Repeated measurements of mite and pet allergen levels in house dust over a time period of 8 years.

BACKGROUND: Studies of the association between indoor allergen exposure and the development of allergic diseases have often measured allergen exposure at one point in time. OBJECTIVE: We investigated the variability of house dust mite (Der p 1, Der f 1) and cat (Fel d 1) allergen in Dutch homes over a period of 8 years. METHODS: Data were obtained in the Dutch PIAMA birth cohort study. Dust from the child's mattress, the parents' mattress and the living room floor was collected at four points in time, when the child was 3 months, 4, 6 and 8 years old. Dust samples were analysed for Der p 1, Der f 1 and Fel d 1 by sandwich enzyme immuno assay. RESULTS: Mite allergen concentrations for the child's mattress, the parents' mattress and the living room floor were moderately correlated between time-points. Agreement was better for cat allergen. For Der p 1 and Der f 1 on the child's mattress, the within-home variance was close to or smaller than the between-home variance in most cases. For Fel d 1, the within-home variance was almost always smaller than the between-home variance. Results were similar for allergen levels expressed per gram of dust and allergen levels expressed per square metre of the sampled surface. Variance ratios were smaller when samples were taken at shorter time intervals than at longer time intervals. CONCLUSION: Over a period of 4 years, mite and cat allergens measured in house dust are sufficiently stable to use single measurements with confidence in epidemiological studies. The within-home variance was larger when samples were taken 8 years apart so that over such long periods, repetition of sampling is recommended.

La mano de DIOS...was the PIAMA intervention study intervened upon?

The PIAMA study evaluates whether application of mite impermeable mattress covers reduces exposure to mite allergen sufficiently to reduce the incidence of asthma and mite allergy. The study started in 1996. Baseline measurements of mite allergen levels were conducted on mattresses of newborn children and their parents, mostly in 1997. Mite allergen levels were surprisingly low in this study compared with previous studies among school children and infants. Mite allergen levels were measured again on mattresses in the PIAMA study in the years 2000/2001 when the children were 4 years old, and in a new study among 6-12 year old school children conducted in the fall of 2001. Data on winter climate were collected as well. In the winters of 1995/1996 and 1996/1997, which preceded and coincided with the PIAMA baseline measurements, temperatures had been extremely low, and precipitation had been extremely low as well. It is likely that these unusual winter weather conditions affected the baseline allergen levels in the PIAMA study so that the effect of the planned intervention (mite impermeable mattress covers) was considerably smaller than it could have been.

An immunoepidemiological approach to asthma: identification of in-vitro T-cell response patterns associated with different wheezing phenotypes in children.

BACKGROUND: Increasing evidence suggests that patterns of T-cell immunity to inhalant allergens in genetically diverse human populations are more heterogeneous than previously assumed, and that covert differences in expression patterns might underlie variations in airway disease phenotypes. We tested this proposition in a community sample of children. METHODS: We analysed data from 172 individuals who had been recruited antenatally to a longitudinal birth cohort study. Of the 194 birth cohort participants, data from the 147 probands (age range 8.6-13.5 years) who consented to blood collection were included along with data from 25 consenting siblings (mean age 11 years [range 7.4-17.4]). We ascertained clinical phenotypes related to asthma and allergy. We measured T-cell responses to allergens and mitogens, together with blood eosinophils and IgE/IgG antibodies, and assessed associations between these indices and clinical phenotypes. FINDINGS: Atopy was associated with allergen-specific T-helper (Th)2 responses dominated by interleukin 4, interleukin 5, interleukin 9, interleukin 13, whereas interleukin 10, tumour necrosis factor alpha, and interferon gamma responses were common to both atopics and non-atopics. The wheal size from skin prick with allergen was positively associated with in-vitro interleukin 5 and interferon gamma responses, and negatively associated with interleukin 10. Asthma, especially in atopics, was strongly associated with eosinophilia/interleukin 5, and bronchial hyper-responsiveness (BHR) was associated with eosinophilia plus polyclonal interferon gamma production. BHR in non-atopics was associated with elevated allergen-specific and polyclonal interleukin 10 production. INTERPRETATION: Parallel immunological and clinical profiling of children identified distinctive immune response patterns related to asthma and wheeze compared with BHR, in atopics non-atopics. Immunological hyper-responsiveness, including within the Th1 cytokine compartment, is identified as a hallmark of BHR. RELEVANCE TO PRACTICE: These findings highlight the heterogeneity of immune response patterns in asthmatic children, including those with seemingly homogeneous Th2-driven atopic asthma. Further elucidation of the covert relationships between wheezing phenotypes and underlying immunophenotypes in this age group will potentially lead to more effective treatments for what is an unexpectedly heterogeneous collection of disease subtypes.

