Patent Application
20080261215
The present invention relates to methods for monitoring the efficacy of an allergen immunotherapy.
An allergy is an immune malfunction wherein an individual is hypersensitised to react immunologically to typically per se harmless substances called allergens. The principal antibody which is involved in allergic reactions is IgE. Every individual has different IgE antibodies and each allergic substance stimulates production of its own specific IgE. An IgE antibody binding a defined allergen will therefore react only against said allergen. The constant region (Fc region) of IgE is able to bind to specific receptors of cells, which are able to release histamine or other inflammatory mediators, cytokines and/or proteases into the surrounding tissue. Histamine releasing cells are mainly mast and basophilic cells. The release of histamine is initiated when cell-bound IgE is contacted and cross-linked by the allergen.
Especially histamine causes the main allergic reactions. Histamine released in the nose, eyes, and sinuses, for example, stimulates sneezing, a runny nose, and itchy eyes; released in the lungs it causes narrowing and swelling of the lining of the airways and the secretion of thick mucus; in the skin, rashes and hives; and in the digestive system, stomach cramps and diarrhea.
Typical allergens are derived from plant pollens, like rye grass, ragweed, timothy grass and birch trees pollens, mold spores, drugs, like penicillins, sulfonamides, salicylates and local anesthetics, foods, like nuts, seafood, egg, peas, beans, peanuts and other legumes, milk, insect products, like bee-sting venom, wasp sting venom, cockroach calyx and dust mites, and animal hair and dander.
There exists a number of medical treatments for allergies. Mainly three methods are regularly used in medical practice: chemotherapy, immunotherapy and alternative medical methods.
In chemotherapy antagonistic drugs are used to block the action of allergic mediators, preventing activation of cells and degranulation processes. They include antihistamines, cortisone, adrenalin (epinephrine), theophylline and Cromolyn sodium. These drugs help alleviate the symptoms of allergy but play little role in chronic alleviation of the disorder. They can play an imperative role in the acute recovery of someone suffering from anaphylaxis.
In alternative medicine, a number of treatments are considered effective by practitioners in the treatment of allergies, particularly traditional Chinese medicine. However, none of these have been backed up by good quality evidence.
The most promising therapy form is probably immunotherapy. In the course of an immunotherapy where an individual is gradually vaccinated against progressively larger doses of the allergen in question. This can either reduce the severity or eliminate hypersensitivity altogether. Alternatively, monoclonal anti-IgE antibodies may be injected. These antibodies bind to free IgE signalling such sources for destruction. They do not bind to IgE already bound to the Fc receptor on basophils and mast cells as this would stimulate the allergic inflammatory response.
The proteins and glycoproteins used in allergen immunotherapy are usually extracted from materials such as pollens, molds, pelt and insect venoms. Based on the clinical evaluation, repeated subcutaneous injections of a solution of the disease-causing allergen or a derivative thereof are done once or twice a week in increasing doses until a maintenance dose is reached. This maintenance dose is then injected every 2 to 4 weeks.
In order to accomplish an immunotherapy in a successful manner monitoring of the progress of said therapy has to be performed.
For instance, in Wantke et al. (Clin Exp Allergy 23 (1993) 992-995) a method for monitoring an immunotherapy for allergic rhinoconjunctivitis is disclosed. Therein the authors analysed the spontaneous histamine release, i.e., the release without addition of allergen, in patients prior and after the immunotherapy and showed that the histamine release into the blood after exposure to the allergen was significantly reduced after four months of treatment. However, this method cannot be used to assess changes in sensitivity towards a particular allergen and specific efficacy of the treatment.
Stephan et al. (Allergy 44 (1989) 453-459) investigated the effect of bee venom immunotherapy over a period of more than five years by analysing the allergen induced histamine release in whole blood. However, the authors of this study did not correlate the results of histamine release with a clinical parameter, e.g., skin sensitivity and hence no data were shown which would justify to use the assay to measure and reflect clinical sensitivity to a given allergen. Furthermore, no samples obtained before and after treatment were compared among each other.
Yuta et al. (Arerugi 51 (2002) 634-648) studied the histamine release from basophilic cells to evaluate an immunotherapy of allergic rhinitis. The authors analysed samples at the beginning of the treatment and at six months after starting immunotherapy and could show the positive effect of the therapy. In this article samples obtained before and after treatment were analysed and the authors could only show that the rush protocol leads to an exhaustion of the cells but does not show a reduction of histamine release. In this context it should be noted that rush immunotherapy works already before “blocking antibodies” are induced by immunotherapy, i.e., sometimes after hours and few days. This may be interpreted as an exhaustion of cells. However, the assessment of the effect of blocking antibodies which appear after several weeks of treatment is important. Hence an assay where the IgG antibodies are still present, e.g. whole blood, has to be used. In contrast thereto, in Yuta et al. the cells were washed and hence the interference of blocking IgG could not be measured.
In addition to histamine release also other methods for the assessment of basophil and mast cell activation are known, which include measuring the release of leutrienes (Van Rooyen & Anderson, R. J. Immunol. Methods 2004, 288, 1-7), tryptase (Taira M et al., J. Asthma 2002, 39, 315-322) and other mast cell or basophil products which are released upon allergen-specific activation of the mast cells and basophils. Furthermore also the upregulation of activation markers such as CD63 and CD203c resulting from the exposure of an individual to an allergen can be measured by flow cytometry (Hauswirth A. W., et al. J. Allergy Clin. Immunol. 2002, 110, 102-109).
