Serum immunoglobulin E levels predict human airway reactivity in vitro

BACKGROUND: Airway hyperresponsiveness to non-specific stimuli is one characteristic feature of airway diseases such as bronchial asthma and chronic bronchitis. Until now, studies aiming to demonstrate a relationship between in vivo conditions associated with airway hyperreactivity and in vitro airway responsiveness have been inconclusive. OBJECTIVE: Since serum immunoglobulin (Ig) E is believed to be one determinant of airway reactivity in vivo, we studied whether in vitro airway reactivity in lung resection material from patients with elevated levels of serum IgE was increased as compared with patients with undetectable IgE. By this approach, we aimed to elucidate the role of circulating IgE for bronchial smooth muscle reactivity in vitro. METHODS: Bronchial rings from nine patients with total serum IgE levels above 200 U/mL and 10 patients with total serum IgE levels below 10 U/mL were passively sensitized, i.e. incubated overnight with buffer or sensitizing serum containing high levels of total IgE (> 250 U/mL). Afterwards, contractile responses to histamine were assessed in the organ bath. RESULTS: Histamine responsiveness was significantly increased in airways obtained from patients with IgE levels above 200 U/mL as compared with airways from patients with IgE levels below 10 U/mL (P < 0.05). Passive sensitization of bronchi from patients with low IgE significantly increased histamine responsiveness, as compared with non-sensitized controls from the same patients (P < 0.05). In contrast, passive sensitization of airways from patients with elevated IgE did not further increase responsiveness. There was no difference in histamine reactivity between non-passively sensitized and passively sensitized tissue preparations from patients with IgE above 200 U/mL and passively sensitized tissues from patients with IgE below 10 U/mL. CONCLUSION: Our findings reveal that elevated levels of serum IgE predict airway hyperresponsiveness to histamine in vitro. At the same time, they indicate that the in vitro model of passive sensitization, in addition to its ability to induce allergen responses, also mimics conditions of non-specific airway hyperreactivity, which are relevant under in vivo conditions.     

Differences in the reactivity of short and giant ragweed with immunoglobulin E antibodies

Currently, the allergens in extracts of SRW and GRW are regarded as equivalent in their ability to react with IgE antibodies. We investigated whether IgE antibodies might recognize differences between these allergens. First, sera from individuals with positive skin tests to both RWs showed higher reactivity with solid-phase SRW in the RAST. Second, immunosorption of 24 sera showed that more than 90% of the IgE antibodies were removed by either SRW or GRW Sepharose. However, all of the sera contained IgE antibodies specific for SRW and six sera contained antibodies specific for both SRW and GRW. Third, the presence of IgE antibodies preferentially reactive with SRW was tested by neutralization of the P-K test. Two individuals were passively sensitized with a serum pool containing IgE antibodies specific for SRW; skin sites were challenged repetitively with an extract of GRW until they no longer reacted with GRW. Challenge of these sites with SRW showed an immediate wheal-and-flare reaction, indicating that antibodies were present in the serum pool which could not be neutralized by GRW and, thus, were specific for SRW. These results indicate that the sera of all RW-sensitive individuals in this study contained IgE antibodies specifically reactive with SRW and many sera contained antibodies specific for GRW. Overall, however, most of the IgE anti-RW reactivity was removed by either GRW or SRW.     

Analysis of skin testing and serum-specific immunoglobulin E to predict airway reactivity to cat allergens

BACKGROUND: When the clinical history is not conclusive, it may be difficult to make an accurate interpretation of the value of skin tests and serum-specific IgE to cat allergens in asthma cases. OBJECTIVE: To analyse the diagnostic efficiency of skin testing (ST) and serum-specific IgE to cat allergens, based on the results of bronchial-specific challenge with cat epithelium. METHODS: Sixty-four asthma patients (49 with cat exposure and 15 without) who did not clearly relate their asthma symptoms to cat exposure and had a positive skin prick testing and/or a positive cat dander-specific IgE determination (CAP-system) underwent intradermal skin tests and specific bronchial challenge with cat epithelium. The results were analysed by receiver operating characteristics curves (ROC curves) and logistic regression. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for different cut-off points. RESULTS: Twenty-seven patients (42.2%) had a positive bronchial-specific challenge. The area under the ROC curve for serum-specific IgE quantification is 0.85, which makes a good diagnostic tool out of this test. Intradermal ST predicts the outcome of the bronchoprovocation test better than skin prick testing (area under the ROC curve of 0.74 vs. area under the ROC curve of 0.54, respectively). The logistic regression analysis shows that the estimated probability of a positive bronchial challenge is > or =93% if CAP values are > or =17 kU(A)/L, whereas if CAP values are less than 0.35 kU(A)/L the estimated probability of a positive bronchial challenge is 16%. When the intradermal skin test is negative, the estimated probability of a positive bronchoprovocation test is 9%, being the test that better identifies patients with a negative bronchoprovocation test. CONCLUSIONS: Levels of serum-specific IgE to cat allergens and intradermal ST can be used to diagnose and treat more accurately asthmatic patients sensitized to cat epithelium when there is uncertainty about cat epithelium causality.     