Differential effect of mattress covers on the level of Der p 1 and Der f 1 in dust.

BACKGROUND: Exposure to house dust mite (HDM) allergens can lead to the development of allergic complaints. Mattress covers seem to be an obvious option for lowering allergen exposure in sensitized individuals. Previous studies have shown that Dermatophagoides pteronissinus was the most prevalent HDM species in the Netherlands. OBJECTIVE: In the present study, we investigated the effect of mattress covers on Der p 1 and Der f 1 concentrations in dust samples in three areas in the Netherlands; Groningen, Utrecht and Rotterdam. METHODS: Dust was obtained from mattresses of 277 patients at the beginning of the study and after 12 months of the placebo-controlled intervention. It was analysed for allergen content by immunoassay. The differential effect of the intervention on Der p 1 vs. Der f 1 was analysed in a subgroup with Der p 1+Der f 1>1 microg/g dust (N=161). It was tested whether the intervention caused a significant change in the Der f 1/Der p 1 ratio. RESULTS: At t=0 we found very similar levels of the group 1 allergens of both species. The relatively high prevalence of D. farinae in our study was geographically restricted: the median Der f 1/Der p 1 ratio was 11.1 in the Rotterdam area compared with 1.32 in the Utrecht area and 0.33 in the Groningen area. Analysis of our data showed that the favourable intervention effect found for the combined allergen data (reduction factor=2.9, P<0.001) is essentially due to a favourable effect of the intervention on the Der f 1 levels only (reduction factor=3.6, P<0.001). The effect on the Der p 1 level was remarkably small (reduction factor: 1.2, P=0.48). In the intervention group, the Der f 1/Der p 1 ratio decreased after 12 months by a factor 2.0, whereas in the placebo group it increased (probability of the intervention effect: P<0.005). CONCLUSION: Mite-impermeable covers are more effective in reducing the level of Der f 1 than that of Der p 1.

Risk factors for atopic dermatitis in infants at high risk of allergy: the PIAMA study.

BACKGROUND: It has been suggested that the period immediately after birth is a sensitive period for the development of atopic disease. OBJECTIVE: We investigated whether birth characteristics and environmental factors are associated with the development of atopic dermatitis in the first year of life. METHODS: Seventy-six children with and 228 without atopic dermatitis, all children of mothers with respiratory allergy or asthma (PIAMA birth cohort study) were included in the study. Atopic dermatitis was defined as a positive history of an itchy skin condition with at least two of the following characteristics: visible dermatitis, history of outer arms/leg involvement, or general dry skin. Multiple logistic regression analysis was performed to study the independent effects of various risk factors. RESULTS: A birth weight >/=4000 g compared to 3000-4000 g was a significant risk factor for atopic dermatitis (odds ratio (OR)=2.4; 95% CI: 1.1-5.1) as was day care attendance (OR=2.9; 95% CI: 1.5-5.9). Exclusive breastfeeding in the first 3 months was negatively associated with atopic dermatitis (OR=0.6; 95% CI: 0.3-1.2), especially with visible dermatitis (OR=0.4; 95% CI: 0.2-1.0). Gender, gestational age, the presence of siblings or pets, and parental smoking were not significantly associated with atopic dermatitis. CONCLUSION: This study shows that a high birth weight and day care attendance increase the risk of atopic dermatitis in the first year of life, while exclusive breastfeeding is a protective factor when dermatitis is found on inspection.

Histamine-releasing factors, a heterogeneous group of different activities.