Therefore it is an object of the present invention to provide in vitro means and methods to monitor as close as possible clinical efficacy and the progress of an allergen immunotherapy and allergen sensitivity of an individual.
Therefore the present invention provides a method for evaluating the allergen sensitivity of an individual and/or the clinical efficacy of an allergen immunotherapy comprising the steps:
The evaluation of the allergen sensitivity of an individual and/or the clinical efficacy as well as the progress of an allergen immunotherapy is important in order to guarantee an effective treatment, e.g. by changing the dose and/or time intervals of the administered allergen. Therefore a reliable method to monitor the immunotherapy is required which directly reflects the sensitivity of an individual for a certain type of allergen prion and in the course of on immunotherapy. The measurement of the amount of IgE binding specifically to an allergen turned out to be not suited to determine the degree of sensitisation of an individual for a certain type of allergen, since there is no direct correlation between the amount of IgE present in an individual and the mediator release from mast and basophilic cells. Therefore the release of mediator of a sample of an individual comprising mediator releasing cells is preferred. It was surprisingly found that the method according to the present invention gave comparable, if not identical, results as the traditionally used skin sensitivity test.
The samples provided by an individual are preferably contacted with the same allergen, which is used for immunotherapy. However, it is also possible to perform the immunotherapy with an allergen extract and to monitor said therapy with substantially purified (“pure”) allergens.
Of course the method according to the present invention may also be used to monitor the progress of an allergen immunotherapy by determining the allergen sensitivity of an individual in the course of the therapy.
“Allergens” according to the present invention are molecules or mixtures of molecules able to induce the production of specific antibodies (IgE) which are responsible to trigger mediator release of a mediator releasing cell and to cause consequently allergic effects in the individual. Of course, “allergens” are also capable to induce the production of antibodies other than IgE (e.g. IgG). However, the allergens used in the method according to the present invention are preferably purified, i.e. the allergens consist substantially of one single allergen molecule, whereby the degree of purity exceeds 90% (w/w), preferably 95% (w/w), most preferably 99% (w/w). Due to the use of substantially purified or isolated allergens it is possible to determine and to dose in a reproducible manner the amount of allergen used in immunotherapy as well as used in a method according to the present invention. In contrast thereto allergen extracts contain varying concentrations of the specific allergen, depending on the specific purification conditions. Furthermore allergen extracts may also contain more than one allergen, which may be present in the extract in different concentrations (the amount of the allergen of interest is not definable in an accurate manner) and may further provoke cross reactions (see for instance Marth K et al. (2004) J. Allergy Clin. Immunol. 113: 470-474; Marth K et al. (2004) XXIII EAACI congress abstract book 597: 181; Akkerdaas H J et al. (2003) Arb. Paul Ehrlich Inst. Bundesamt Sera Impfstoffe Frankf. a. M. 94: 87-95). In addition, allergen extracts may contain contaminations or substance which may influence the stability of the extract. This problem can also be avoided by using substantially purified or “pure” allergens.
The term “derivative” allergen as used herein refers to modified (deleted, point mutated, truncated etc.) allergens which still exhibit the same antigenic and IgE binding properties as the native allergen from which they are derived from.
According to a preferred embodiment of the present invention the mediators are selected from the group consisting of histamine, tryptase, prostaglandins, leukotrienes, especially cysteinyl leukotrienes, eosinophil cationic protein, cytokines, like interleukins (IL), especially IL-2R, CD63, CD203c and combinations thereof.
The allergic response of an individual after the exposure of said individual to an allergen is primarily caused by the release of mediators by mast cells. These mediators produce the early symptoms of an allergic reaction (e.g. sneezing, itching) and stimulate the production and infiltration into local tissue of circulating leukocytes (e.g. eosinophils). The mediators can be released from the cells by degranulation (histamine and proteases) or after neosynthesis of said mediators (Quraishi S. A. et al., JAOA Supplement 5, 104:S7-S15). According to the present invention also activation markers—besides mediators—can be determined (e.g. Yoshimura C., et al., (2002) J Allergy Clin Immunol. 109:817-23).
The sample is blood or fractions thereof (e.g. plasma, serum), connective tissue, nasal, bronchial, skin or gut biopsy material.
Mediator releasing cells can be found in blood and fractions thereof, in connective and several other tissues. It was surprisingly found that the method according to the present invention closely mirrors cutaneous sensitivity when using pure allergens, especially when whole blood is used. In contrast thereto, measurements of specific IgE did not correlate with cutaneous sensitivity. Therefore the sample to be used in a method according to the present invention may be a blood sample (preferably heparinised blood) or connective tissue.
The mediator releasing cells used in the method according to the present invention may be isolated from the sample. Due to this isolation other possibly disturbing substances present in the sample may be removed. Especially considering that blood, for instance, may contain released mediator providing a high background during the determination of the amount of mediator released into the sample upon contact with an allergen. This problem may be avoided by measuring the amount of mediator present in the sample prior its exposure to the allergen. On the other hand experimental data revealed that substantially no correlation between histamine release and skin sensitivity, for instance, exists. Therefore, the samples to be used according to the present invention are not isolated or washed prior contacting the sample with the allergen or derivative thereof. This may be reasoned by the fact that when mediator releasing cells are washed all antibodies including those IgG antibodies which should be induced in the course of an allergen therapy and which would act as blocking antibodies in order to reduce the amount of IgE-allergen complexes (due to competition with IgE molecules) in the sample are removed (see e.g. Stahl-Skov et al. (1977) Clin. Exp. Immunol. 27: 432-439)
Preferably said cells are mast and/or basophilic and/or eosinophilic cells.