Functional analysis of cross-reactive immunoglobulin E antibodies: peanut-specific immunoglobulin E sensitizes basophils to tree nut allergens

BACKGROUND: Peanut and tree nuts are a major cause of food-induced anaphylaxis with an appreciable mortality. Co-sensitization to peanuts and tree nuts is a common clinical observation and may be because of peanut-specific serum IgE antibodies that cross-react with tree nut allergens. It is, however, unclear whether these cross-reactive IgE antibodies are involved in effector-cell activation. OBJECTIVE: To determine if cross-reactivity of peanut-specific IgE antibodies with tree nuts can cause effector cell activation using an in vitro basophil activation assay. METHODS: Two peanut allergic subjects with positive specific IgE for peanut and tree nuts (as measured by CAP-FEIA) were studied. Basophil activation to peanut and tree nuts, as indicated by CD63 expression, was assessed by flow cytometry to confirm co-sensitization to peanut and tree nuts. Inhibition ELISA using sera from the subjects was performed to detect peanut-specific IgE antibodies that cross-reacted with tree nut proteins. To determine whether cross-reactive tree nut allergens can induce effector-cell activation, peanut-specific antibodies were affinity purified from the subject sera and used to resensitize non-peanut/tree nut allergic donor basophils stripped of surface IgE. Basophil activation was then measured following stimulation with peanut and tree nut extracts. RESULTS: The two peanut allergic subjects in this study showed positive basophil activation to the peanut and tree nut extracts. Inhibition ELISA demonstrated that pre-incubation of the peanut allergic subject sera with almond, Brazil nut and hazelnut extracts inhibited IgE binding to peanut extract. IgE-stripped basophils from non-peanut/tree nut allergic subjects resensitized with affinity-purified peanut-specific antibodies from the peanut allergic subject sera became activated following stimulation with peanut, almond and Brazil nut extracts, demonstrating biological activity of cross-reactive IgE antibodies. CONCLUSION: Peanut-specific IgE antibodies that cross-react with tree nut allergens can cause effector-cell activation and may contribute to the manifestation of tree nut allergy in peanut allergic subjects.     

Quantitative analysis of immunoglobulin E reactivity profiles in patients allergic or sensitized to natural rubber latex (Hevea brasiliensis)

BACKGROUND: Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology. RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy.     

The inheritance of immunoglobulin E: genetic linkage analysis

Linkage analyses between 21 genetic markers including HLA-A, B, and the postulated locus for determining total serum IgE levels were done to try to clarify the inheritance of total IgE levels and to map the locus. A total of 316 individuals from five Mormon kindreds were studied, and data from an additional 204 Amish individuals from 11 families were analyzed for possible HLA linkage. Segregation analyses of both data sets did not give clear definition of the mode of inheritance of total IgE levels, but purely environmental models were rejected. Linkage analyses gave significant evidence against HLA linkage with the codominant, recessive, or dominant model of inheritance for total IgE levels. No significant evidence for linkage with any of the genetic markers was obtained. Since total serum IgE levels are correlated with allergies, understanding the genetics of total IgE levels is important to understanding the genetics of allergic disease in man.     