Histamine-releasing factor or HRF is a collective term used for a heterogeneous group of factors with different modes of action. The current review is focussed on IgE-dependent HRF that require the presence of certain types of IgE (designated IgE+) to induce histamine release. IgE+ might be a structurally different IgE molecule, or, alternatively, autoreactive IgE. A subgroup of IgE-dependent HRF does not bind to IgE, such as cloned HRF p23. This factor turned out to be a basophil-priming cytokine. Alternatively IgE-dependent HRF might be an autoallergen. Several groups demonstrated IgE antibodies to human proteins. However, not all IgE autoallergen-containing extracts induce histamine release of appropriately sensitized basophils. In culture supernatants of human mononuclear cells an autoallergenic activity (Agmn) is found, but no binding to IgE+ was found yet. Agmn might be an autoallergen, since it is cross-reactive with a grass pollen allergen in the stripped basophil assay. IgE-dependent HRF and IgE+ may play a role in the late allergic reaction (LAR). However, IgE+ responsiveness to Agmn (IgEmn+) was not required for a bronchial LAR. IgEmn+ is associated with chronic allergic disease, since the prevalence of IgEmn+ is high in the serum of severe asthmatics and atopic dermatitis patients. Our hypothesis is that exogenous allergens induce IgE antibodies cross-reactive with an endogenous protein. During a LAR, these endogenous proteins are released and the subsequent IgE-mediated reaction prolongs and aggravates the allergic and/or asthmatic symptoms. In conclusion, HRF is a confusing term since it is used for different activities. It might be better to avoid this terminology on and just describe the activity of the factors. Autoallergenic activity is likely to explain most, if not all, IgE-dependent activity.

Mattress encasings and mite allergen levels in the Prevention and Incidence of Asthma and Mite Allergy study.

BACKGROUND: Reduction of allergen exposure from birth may reduce sensitization and subsequent allergic disease. OBJECTIVE: To measure the influence of mite allergen-impermeable mattress encasings and cotton placebo encasings on the amount of dust and mite allergen in beds. METHODS: A total of 810 children with allergic mothers took part in the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) study. Allergen-impermeable and placebo mattress encasings were applied to the childrens' and the parents' beds before birth. Dust samples were taken from the beds of children and their parents before birth and 3 and 12 months after birth. Extracts of dust samples were analysed for mite allergens (Der p 1 and Der f 1). RESULTS: Active mattress encasings were significantly more effective in reducing dust and mite allergen levels than placebo encasings. Mite allergen levels were low in general and the treatment effect was modest. Twelve months after birth, mattresses with active mattress encasings had about half the amount of Der 1 (Der p 1 + Der f 1)/m2, compared to mattresses with placebo encasings, for the child's and the parental mattress. CONCLUSION: This study shows that mite-impermeable mattress encasings have a significant but modest effect on dust and mite allergen levels of mattresses with low initial mite allergen levels, compared to placebo.

Use of a chimeric ELISA to investigate immunoglobulin E antibody responses to Der p 1 and Der p 2 in mite-allergic patients with asthma, wheezing and/or rhinitis.

BACKGROUND: Sensitization to indoor allergens, particularly to dust mites, is a strong risk factor for asthma in children and adults. Assessment of sensitization is carried out using in vivo and in vitro tests to detect specific IgE antibodies. OBJECTIVE: To investigate IgE antibody responses to mites in patients with asthma, wheezing and/or rhinitis, using chimeric ELISA to measure specific IgE antibodies to mite allergens Der p 1 and Der p 2. METHODS: Specific IgE antibodies to Der p 1 and Der p 2 were quantified by chimeric ELISA, and compared with IgE to Dermatophagoides pteronyssinus (Dpt) measured using the CAP system (Pharmacia). A panel of sera from 212 patients with asthma, wheezing and/or rhinitis and 11 controls was analysed. RESULTS: There was a significant correlation between IgE to Dpt measured by CAP and IgE to Der p 1 (r = 0.81, P < 0.001), Der p 2 (r = 0.79, P < 0.001) and combined Der p 1 and Der p 2 (r = 0.86, P < 0.001). Seventy per cent of all patients had IgE to Dpt, and of those, 76.5% had IgE to Der p 1, 79.2% had IgE to Der p 2 and 83.1% had IgE to Der p 1 and Der p 2 combined. Considering the cut-off level of 2 IU/mL of IgE to either Der p 1 or Der p 2, the predictive value for a positive IgE to Dpt by CAP was greater than 95%. CONCLUSIONS: The chimeric ELISA allowed accurate quantification of IgE antibodies to Dpt allergens Der p 1 and Der p 2, and it could be useful for studying immune responses to mites in patients with asthma and/or rhinitis.