Mast and basophilic cells are those cells which release most of the mediators, especially histamine, when exposed to an allergen. Mast cells are found in connective tissues of the skin, lung and gastrointestinal tract, whereas basophilic cells are found in blood. These cells can be isolated by known methods and be used in a method according to the present invention. Isolation protocols for mast cells can be found in Jamur M C et al. (J Histochem Cytochem. 1997 45:1715-1722), Massey W A (J. Immunol. 1991 147:1621-7), isolation protocols for basophilic cells in Valent P. (Proc. Natl. Acad. Sci USA 1989, 86, 5542-5546).
According to a preferred embodiment of the present invention the sample further comprises immunoglobulins (Ig), especially immunoglobulin G (IgG).
The procedure should preferably be carried out with samples containing IgG, e.g. whole blood samples. The presence of IgG in such samples is preferred since it allows the measurement of the interference of blocking IgG during the exposure of said cells to the allergen. In the course of an allergen immunotherapy IgGs directed to said allergen are produced. These IgGs bind to the allergen when an individual is contacted with said allergen and prevent that the allergen binds to IgE. Since the production of allergen binding IgGs is therefore directly involved in the response of an individual to an allergen and thus influencing the allergen sensitivity of an individual, the sample should preferably contain IgGs.
In order to evaluate the allergen sensitivity of an individual or the clinical efficacy of an allergen immunotherapy the samples are preferably provided before and after subjecting said individual to an immunotherapy.
To monitor and to evaluate the efficacy of an immunotherapy it is necessary to determine the sensitivity of an individual to an allergen prior and in the course of the therapy. Therefore the mediator release is determined at various stages of the therapy. In the course of the therapy the sensitivity to an allergen ideally decreases. Furthermore, the determination of the mediator release at one or more time points before the immunotherapy may be useful for dosing the allergen in the course of the therapy.
According to another preferred embodiment of the present invention the samples are provided after subjecting said individual to an immunotherapy.
Of course an immunotherapy may also be evaluated solely by analysing samples after the first administration of a medicament comprising an allergen.
Preferably the at least one sample is provided after a maximum of 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 5 days, 10 days, 4 weeks, 6 months, 12 months, 24 months and 36 months, after subjecting said individual to an immunotherapy.
The sample to be analysed may be provided after a defined time period after the first administration of the allergen. Also the time intervals in between the single determinations of the mediator release may be preferably varied within the range of 1 hour, 2 hours, 6 hours, 12 hours, 2 days, 5 days, 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, 12 months and 24 months.
According to a preferred embodiment said allergen is recombinantly produced.
An efficient allergen immunotherapy and an accurate method to determine the release of mediator is preferably conducted with an allergen, which is recombinantly produced. Due to genetic engineering it is possible to produce a specific allergen in a high amount and to isolate said allergen. Allergens are usually isolated directly from the source which contains the allergen (e.g. pollen) and since the allergen is contained in an extract, said allergen is always part of a mixture of different allergenic and potential allergenic substances. Even purified “natural allergens” consist of several isoforms, some of them which may be even hypo or non-allergenic and hence give false test results (Ferreira F., et al., J. Exp. Med. 1996, 183, 599-609). This problem can be avoided by the recombinant production of allergens. The allergen used for the administration to an individual may also be used in a method according to the present invention.
Said allergen comprises preferably at least one deletion, at least one substitution or at least one insertion.
Also hypoallergenic allergen or derivatives thereof can be used when it comes to the question whether the patient may become sensitised to these derivatives during treatment.
According to a preferred embodiment of the present invention said allergen is modified by reshuffling the fragments of said allergen by genetic engineering.
The sample is preferably contacted with varying concentrations of said allergen.
The amount of mediator released from a mediator releasing cell depends on the concentration of the allergen employed in the method according to the present invention. The higher the concentration of the allergen used to induce the release a distinct amount of mediator is, the lower is the sensitivity of the cells provided from an individual and vice versa. Therefore the determination of the amount of mediator released requires the use of varying concentrations of allergen.
Preferably the concentration of said allergen is selected within the range of 1 ng/ml to 100 μg/ml, preferably within the range of 1 pg/ml to 10 μg/ml.
According to a preferred embodiment the total amount of mediator of the cells contained in the sample provided by an individual is determined.
In order to determine the amount of total mediator present in the cells, these cells are lysed e.g. by several thawing and freezing cycles. The determined amount of mediator indicates the mediator potentially releasable by said cells, which value may be employed to determining the degree of cellular sensitisation of the cells to a certain allergen.
A degree of cellular sensitisation is preferably defined by determining the concentration of said allergen inducing the release of 10%, preferably 30%, of the total amount of mediator of said cells.