Physico‐chemical features of the environment affect the protein conformation and the immunoglobulin E reactivity of kiwellin (Act d 5)

Background Allergy diagnostic systems sometimes give false positive or negative results. In this respect, the influence of protein conformational changes on the allergen–IgE interaction sites is worthy to be investigated. Objective To investigate the influence of different experimental conditions on the structural properties and IgE reactivity of kiwellin (Act d 5) as a model system. Methods Act d 5 was purified from the natural source. To study its conformational features, experiments of circular dichroism (CD) in different media were performed. The IgE reactivity was investigated by skin testing, immunoblotting and ISAC microarray system, in a population of kiwifruit allergic subjects. Results CD experiments indicated that Act d 5 has a mainly helical structure and the conformation is strongly affected by the experimental conditions. The protein is more structured in low polarity media and at acidic pH values, similar to those of the natural source. Eleven subjects of 29 (38%) allergic to kiwifruit were positive to purified natural Act d 5 by skin test. Among them, three patients (10%) showed a reaction only to Act d 5 at pH 4.5, and three (10%) showed a reaction only to the allergen in standard neutral conditions. No one of the 11 subjects with positive skin test recognized Act d 5 immobilized on the ISAC system. Eight of nine subjects detected Act d 5 by IgE immunoblotting. One subject did not recognize the sequence epitopes of Act d 5 in IgE immunoblotting experiments and reacted to the skin test only when the allergen was in acidic conditions. Conclusions and Clinical Relevance The conformation and IgE reactivity of Act d 5 are affected by the physico‐chemical characteristics of the solvent. These findings suggest that the assay conditions influence the results of the diagnostic systems by modulating the pattern of exposed antigenic epitopes. Cite this as: M. L. Bernardi, D. Picone, L. Tuppo, I. Giangrieco, G. Petrella, P. Palazzo, R. Ferrara, M. Tamburrini, A. Mari and M. A. Ciardiello, Clinical & Experimental Allergy, 2010 (40) 1819–1826.     

An epidemiologic study of the interrelationships of total serum immunoglobulin E,allergy skin-test reactivity,and eosinophilia

In a general population sample from Tucson, Arizona, we examined the effects of age, sex, and smoking habits on the interrelationships of total serum IgE, allergy skin-test reactivity, and peripheral blood eosinophilia. Allergy skin-test reactivity showed a direct linear relationship to total serum IgE, but this relationship was differentially affected by age and smoking habits. The frequency of positive skin-test reactivity for a given level of IgE was greater among females than that among males, but a comparison of female and male nonsmokers indicated that this difference was attributable to differences in smoking habits rather than sex. Subjects less than 16 and those over 45 yr of age demonstrated reduced skin-test reactivity for a given level of IgE when compared with subjects 16 to 45 yr of age. Restricting the analysis to nonsmokers diminished (but did not obliterate) this age effect. Those over 45 yr of age (but not those less than 16) were shown to have reduced levels of IgE specific for Bermuda grass as compared with the 16 to 45 yr age group. The skin of subjects less than 16 and those over 65 yr of age was reduced in its capacity to respond to histamine. Thus the reduction in the skin-test reactivity of subjects less than 16 yr of age appears to involve a reduction in their skin reactivity to mediators, whereas for those over 45 it is related to a reduction in IgE specific for common aeroallergens, and for those over 65 it is apparently related to both of these causes. The frequency of peripheral blood eosinophilia was also found to be directly related to total serum IgE. Skin test-negative subjects had the same frequency of eosinophilia as that in skin test-positive subjects at any given level of serum IgE. Males had higher rates of eosinophilia than did females for a given level of IgE, but this difference also became insignificant if the analysis was restricted to nonsmokers. The increase in eosinophilia among smokers as compared with nonsmokers with equivalent serum IgE levels implies that smoking may trigger immunologic reactions associated with eosinophilia.     

Food allergy to wheat: identification of immunogloglin E and immunoglobulin G-binding proteins with sequential extracts and purified proteins from wheat flour.

BACKGROUND: Cereal-associated allergy is particularly considered a serious problem, because cereals are essential in our daily diet. Wheat proteins are classified into albumins, globulins and prolamins (insoluble gliadins and glutenins). OBJECTIVES: Our objectives were to study the involvement in food allergy to wheat of these different protein types by using purified fractions and to identify those binding IgE and IgG antibodies. METHODS: Sera were obtained from 28 patients with food allergy to wheat. Albumins/globulins, gliadins and glutenins were obtained by sequential extraction based on differential solubility; alpha-, beta-, gamma- and omega-gliadins and low molecular weight (LMW) and high molecular weight (HMW) glutenin subunits were purified by chromatography. IgE binding to these extracts and fractions were analysed by radioallergosorbent test (RAST), and immunoblotting; IgG binding was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: In RAST, 60% of sera were shown to have specific IgE antibodies against alpha-, beta-gliadins and LMW glutenin subunits, 55% to gamma-gliadins, 48% to omega-gliadins and 26% to HMW glutenins. Immunoblotting analysis confirmed results obtained in RAST concerning LMW and HMW glutenin subunits and showed that 67% of patients have IgE antibodies to the albumin/globulin fraction. CONCLUSION: Results obtained in the different tests showed common features and in agreement with other studies indicated the presence of numerous allergens in food allergy to wheat; alpha-, beta-, gamma- and omega-gliadins, LMW glutenin subunits and some water/salt-soluble proteins appeared as major IgE binding allergens, whereas HMW glutenins were only minor allergens. The same type of antigenic profile against gliadins and glutenins was observed with IgG antibodies. Important sequence or structural homologies between the various gliadins and LMW glutenin subunits could certainly explain similarity of IgE binding to these proteins.     