The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds.

Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In a previous paper, we showed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4-a large fraction has two antigen-binding sites resulting in bispecificity. We postulated that the generation of bispecific antibodies was caused by a post-secretion mechanism, involving the exchange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypothesis was based on the observed instability of the inter-heavy chain disulfide bonds of IgG4. To investigate this instability, we constructed IgG4 mutants and analyzed the covalent interaction between the heavy chains by sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a more stable rather than a more labile inter-heavy chain linkage. Moreover, we confirmed that mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy-chains. These two observations suggested an explanation for the observed instability of the inter-heavy chain disulfide bonds: the formation of an alternative, intra-chain cystine. Obviously, this intra-chain cystine cannot be formed in the mutant where Cys226 is replaced by Ser, and cannot easily be formed in the mutant with the IgG1 hinge sequence (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. We, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intra-chain cystines. Based upon the published structure of the IgG4-related hinge-deleted IgG1 myeloma protein Mcg, we propose a model for the two forms of IgG4 and for the half-molecule exchange reaction, which might result in the formation of bispecific IgG4 antibodies.

Reactivity to IgE-dependent histamine-releasing factor is due to monomeric IgE.

BACKGROUND: IgE-dependent histamine-releasing factor (HRF) can distinguish between IgE+ and IgE-. In contrast to IgE-, IgE+ sensitizes basophils to release histamine in response to HRF. But we do not know what particular feature distinguishes IgE+ from IgE-. The objective was to investigate the hypothesis that IgE+ is polymeric IgE. METHODS: IgE+ plasma was separated by size-exclusion chromatography. The basophil-sensitizing capacity of the fractions was analyzed in response to HRF produced by mononuclear cells. RESULTS: We showed that monomeric IgE sensitized basophils to release histamine in response to HRF and to house-dust mite, whereas no enhanced reactivity was found in the fractions containing polymeric IgE. CONCLUSIONS: HRF reacts with monomeric IgE, and not (exclusively) with polymeric IgE.

Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens.

BACKGROUND: Cockroaches produce several proteins that induce IgE antibody responses. Although cockroaches are abundant in warm and humid areas, sensitization to cockroach allergens has not been investigated in Brazil. OBJECTIVE: The aims of this study were to investigate the frequency of cockroach allergy among patients with asthma, rhinitis, or both in Brazil and to identify American cockroach allergens. METHODS: Skin tests using cockroach extracts were performed on children and young adults with asthma, rhinitis, or both. A Periplaneta americana complementary (c)DNA library was screened by using IgE antibodies from Brazilian patients allergic to cockroaches. Reactivity of an mAb directed to Dermatophagoides pteronyssinus tropomyosin against cockroach tissue was examined by immunofluorescence. RESULTS: Cockroach allergy was present in 55% and 79% of the patients, as determined by using skin prick tests alone or combined prick and intradermal tests, respectively. Five cDNA clones reacted with IgE antibody and contained the same sequence. A representative clone (1300 bp), pa 12, coded for a protein that reacted with 50% of the sera from patients allergic to cockroaches on plaque immunoassay and showed a high degree of homology to tropomyosins, particularly those from invertebrates. P americana tropomyosin showed 80%, 81%, and 82% sequence identity to tropomyosins from D pteronyssinus, D farinae, and shrimp, respectively, which have been previously defined as important allergens. An mAb directed against D pteronyssinus tropomyosin, which also recognizes shrimp tropomyosin, showed binding to cockroach striated muscle. CONCLUSION: Our results support the recommendation that cockroach extracts should be routinely used for the evaluation of patients with asthma, rhinitis, or both in Brazil. The identification of P americana tropomyosin as an important allergen will make it possible to investigate cross-reactivity among cockroaches, mites, and food derived from invertebrates.

Reactivity to IgE-dependent histamine-releasing activity in asthma or rhinitis.