The degree of cellular sensitisation is an indicator of the progress of the immunotherapy because it reveals the concentration, at which a cell releases 10%, preferably 20%, 25%, 30%, of the total amount of mediator present in the mediator releasing cell. In the course of a successful allergen immunotherapy the concentration of the allergen employed should increase because a high concentration of allergen releasing a certain amount of mediator from said cells indicates that the cells are less sensitive than in a previous measurement. Also the dose inducing maximum release of the mediator may be evaluated. This allows to create a dose response curve and to measure the shifting of said curve in the course of an allergen immunotherapy.
Therefore, the allergen sensitivity of an individual and/or the clinical efficacy of the allergen immunotherapy is preferably evaluated by observing the degree of cellular sensitisation in the course of said immunotherapy.
According to a preferred embodiment of the present invention the mediator in the sample is determined by an immunological and/or a chromatographical method, preferably the method is selected from the group consisting of radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC), reverse transcriptase polymerase chain reaction, immunofluorescence flow cytometry and combinations thereof.
All of these methods have been established to come closer to clinical sensitivity. However, none of these methods has been used to look at a pure allergen in serology, basophil activation and skin sensitivity (e.g. Pierkes M. et al., J Allergy Clin Immunol. (1999) 103:326-32; Di Lorenzo G. et al., J Allergy Clin Immunol. (1997) 100:832-7).
Preferred allergens to be used by the present invention include all major protein allergens available e.g. under www.allergen.org/List.htm. Specifically preferred groups of allergens according to the present invention include major allergens such as major birch pollen allergens, e.g. Bet v 1, major timothy grass pollen allergens, e.g. Phl p 1, Phl p 2, Phl p 5 and Phl p 6, major house dust mite allergens, e.g. Der p 1, Der p 2, major cat allergen, e.g. Fel d 1, major bee and wasp allergens (see list), other profilins, especially Phl p 12, other birch allergens, especially Bet v 4, storage mite allergens, especially Lep d 2, and the allergens listed in table 1.
Ambrosia artemisiifolia
Ambrosia trifida
Artemisia vulgaris
Helianthus annuus
Mercurialis annua
Caryophyllales
Chenopodium album
Salsola kali
Humulus japonicus
Parietaria judaica
Parietaria officinalis
Cynodon dactylon
Dactylis glomerata
Festuca pratensis
Holcus lanatus
Lolium perenne
Phalaris aquatica
Phleum pratense
Poa pratensis
Sorghum halepense
Phoenix dactylifera
Alnus glutinosa
Betula verrucosa
Carpinus betulus
Castanea sativa
Corylus avellana
Quercus alba
Fraxinus excelsior
Ligustrum vulgare
Olea europea
Syringa vulgaris
Plantaginaceae
Plantago lanceolata
Cryptomeria japonica
Cupressus arisonica
Cupressus sempervirens
Juniperus ashei
Juniperus oxycedrus
Juniperus sabinoides
Juniperus virginiana
Platanaceae
Platanus acerifolia
Acarus siro
Blomia tropicalis
Dermatophagoides farinae
Dermatophagoides microceras
Dermatophagoides pteronyssinus
Euroglyphus maynei
Glycyphagus domesticus
Lepidoglyphus destructor
Tyrophagus putrescentiae
Bos domesticus
Canis familiaris
Equus caballus
Felis domesticus
Cavia porcellus
Mus musculus
Rattus norvegius
Alternaria alternata
Cladosporium herbarum
Aspergillus flavus
Aspergillus fumigatus
Aspergillus niger
Aspergillus oryzae
Penicillium brevicompactum
Penicillium chrysogenum
Penicillium citrinum
Penicillium oxalicum
Fusarium culmorum
Trichophyton rubrum
Trichophyton tonsurans
Candida albicans
Candida boidinii
Psilocybe cubensis
Coprinus comatus
Rhodotorula mucilaginosa
Malassezia furfur
Malassezia sympodialis
Epicoccum purpurascens
Aedes aegyptii
Apis mellifera
Bombus pennsylvanicus
Blattella germanica
Periplaneta americana
Chironomus kiiensis
Chironomus thummi thummi
Ctenocephalides felis felis
Thaumetopoea pityocampa
Lepisma saccharina
Dolichovespula maculata
Dolichovespula arenaria
Polistes annularies
Polistes dominulus
Polistes exclamans
Polistes fuscatus
Polistes gallicus
Polistes metricus
Vespa crabo
Vespa mandarina
Vespula flavopilosa
Vespula germanica
Vespula maculifrons
Vespula pennsylvanica
Vespula squamosa
Vespula vidua
Vespula vulgaris
Myrmecia pilosula
Solenopsis geminata
Solenopsis invicta
Solenopsis saevissima
Triatoma protracta
Gadus callarias
Salmo salar
Bos domesticus
Gallus domesticus
Metapenaeus ensis
Penaeus aztecus
Penaeus indicus
Penaeus monodon
Todarodes pacificus
Helix aspersa
Haliotis midae
Rana esculenta
Brassica juncea
Brassica napus
Brassica rapa
Hordeum vulgare
Secale cereale
Triticum aestivum
Zea mays
Oryza sativa
Apium gravaolens
Daucus carota
Corylus avellana
Malus domestica
Pyrus communis
Persea americana
Prunus armeniaca
Prunus avium
Prunus domestica
Prunus persica
Asparagus officinalis
Crocus sativus
Lactuca sativa
Vitis vinifera
Musa x paradisiaca
Ananas comosus
Citrus limon
Citrus sinensis
Litchi chinensis
Sinapis alba
Glycine max
Vigna radiata
Arachis hypogaea
Lens culinaris
Pisum savitum
Actinidia chinensis
Capsicum annuum
Lycopersicon esculentum
Solanum tuberosum
Bertholletia excelsa
Juglans nigra
Juglans regia
Anacardium occidentale
Ricinus communis
Sesamum indicum
Cucumis melo
Anisakis simplex
Argas reflexus
Ascaris suum
Carica papaya
Dendronephthya nipponica
Hevea brasiliensis
Homo sapiens
Triplochiton scleroxylon
The knowledge of the nucleic acid sequences encoding these allergens allows their recombinant production. Therefore especially these allergens are preferably used in immunotherapies and in methods according to the present invention.