Primary versus secondary immunoglobulin E sensitization to soy and wheat in the Multi-Centre Allergy Study cohort

Background IgE sensitization to soy and wheat is classified as ‘primary’ when generated by food ingestion and ‘secondary’ when it as a consequence of primary sensitization to cross‐reacting pollen antigens via inhalation. The age‐specific relevance of these categories of sensitization throughout childhood is unknown. Objective To monitor the natural course of IgE sensitization against common food allergens in childhood in relation to sensitization against cross‐reactive airborne allergens. Methods The German Multi‐Centre Allergy Study with follow‐up from birth to age 13 recruited initially 1314 children. IgE antibody levels against cow's milk, hen's egg, soy, wheat, mites, cat and dog dander, birch and grass pollens were tested. Longitudinal data were analysed from the 273 children with sera obtained at age 2, 5, 7 and 10 years of age. Results The point prevalence of sensitization (>1.0 kU/L) to milk and egg allergens progressively decreased from about 4% at 2 years to <1% at 10 years. By contrast, the prevalence of IgE to wheat and soy progressively increased with age, from 2% to 7% (soy) and from 2% to 9% (wheat). At 10 years of age, IgE to grass pollen was detected in 97% and 98% of the children reacting against soy and wheat, respectively; IgE to birch pollen was observed in 86% and 82% of the children reacting against soy and wheat, respectively. Early IgE sensitization to soy or wheat preceded that to grass or birch pollen in only 4% and 8% of participants sensitized to soy and wheat, respectively. Conclusion IgE sensitization to soy and wheat is relatively uncommon and mostly primary in early infancy, more frequent and mostly secondary to pollen sensitization at school age. Clinical Implications Awareness should be raised to avoid unnecessary diet restrictions due to the high frequency of clinically irrelevant, secondary sensitization to soy and wheat in schoolchildren with pollinosis.     

gammadelta T cells contribute to the systemic immunoglobulin E response and local B-cell reactivity in allergic eosinophilic airway inflammation

Allergic airway inflammation induced in mice is T-cell dependent and recruitment of eosinophils to airspaces requires both alphabeta and gammadelta T cells. From previous studies it is evident that alphabeta T cells are essential for the allergic T helper type 2 (Th2)-like response, while the mechanistic contribution of gammadelta T cells is still unclear. In this study, we have investigated the role of gammadelta T cells in allergic airway eosinophilia induced by ovalbumin hypersensitivity. By comparing the responsiveness to sensitizing allergen of wild-type mice with that of T-cell receptor gammadelta knockout mice (TCRgammadelta KO) we demonstrated that mice lacking gammadelta T cells are defective in the systemic ovalbumin-specific immunoglobulin E (IgE) response. Furthermore, after aerosol challenge with allergen, gammadelta T-cell deficient mice exhibited a significantly decreased migration of B cells and natural killer cells to airways and reduced levels of allergen-specific IgG and IgA in bronchoalveolar lavage fluid. The role for B cells in the airway inflammation was indicated by the impaired ability of mice lacking functional B cells to evoke an eosinophilic response. The diminished eosinophilia in TCRgammadelta KO mice could not be explained by a defective Th2 activation since these mice displayed a normal IgG response in serum and an unaffected IG2b/IgG1 ratio in airways. Analysis of immunoregulatory cytokines in isolated lung tissue, thoracic lymph nodes and spleen further supported the notion that these mice are able to evoke a sufficient activation of T helper cells and that gammadelta T cells are not required for maintaining the Th2 profile. These results indicate that gammadelta T cells contribute to allergic airway inflammation by pathways separate from classical Th2 immune activation.     