We investigated the relation between IgE reactive with histamine-releasing factor (HRF) and clinical status in patients with asthma or rhinitis. Sera were used to passively sensitize purified, lactic-acid treated basophils. IgE-independent HRF due to chemokines was removed from mononuclear cell supernatants with heparin-Sepharose. IgE-dependent HRF was determined by measuring the increase in histamine release between 1 min and 60 min, which was designated delta HRF. HRF-reactive IgE was demonstrable in nine of 18 patients with allergic asthma, three of 19 patients with nonallergic asthma, five of 17 patients with allergic rhinitis, and none of 19 control patients. The presence of HRF-reactive IgE was associated with: (1) IgE to inhalant allergens; 40% of radioallergosorbent test (RAST)-positive individuals versus 8% of RAST-negative individuals were positive (OR = 7.8, p < 0.005); (2) bronchial sensitivity to histamine in all asthmatic patients (geometric mean PC20: 1.50 versus 0.51 mg/ml; p < 0.005); and (3) bronchial sensitivity to histamine in allergic asthmatic patients (geometric mean PC20: 1.27 versus 0.37 mg/ml, p < 0.02). These findings support the hypothesis that IgE-dependent HRF might contribute to the chronic allergic reaction.

IgE antibodies reactive with silverfish, cockroach and chironomid are frequently found in mite-positive allergic patients.

Approximately 30% of the house dust mite allergic patients in The Netherlands have IgE antibodies reactive with silverfish, cockroach and/or chironomid. In allergic patients without IgE antibodies against Dermatophagoides pteronyssinus less than 5% have IgE antibodies reactive with these insects. By means of RAST inhibition studies it is shown that cross-reactivity exists between D. pteronyssinus and silverfish, cockroach or chironomid. This means that a positive RAST for silverfish, cockroach, chironomid or D. pteronyssinus cannot be taken as evidence for exposure.

IgE and IgG4 binding to synthetic peptides of the cat (Felis domesticus) major allergen Fel dI.

Specificities of IgE and IgG4 antibodies in 12 cat-allergic patients were compared with respect to their reactivity towards 3 IgE-binding synthetic peptides of Felis domesticus allergen 1 (Fel dI): peptides 25-38 and 46-59 of chain 1 and peptide 15-28 of chain 2. Peptides were coupled to Sepharose and anti-Fel dI antibodies were isolated by affinity chromatography. Fel dI-specific IgE- and IgG4 antibody activity in the peptide eluates was measured using Fel dI binding assays. Fel dI-specific IgE/IgG4 ratios in the eluates from peptide-Sepharose were determined and compared with the IgE/IgG4 ratios in the eluates from Fel dI-Sepharose. The mean ratio Fel dI-specific IgE/IgG4 in the peptide eluates (0.84; range 0.06-4.6) was significantly higher than the mean ratio in the eluates from Fel dI-Sepharose (0.31; range 0.13-1.1), demonstrating a higher reactivity of IgE antibodies with the peptides, compared to IgG4. These results indicate differences between the B cells producing IgE antibodies and the B cells producing IgG4 antibodies.

Variability of IgE-dependent histamine-releasing activity in supernatants of human mononuclear cells.

Histamine-releasing factors (HRF) that release mediators from human basophils by interacting with IgE have been identified from different cell sources, including lymphocytes, monocytes, thrombocytes and endothelial cells. These factors are studied in view of their potential importance as a stimulus in chronic inflammation. In this report we investigated the qualitative variability of the histamine-releasing activity in the supernatants of activated mononuclear cells. Purified human mononuclear cells of 8 donors were activated with streptokinase/streptodornase (SK/SD) and the supernatants (HRF-MN) were tested for histamine-releasing activity (HRA) in both allergic (RAST positive for inhalant allergens) and nonallergic individuals. Four of the eight HRF-MN supernatants were discriminating, i.e. showing no histamine-release response with nonallergic individuals, whereas four supernatants were not. Two of the HRF-MN supernatants that exhibited discriminating properties were studied in more detail. The response to HRF-MN was tested (1) in a direct bioassay on basophils of allergic (RAST positive for inhalant allergens) and nonallergic individuals and (2) in an indirect bioassay with 70% pure basophils of RAST-negative donors after passive sensitization with sera of allergic donors. An association was found between the response to HRF-MN and the RAST for inhalant allergens: none (0/12) of the RAST-negative but 15/22 of the RAST-positive individuals were HRF-MN responders. The IgE dependency of HRF-MN was shown e.g. by inhibition of passive sensitization by preincubating a responder serum with monoclonal antibody (moAb) anti-IgE MH25-1. Our results are in contrast with findings of other investigators who use pooled supernatants and demonstrated HRF-MN responsiveness with both allergic and nonallergic donors.(ABSTRACT TRUNCATED AT 250 WORDS)