Another aspect of the present invention relates to a method for evaluating the allergen sensitivity of an individual and/or the clinical efficacy of an allergen immunotherapy comprising the steps:
The cells which are capable of releasing mediators comprise normally IgE molecules bound thereto. Such cells can be isolated from samples which are obtained from the individual subjected to the method according to the present invention or from other individuals. Of course, it is also possible to use cell lines capable of binding IgE in a method according to the present invention.
The method according to the present invention is especially suited for the determination of the allergen sensitivity of an individual because it allows to determine the ratio between the allergen specific IgE and IgG molecules in the plasma and serum of said individual. Since only IgE-allergen complexes and not free IgE are able to induce the release of mediators from mediator-releasing cells like leukozytes the level of released mediator correlates with the amount of IgE-complex present in the sample. In turn the amount of IgE-complex in said sample correlates with the amount of allergen specific IgE, allergen and allergen specific antibodies other than IgE such as IgG, IgA or IgM which compete with IgE for the free allergen and consequently inhibits the formation of an IgE-allergen complex. This means that a low level of allergen specific IgE or a high level of allergen specific IgG leads to the formation of a low number of IgE complex and thus to a reduced mediator release.
The concentration of allergen in said serum and/or plasma is preferably within 1 ng/ml to 100 μg/ml, more preferably within 1 pg/ml to 10 μg/ml.
Another aspect of the present invention relates to a kit for evaluating the allergen sensitivity of an individual and/or the clinical efficacy of an allergen immunotherapy for at least one allergy comprising
The kit provided herein comprises at least one allergen, which can be used to induce the release of a mediator from mediator releasing cells contained in a sample. The released mediator is then detected directly or preferably—after the removal of solid parts of the sample—in the supernatant of the reaction mixture. Optionally also means for the detection of IgE molecules binding said allergen are enclosed in the kit according to the present invention. IgE is able to bind a distinct allergen and to mediate, when bound to a mediator releasing cell and the allergen, the release of mediator from said cells. However, IgE specific for an allergen is not normally detected in the blood and is only produced when a person becomes sensitised to an allergen. In order to accurately determine the amount of mediator in the sample (for the provision of a standard curve) a mediator standard may be optionally part of the kit.
Preferably the cells are mast and/or basophilic and/or eosinophilic cells.
According to another preferred embodiment of the present invention the allergen is selected from the group consisting of major birch pollen allergens, in particular Bet v 1 and Bet v 4, major timothy grass pollen allergens, in particular Phl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p 7, major house dust mite allergens, in particular Der p 1 and Der p 2, major cat allergen Fel d 1, major bee allergens, major wasp allergens, profilins, especially Phl p 12, and storage mite allergens, especially Lep d 2 and the allergens listed in table 1.
The means for detecting mediators are preferably antibodies.
A mediator, as outlined above, is preferably detected by immunological methods. Therefore the kit may provide at least one antibody which is able to bind specifically mediator. Preferably enzyme linked immuno sorbent assays (ELISA), radio immuno assays (RIA) or lateral flow devices are employed.
Another aspect of the present invention relates to a kit for evaluating the allergen sensitivity of an individual or the clinical efficiency of an allergen immunotherapy for at least one allergy comprising at least two of the following components:
The present invention is further illustrated by the following figures and example, without being restricted thereto.