Protein sequence analysis of a novel 103-kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients

BACKGROUND: House dust mites are regarded as important indoor allergens. While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned. We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11. OBJECTIVE: The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients. METHODS: The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR. The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema. RESULTS: Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein. The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins. The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%). A high frequency (five out of eight) of MAST(Dp)- allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative. CONCLUSION: The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.     

Cow's milk-specific immunoglobulin E levels as predictors of clinical reactivity in the follow-up of the cow's milk allergy infants

Background IgE‐mediated cow's milk proteins (CMPs) allergy shows a tendency to disappear with age. The sooner tolerance is detected, the earlier the substitute diets can be suspended and the quicker family emotional hardship is alleviated. Objective To analyse the specific IgE levels to cow's milk and its proteins, which help to separate tolerant from no tolerant children in the follow‐up of infants with allergy to cow's milk. Patients and methods Sixty‐six infants diagnosed with IgE‐mediated allergy to CMPs were included in this prospective follow‐up study. Periodic reassessments were carried out every 6 months until they were 2‐years old and then, annually, until tolerance arose or until the last reassessment in which tolerance had not been achieved. Non‐tolerant infants were followed, at least, for a period of 3 years. In each visit, the same skin tests and determination of specific IgE (CAP System FEIA) for milk and its proteins were carried out. The open challenge test was repeated unless a clear transgression to milk, which came to be positive, had taken place within the previous 3 months in each of the follow‐up visits. Specific IgE levels to milk and its proteins, in different moments of the follow‐up were analysed by means of the receiver‐operating characteristic curve to predict clinical reactivity. Results Throughout the follow‐up 45 (68%) infants became tolerant. The follow‐up mean for tolerant infants was 21.2 months whereas for non‐tolerant infants it was 58 months. The specific IgE levels which were predictors of the clinical reactivity (positive predictive value (PPV)90%), grew as the age of the infants increased: 1.5, 6 and 14 kU_A/L for milk in the age range 13–18 and 19–24 months and in the third year, respectively. Specific IgE levels to casein: 0.6, 3 and 5 kU_A/L, respectively, predicted clinical reactivity (PPV90%) in the different analysed moments of the follow‐up. The cut‐off points: 2.7, 9 and 24 kU_A/L for milk and 2, 4.2 and 9 kU_A/L for casein, respectively, predicted clinical reactivity with an accuracy 95% corresponding to a specificity of 90%. Conclusions Monitorization of specific IgE concentration for milk and casein by means of the CAP system in allergic children to CMPs allows us to predict, to a high degree of probability, clinical reactivity. Age factor must be taken into account to evaluate the specific IgE levels which are predictors of tolerance or clinical reactivity.     

Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins: an experimental and structure-based analysis

BACKGROUND: Profilins are cross-reactive plant allergens responsible for multiple pollen sensitization and pollen-associated food allergy. While it is assumed that profilins from different species are immunologically equivalent, some studies suggest partial or even lacking IgE cross-reactivity between certain profilins. OBJECTIVE: We aimed to obtain a semi-quantitative assessment of the contributions of conserved and species-specific epitopes to IgE binding of plant profilins. METHODS: We compared model structures of profilins from timothy, mugwort, celery and bell pepper with crystal structures of birch and latex profilins. We predicted potential conformational epitopes that consisted of contiguous patches of at least 20% surface-exposed residues. Celery and timothy profilins were purified from their natural sources, and profilins from birch, mugwort, bell pepper and latex were expressed in Escherichia coli. The structural integrity of all purified proteins was confirmed by circular dichroism spectroscopy. IgE ELISAs and ELISA inhibitions using sera from 22 profilin-sensitized allergic patients were carried out. RESULTS: Peptide backbone conformations of all six profilins were highly similar. Nine variable epitopes and two containing high proportions of conserved residues were predicted. IgE from all sera bound to all tested profilins and the amounts were highly correlated. However, IgE inhibition experiments revealed that up to 60% of total IgE binding was mediated by species-specific epitopes. The extent of cross-reactivity among profilins from timothy, birch, latex and celery was greater than cross-reactivity to mugwort and bell pepper profilins. This pattern was mirrored by sequence similarities among one of the predicted variable epitopes. Patients with IgE to cross-reactive epitopes displayed allergic reactions to a greater number of plant foods than patients having IgE directed to species-specific epitopes. CONCLUSION: The large extent of cross-reactivity among plant profilins justifies using a single profilin for diagnosis. However, the fine specificity of IgE directed to variable epitopes may influence the clinical manifestation of profilin sensitization.