4. In addition, it was shown in mamals that chymase raise microvasuclar permeability and increase the maturation of inflammatory cells . It was found that mMCP-4 (mouse mast cell protease) had highest similarity to the human chymase. Studies with special designed mMCP-4 knock out mouse showed that chymase maybe increase the IgE and IgG level. Chymase can also play a role in regulation of bacterial infections . It also digests endogenous and exogenous targets like birch pollen profilin which is an allergen, angiotensin I which is an endogenous peptide, hepatocyte growth factor and endogenous immune proteins such as cytokines IL-6 and IL-13 . In knockout models, mMCP-4 was investigated for the influence of chymase in the extra cellular matrix. It was found that chymase induces the pro-matrix metalloproteinase 9 which leads to a reduction 0of the extra/cellular matrix.
A chymase inhibitor is available and has showen good results, especially in abdominal aortic aneurysm injuries in which a decrease was detected due to tye involvement of chymase inhibitors .
Next to cymase, basophils, histamine, tryptase, carboxypetidase, and DPP1, other mast cell and basophils products are released after activation. Heparin belongs to proteoglycans which are set free after activation. It was shown that heparin can lead to contraction of smooth muscle and rise vascular permeability . Cathepsin G is also a protease and is released from mast cells and basophils. It is also expressed in some leukocytes such as neutrophils, monocytes and dendritic cells. In mice it was found that cathepsin G might be involved in defence against bacteria and fungi. In addition to chymase, cathepsin G can also convert angiotensin I to II. In contrast to chymase, cathepsin G can cleave tryptic and chymotryptic substrates and has a decreased chymotryptic activity than chymase .
Another important group of mediators are cytokines. They are responsible for the induction of the innate immune response, process of antibodies but also play a role in many other immune response . In mast cells, TNF-α is a major cytokine. Endothelial and epithelial adhesion molecule expression is increased by TNF-α. Eosinophil development and survival are under control of IL-3, GM-CSF, and IL-5. It is also known that mast cells release some chemokines such as CXCL8 and CCL3. Basophils release IL-4, IL-13 and CD40L which could be involved in the amplification of IgE synthesis .
Basophils and mast cells also release lipid mediators and other enzymes such as β-hexosaminidase which can be measured after cell challenge.
Diagnosis of allergic drug reactions
The detection of allergic reactions to medications is limited. A few tests are available to measure drug-specific antibodies, drug-specific T lymphocytes or mediator of basophil/mast cells . Histamine, tryptase and IgE were measured to identify drug allergy . Histamine and tryptase measurement is general used to detect anaphylaxis.
Oral food challenge (OFC) can be avoid because of the patient's age, clinical history or physical condition. This allergy test is mostly for food allergy . Skin test and intradermal test should be carried out after an allergic reaction. Positive and negative controls should be always carried out . The trigger agent is put directly to the skin and picked with a lancet under the skin surface. The result can be interpreted and compared with the controls after 15-20 min . For intradermal test, the injection of 0.02 - 0.05 mL of diluted medication is carried out . In some cases patch tests are performed. Torres et al. has drawn attention to the fact that in the diagnosis of immediate reactions to beta lactams are not clear . Some medication allergens are not available for skin testing and the reaction has to be IgE mediated . Special when the antigen is an unidentified breakdown product or a metabolite of a medication. Β-lactam and a rare other medications are available for skin test . However, skin test is a safe procedure and can be used to detect drug allergy.
Histamine and total tryptase can be measured of the biomarkers of mast cell and basophil in clinical laboratories [11, 13]. The measurement of the histamine concentration should be taken place after 15-60 minutes when the first symptoms are appeared. In contrast to histamine, serum total tryptase levels can be still detected after 3 hours . However, tryptase levels are rarely raised in food-introduced anaphylaxis . It also an immunoCAP test available which tests in vitro the serum of the patient for allergen-specific IgE . This test is very sensitive, easy and automated. However, the result does not give always a clear diagnosis about the allergy special in food allergy cases .
Possible mast cell activation products are existed such as carboxypeptidase A3, chymase, plate-let-activating factor, and cytokines. Therefore, it is an aim to develop timeless, sensitive, and specific laboratory tests which can confirm diagnosis of anaphylaxis .
PhD project: Allergic drug reactions and the identification of those at risk of severe symptoms: Mast cells and basophils activation as key cellular mechanisms
We hypothesise that the level of mast cell and basophil mediators are increased in serum after an allergic drug reaction. The project investigates the non IgE mediated and IgE mediated allergic drug reaction of different drugs by assignment of human mast cell line LAD2 cell and already available ELISA assays for measurement of allergic markers .
An in vitro cell assay will be developed to measure different allergic markers after drug challenge. The developed assay will be used to investigate the non IgE mediated and IgE mediated drug allergy. Patient serum will be used to sensitise the cells and to challenge with different drugs and drug concentrations. The serum has been and will be collect from the drug allergy skin test centre.
This assay can help to assess drug allergy without using skin testing which can causes allergic reaction or life threating reactions such as anaphylaxis. A laboratory test could avoid this. Blood could be taken from the patient without exposure to any danger.