The cross-linking of effector cell (mast cells and basophils)-bound IgE antibodies by allergens is a crucial event for the induction of the immediate symptoms of type I allergy (Kawakami T, et al., Nat Rev Immunol (2002) 2:773-86). As described in the classical experiments by Prausnitz and Küstner (Prausnitz C, at al., Centralbe F Bact 1 Abt Orig (1921) 86:160-8), this event depends on three major factors, i.e. allergen-specific IgE antibodies, effector cells and allergens. Because the characterisation of IgE antibodies and the development of diagnostic tests capable of measuring the precise amount of allergen-specific IgE antibodies, several studies have investigated the association of allergen-specific serum IgE levels and biological sensitivity to allergens in allergic patients (Stenius B, et al., Clin Allergy (1971) 1:37-55; Bryant D H, et al., Clin Allergy (1975) 5:145-57; Pauli G, et al., Clin Allergy (1977) 7:337-46; Bousquet J, et al., Clin Allergy (1987) 17:529-36; Witteman A M, et al., J Allergy Clin Immunol (1996) 97:16-25; Niederberger V, et al. J Invest Dermatol (2001) 117:848-51; Norman P S, et al., J Allergy Clin Immunol (1973) 52:210-24; Lichtenstein L M, et al. J Allergy Clin Immunol (1971) 47:103 (A37)). It is well established that the presence of allergen-specific serum IgE is a pre-requisite for the occurrence of an immediate type of reaction, but whether the amount of allergen-specific IgE correlates with immediate type sensitivity to the given allergen has been a matter of great debate. To address the problem almost all of the investigations carried out in the past have used allergen extracts, i.e. mixtures of allergens and non-allergenic molecules (Stenius B, et al., Clin Allergy (1971) 1:37-55; Bousquet J, et al., Clin Allergy (1987) 17:529-36; Norman P S, et al., J Allergy Clin Immunol (1973) 52:210-24; Lichtenstein L M, et al. J Allergy Clin Immunol (1971) 47:103 (A37)). This is the reason why these studies could not analyse the association between allergen-specific IgE levels and biological activities at molecular levels. Recent studies using purified natural and recombinant allergens to re-investigate the relation between skin sensitivity and allergen-specific IgE levels report considerable discrepancies between these parameters (Witteman A M, et al., J Allergy Clin Immunol (1996) 97:16-25; Niederberger V, et al. J Invest Dermatol (2001) 117:848-51). In this example, purified recombinant Bet v 1, the major birch pollen allergen, was used as a paradigmatic tool to further investigate the association between allergen-specific IgE levels, effector cell responses and in vivo sensitivity. In a population of 18 birch pollen-allergic patients, selected on well-defined clinical criteria, and out of the pollen season, skin sensitivity and basophil degranulation in response to defined amounts of structurally folded recombinant Bet v 1 was quantified. The results of the biological and of the serological tests were compared. For the measurement of Bet v 1-specific IgE antibody levels two different assays were used: one to detect any Bet v 1-specific IgE, and the other to detect Bet v 1-specific IgE able to bind to effector cells.
Material and Methods
Study Population
The examination of the patients was performed between January and April before the beginning of the birch pollen season. Eighteen patients, eight women and 10 men aged between 28 and 58 years (mean age: 45.6 years), were included in the study on the basis of clinical history of birch pollinosis and positive skin prick tests to birch pollen extract. All patients had moderate to severe rhino-conjunctivitis first diagnosed at least 3 years before. Five patients had mild asthma during birch pollen season and 12 patients had oral allergy syndrome with fruits of the Rosaceae family (apple, peach, apricot and almonds) and vegetables from Solanaceae (potato, tomato) and Apiaceae family (celery, carrot). Skin prick tests with a standard panel of respiratory allergens (Stallergènes, France) consisting of house dust mites, mixtures of fungal allergens, dog and cat dander, cockroach, grass, trees (including birch, olive and ash) and weed pollens were performed to identify the sensitisation profile. Patients' characteristics are displayed in the following Table 1.
Study Design
To analyse the possible association between allergen-specific IgE levels, skin sensitivity, and basophil degranulation, patients were bled and their skin was tested on the same day. The analyses were carried out strictly out of the birch pollen season to exclude effects because of seasonal allergen contact. Patients were not allowed to take anti-allergic or anti-inflammatory medication at least 1 week before the study was performed. None of the patients had received allergenspecific immunotherapy over the last 5 years. After informed consent was given, blood was collected for basophil histamine release and for serum sampling. Immediately thereafter, intradermal skin tests were performed using the end-point titration method (Grammer L C, et al., J Allergy Clin Immunol (1985) 76:123-7).
Detection and Quantification of Allergen-Specific Antibodies
Allergen-specific IgG1 to IgG4 subclass levels as well as allergen-specific IgM and IgA levels were measured by ELISA using isotype-specific monoclonal antibodies as described (Vrtala S, et al., J Allergy Clin Immunol (1996) 97:781-7). Results represent means of duplicate determinations and are shown as OD values corresponding to the amount of bound antibodies.
Basophil Histamine Release Test
The challenge of whole blood with rBet v 1 and anti-IgE as a positive control was performed in a dose response fashion according to the method described by Tanisaki et al. (Tanisaki Y, et al., Int Arch Allergy Appl Immunol (1984) 73:141-5). Ten millilitres of venous blood was drawn into a plastic syringe containing 1 ml of heparin. 250 μl of different concentrations of rBet v 1 (from 10−4 to 10 mg/ml) or anti-IgE (from 10−4 to 10−3; e-specific, Dako, Glostrup, Denmark) were added to the test tubes containing 500 ml of whole blood diluted 1:4 in Tris buffer (10 mmol/l Tris, 136 mmol/l NaCl, 2.7 mmol/l KCl, 0.23 mmol/l MgCl2, 1.8 mmol/L CaCl2, 5.5 mmol/l glucose; pH 7.3). The mixed solution was incubated for 30 min at 37° C. The reaction was stopped, and the cells were separated by cold centrifugation (4° C.) at 375×g
for 5 min. 200 μl of the cell-free supernatant was used for histamine quantification in a radioimmunoassay with acylated histamine monoclonal antibodies (Immunotech, Marseille, France) as described previously (Morel A M, et al. J Allergy Clin Immunol (1988) 82:646-54). Total histamine was measured after cell lysis by repeated thawing and freezing. All experiments were performed in duplicate. The parameter used to describe the degree of basophil sensitivity was the lowest allergen concentration inducing 30% of total histamine release.
Intradermal Testing
Threshold intradermal skin tests were performed by injection of 0.03 ml of 10-fold dilutions of rBet v 1 on the lateral part of the arm. Serial dilutions were prepared from a solution of 1000 mg/ml and the first dilution tested was 10 mg/ml. The tests were read 15 min after injection. The area of weal and erythema was recorded. The test was considered positive when the induced weal area exceeded that of the weal induced by injection and the lowest concentration of allergen inducing a positive test result was used for comparison with other parameters (Grammer L C, et al., J Allergy Clin Immunol (1985) 76:123-7).
Statistical Analysis of the Data
Correlation between different parameters was tested by Spearman s non-parametric tests using VisualStats Professional software (version 2003).
Results
Poor Association Between rBet v 1-Specific Immunoglobulin E Levels and Skin Sensitivity to rBet v 1
To compare rBet v 1-specific IgE levels and skin sensitivity, rBet v 1-specific IgE levels were measured by CAP and correlated with the threshold concentration of rBet v 1 inducing a positive intradermal test weal reaction.
Poor Association Between rBet v 1-Specific Immunoglobulin E Levels and rBet v 1-Related Basophil Sensitivity
Association Between rBet v 1-Induced Basophil Histamine Release and Skin Sensitivity
Measurements of rBet v 1-Specific Immunoglobulin G Subclasses, Immunoglobulin A and Immunoglobulin M
It has been described that Bet v 1-allergic patients' sera contain Bet v 1-specific IgG antibodies that may interfere with IgE binding to Bet v 1 or recognise epitopes on the Bet v 1 molecule other than IgE and hence have no effect on IgE binding to Bet v 1 (Visco V, et al. J Immunol (1996) 157:956-62; Denepoux S, et al. FEBS Lett (2000) 465:39-46). Therefore the levels of rBet v 1-specific IgG were determined (IgG1 IgG4; Table 1). The patients exhibited varying rBet v 1-specific IgG1 IgG4 subclass responses with most pronounced responses in the IgG1 and IgG4 subclasses. No significant levels of rBet v 1-specific IgA and IgM antibodies were detected in the sera, excluding the possibility that these antibody classes may influence IgE recognition of Bet v 1.
Evaluation of Bet v 1-Specific Immunoglobulin E as a Percentage of Total Immunoglobulin E
If Bet v 1-specific IgE only accounts for a low percentage of total IgE, poor histamine release and skin reactivity might be explained by the fact that basophils and mast cells are primarily occupied by IgE directed against other allergens. Therefore the total IgE values were determined and the percentage of Bet v 1-specific IgE was calculated. The patients in this example had relatively low total IgE values (<168 kU/L) and no association between a low percentage of Bet v 1-specific IgE and poor biological responses was found. For example, in patient 11, who showed high sensitivity, Bet v 1-specific IgE only accounted for 20% of the total IgE. On the other hand, patient 13 was less sensitive, although 62.6% of the total IgE was directed against Bet v 1 (Table 1).
Discussion
The question of whether allergen-specific IgE antibody levels, effector cell sensitivities, and clinical sensitivity correlate remains a matter of controversy. Several studies have shown a significant correlation of allergen-specific serum IgE antibodies with allergen-induced immediate type reactions even when using a complex mixture of various allergenic and non-allergenic components, which may make it difficult to compare skin tests and RAST (Stenius B, et al., Clin Allergy (1971) 1:37-55; Bousquet J, et al., Clin Allergy (1987) 17:529-36; Norman P S, et al., J Allergy Clin Immunol (1973) 52:210-24; Lichtenstein L M, et al. J Allergy Clin Immunol (1971) 47:103 (A37)). Recently, other studies using purified allergens (Witteman A M, et al., J Allergy Clin Immunol (1996) 97:16-25) and recombinant allergens (Niederberger V, et al. J Invest Dermatol (2001) 117:848-51) have demonstrated considerable discrepancies between antibody levels and biological sensitivity.
A clinical study using a defined purified and structurally folded allergen (i.e. the major birch pollen allergen, Bet v 1) to investigate the relation between specific IgE, basophil degranulation, and skin sensitivity at a molecular level was performed. Good agreement between the three methodologies and clinical relevance of birch sensitivity was found; however, strong discrepancies were noted between the levels of allergen-specific IgE, the basophil sensitivity and in vivo sensitivity (i.e. skin sensitivity as determined by end-point titration). In certain patients, very low specific IgE levels but high sensitivity in basophil degranulation and skin tests and vice versa was observed. A review of the literature reveals the scarcity of studies comparing skin tests, basophil histamine release and specific IgE levels. The few available studies showed greatly varying results and were performed with crude allergen extracts. For example, Norman et al. (Norman P S, et al., J Allergy Clin Immunol (1973) 52:210-24) found that the three tests were in good agreement with each other in the diagnosis of ragweed hayfever. Lichtenstein et al. (Lichtenstein L M, et al. J Allergy Clin Immunol (1971) 47:103 (A37)) found a quantitatively significant relationship between skin tests and histamine release. However, no measurement of specific IgE was performed in this example. The response of sensitised leucocytes and mast cells to antigen can depend on a great variety of factors.