Experimental work carried out so far
The human mast cell line LAD2 (generated by A.S. Kirshenbaum et al. ) was used to set up a drug challenge experiment. The challenges were carried out in triplicates and a sterile, V-bottomed, 96 well polystyrene cell culture microplate was used. The β-hexosaminidase assay was carried out with Chlorhexidine, Paracetamol and ampicillin which belongs to beta-lactam antibiotics group. 1 x 10-6 to 1 x 10-3 medication concentration was tested. Calcium ionophore A23187 which raises the intracellular calcium flux and activates degranulation was used as a positive control. In each well, 1:100 dilution (90 μL cells in tyrode buffer plus 10 μL of diluted medication/calcium ionophore) was applied. The LAD2 cells were challenged for 45 minutes at 37 °C. 30 μL of supernatant was transferred and incubated with substrate solution at 37 °C for one hour (1.3 mg/mL in 0.1 M Na2HPO4). The development was stopped with 2 M glycine and the plate was read at 410 nm.
The paracetamol, chlorhexidine, ampicillin and calcium ionophore A23187 was diluted in tyrode buffer which included the metabolic inhibitors 2-deoxy-D- glucose (10mM) and antimycin A. Same conditions and dilution like above described was applied to this inhibition assay.
It was investigate the non-IgE mediated reaction of ampicillin, chlorhexidine and paracetamol.
Cell lysate assay
It was first carried out a LAD 2 cell lysate to find out the correct concentration of cells per mL for the β-hexosaminidase assay. The different cell concentrations were lysate with 1% Triton-X.
Figure :LAD2 cell lysate for examination the cell number per mL which had the maximum of total release
The curve showed a maximum total release of around 3.5 at 0.5x106, 1x106, and 2x106 cells. This result suggests that 0.5 x 106 cells per ml are enough cells to reach the highest total release.
In vitro β-hexosaminidase release was not found after challenging with paracetamol and ampicillin. In contrast to paracetamol and ampicillin, chlorhexidine showed a net release around 10 % at the concentration of 0.3 mM and a net release of approximately 30 % at the concentration of 1 mM.
Figure : β-hexoaminidase release from LAD2 cells induced by paracetamol (blue line) chlorhexidine (black line) and ampicillin (red line).The concentration of 1,0.3,0.1,0.03,0.01,0.003, and 0.001 mM was applied. Data are present 4 experiments.
The result of chlorhexidine raises the question if the solvent (70% isopropyl alcohol) of chlorhexidine accounts for the release. It was carried out an experiment with investigated the effect of 70 % isopropyl alcohol to LAD2 cells. The70 % isopropyl alcohol dilution was prepared after the chlorhexidine dilution and had the final molar concentrations of 2, 0.6, 0.02, 0.06, and 0.02 mM. The same experiment condition was carried out like before.
Figure : β-hexoaminidase release from LAD2 cells induced by 70% is propyl alcohol. It was used the concentration of 2,0.6.0.02,0.06, and 0.02 mM.
70 % isopropyl alcohol showed a release under 5% on the lowest concentration. It could be that 70 % isopropyl alcohol stimulates LAD2 so that it promotes a release of β-hexosaminidase. However, it is more like that it could be subject of photometric accuracy.
Calcium ionophore A23187 as positive control showed a constantly curve with the highest net release at 1 μM.
Figure : β-hexoaminidase release from LAD2 cells induced by calcium ionophore. It was used the concentration of 1, 0.1, and 0.01 μM.
β-hexosaminidase assay with inhibitor 2-deoxy-D-glucose and antimycin A
The highest concentration of chlorhexidine which is used in skin testing is less than 1mM. Therefore, the cytotoxic effect of chlorhexidine on the LAD2 cells was investigated to prove if the highest concentrations of chlorhexidine (1 mM and 0.3 mM) are toxic to the cells.
It was found that 1 mM and 0.3 mM of chlorhexidine have a higher net per centage release than without metabolic inhibitors. The result suggests that with these concentrations of chlorhexidine are toxic to LAD 2 cells.
Figure : β-hexoaminidase release from LAD2 cells induced by chlorhexidine in the pressence (-- ) or (---) of metabolic inhibitors (2-deoxy-D-glucose 10 mM and antimycin A)
Calcium ionophore A23187 showed a lower curve with the metabolic inhibitors than without. These results suggest that calcium ionophore A23187 has almost no cytotoxic effect to the cells.
Figure :β-hexoaminidase release from LAD2 cells induced by calcium ionophore in the pressence ( ) or (---) of metabolic inhibitors (2-deoxy-D-glucose 10 mM and antimycin A)
New rat mast cell line (RS-ALT8 ) is used for cell challenging with medications such as β-lactam antibiotics, neuromuscular blocking agents, and chlorhexidine. The cells will be sanitises with patient serum and challenged with different drug concentration. The supernatant of the cell challenge will be used to measure different mast cell biomarkers. In addition, the RS-ALT8 cells contain an IgE crosslinking-induced luciferase expression . The result could be helpful in understanding the non-IgE and IgE mediated pathway. In addition, it might be a possibility for a new in vito technique which could give quick and accurate diagnosis of allergy.
The link between the clinical picture of allergy and concentration of the biomarkers of mast cell and basophil will be investigated. In addition, the results of the in vitro challenge and the outcome of the clinical diagnostics will be compared. These results could give a brighter picture of risk factors.