One possibility for low sensitivity and poor release of histamine would be that only a small proportion of the total serum IgE accounts for allergen-specific IgE. Therefore the total IgE levels were determined and the percentage of allergen-specific IgE was calculated. However, an association between low percentages of allergen-specific IgE responses and poor biological activity was found. The possibility that a low percentage of specific IgE out of the total IgE may be responsible for poor biological responses towards the given allergen may be of greater importance in polysensitised subjects (Norman P S, et al., J Allergy Clin Immunol (1973) 52:210-24; Conroy M C, et al. J Immunol (1977) 118:1317-21; MacGlashan D W Jr, et al., J Immunol (1986) 136:2231-9).
There are several other factors that may be responsible for the discrepancy between allergen-specific IgE levels and biological responses but they cannot be addressed even in a system using purified allergens. They include interindividual differences in basophil and mast cell sensitivities because of variability in IgE-receptor cell surface density, a parameter that is regulated by serum IgE levels (Conroy M C, et al. J Immunol (1977) 118:1317-21; Malveaux F J, et al., J Clin Invest (1978) 62:176-81; Dembo M, et al., J Immunol (1978) 121:345-53; MacGlashan D W Jr, et al. J Allergy Clin Immunol (1999) 104:492-8). Different cell sensitivities have been demonstrated by variable shifts of the dose response curves (measured by 50% or 30% sensitivity) in case of similar total and antigen-specific IgE serum concentrations (Conroy M C, et al. J Immunol (1977) 118:1317-21; MacGlashan D W Jr., J Allergy Clin Immunol (1993) 91:605-15).
Furthermore, it has been shown that persons with equivalent numbers of IgE molecules on basophils may release 0-100% of their histamine content (Conroy M C, et al. J Immunol (1977) 118:1317-21). The same has been observed for cutaneous mast cells (Petersen L J, et al., J Allergy Clin Immunol (1996) 97:672-9; Bordignon V, Pet al., Invest Allergol Clin Immunol (2000) 10:78-82). In addition, it has been shown that early signal events occur involving sykkinase and IP3 products, which are not linked to the level of specific IgE or basophil sensitivity (MacGlashan D W Jr., J Allergy Clin Immunol (1993) 91:605-15; Miura K, et al., J Immunol (2001) 167:7027; MacGlashan D W Jr., J Immunol (2003) 170:4914-25).
Recent evidence indicates that mast cells may also be influenced via Toll-like receptors (Marshall J S, et al., Int Arch Allergy Immunol (2003) 132:87-97). However, the rBet v 1 preparation used for the experiments did not contain endotoxins.
Finally, it is possible that the presence of IgE antibodies with varying affinities or binding specificities for epitopes inducing varying anaphylactic activity may have influenced serological and biological test results.
In conclusion, this study demonstrates on a molecular level that allergen-specific serum IgE levels are not necessarily related to the biological sensitivity as determined by cellular and in vivo tests. A moderate association was, however, found between the cutaneous tests and the basophil histamine release tests.
To determine the sensitivity of a patient before therapy to allow the choice of the correct dose a whole blood basophil histamine release test is used. Patients with high sensitivity will be injected smaller doses than less sensitive patients. Before treatment a dose response curve will be established with purified allergen. In parallel, cells will be stimulated with anti-IgE to determine overall cell sensitivity which may affect sensitivity to the allergen. Success of treatment should be controlled after IgG antibodies against the allergen become detectable which is usually the case after 4-8 weeks of treatment. Since blocking of IgG antibodies may be responsible for the reduction of sensitivity it may be useful to determine in parallel IgG levels to the given allergen. Again a dose response is determined with the purified allergen and anti-IgE. Either the dose giving maximal cell activation (i.e., maximal histamine release or CD203c upregulation) is compared or the dose giving a certain degree of activation is determined and compared with the test result obtained before treatment. Materials and methods are as described in example 1.
When basophil histamine release experiments were performed with washed granulocyte preparations as described (Stahl-Skov et al. 1977. J Exp Immunol 27: 432-439) no correlation between histamine release data and skin sensitivity was found.
Histamine release was done using basophils from allergic patients. They were enriched by Dextran sedimentation, isolated, washed, re-suspended in histamine release buffer, and exposed to different concentrations of recombinant Bet v 1 (10−5, 10−4, 10−3, 10−2, 10−1, 1 μg/ml) or anti-IgE mAb E-124-2-8 (1 μg/ml) in 96-well microtiter plates (TPP, Trasadingen, Switzerland) for 30 minutes at 37° C. After incubation, cells were centrifuged. Cell-free supernatants were recovered and analyzed for histamine content by using a commercial radioimmunoassay (Immunotech, Marseille, France). Histamine release was expressed as a percentage of total histamine measured in cell lysates (Valent et al., 1989, Proc Natl Acad Sci USA 86: 5542-5546).
Skin prick tests were performed with serial dilutions (1:2) of recombinant Bet v 1 as described (Pauli et al., 1996, J Allergy Clin Immunol 97: 1100-1109).
Maximal histamine released from basophils exposed to recombinant Bet v 1 (HR %-max) did not correlate with skin prick test reactions (mm2) (SPT 2 μg/ml) (r=0.224, P=0.342) (
| Number | Date | Country | Kind |
|---|---|---|---|
| A 214/2005 | Feb 2005 | AT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AT2006/000050 | 2/9/2006 | WO | 00 | 8/8/2007 |