IgE epitopes on the cat (Felis domesticus) major allergen Fel d I: a study with overlapping synthetic peptides.

BACKGROUND: The major cat allergen Fel d I is composed of two disulfide-linked polypeptide chains, chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Reduction and alkylation of Fel d I eliminates almost all antigenic and allergenic activity, and detection of linear epitopes with synthetic peptides is therefore not expected. METHODS: We synthesized synthetic peptides of both chains of about 14 amino acid residues, overlapping by 7 residues. The peptides were coupled to Sepharose (Pharmacia, Uppsala, Sweden) and tested with sera of patients with cat allergy. RESULTS: Three peptides showed specific binding of human IgE, residues 25-38 and 46-59 of chain 1 and residue 15-28 of chain 2. IgE binding was inhibited by Fel d I and the corresponding peptide. Of 61 patients with cat allergy tested, 65% showed IgE binding to at least one of the peptides; 46% showed IgE binding to peptide 25-38, 11% to peptide 46-59, and 28% to peptide 15-28. Each peptide was recognized by only one of the 78 patients with negative RAST results. By affinity chromatography with peptide-Sepharose anti-Fel d I antibodies were isolated, also confirming the specificity of IgE binding to the peptides. The percentage of IgE antibodies against Fel d I reactive with the peptides varied with the serum and the peptide-Sepharose used and ranged from 2% to 55%. CONCLUSIONS: Because the affinity of IgE binding to the peptides was very low and only serum samples with high titers of Fel d I-specific IgE antibodies (RAST 4+/5+) showed significant binding, these peptides are not suitable for diagnostic purposes. However, the peptides are useful tools for comparing IgE and IgG responses and for studying the relationship to the T-cell epitopes on this molecule.

T cell epitopes of house dust mite major allergen Der p II.

Recent work has indicated the significance of IL-4- and IL-5-secreting allergen-specific human Th2 lymphocytes in the control of immune responses to allergens in atopic individuals. The precise allergenic epitopes that activate these allergen-specific Th2 cells are, however, hardly known. We analyzed the epitope-specificity of T lymphocytes specific for Der p II, one of the major allergens of house dust mite Dermatophagoides pteronyssinus. Using a panel of overlapping synthetic peptides that span the entire Der p II molecule, we could demonstrate that polyclonal Der p II-specific T cell lines prepared from the peripheral blood of five atopic patients can react with at least 10 different epitopes of the molecule. Each donor showed a different pattern of reactivity with the synthetic peptides, suggesting that Der p II contains multiple T cell epitopes that may differ from individual to individual. We studied the specificity of the T cell response to Der p II in more detail in one atopic patient using a short term polyclonal T cell line that strongly reacted to one single peptide (116-129) of the allergen. From this patient we established a panel of 11 Der p II-specific TLC. Ten TLC were of the CD3+ CD4+ phenotype and showed a high IL-4/IFN-gamma production ratio, whereas another TLC expressed CD3 and CD8 and failed to secrete substantial IL-4 and IFN-gamma. The use of at least four different TCR V beta gene segments was shown within this panel TLC. All TLC tested recognized the allergen in an HLA-DR1-restricted manner. Although this patient reacted to only one peptide on the polyclonal level, two T cell epitopes were identified on the clonal level by using synthetic peptides and autologous APC to stimulate the TLC. Combining data of CD4/CD8 expression, TCR V beta usage, and epitope specificity, at least six different types of Der p II-specific TLC could be identified within this patient. Binding of IgE to all synthetic peptides of Der p II is low and of low affinity, which may be of particular importance with respect to possible desensitization protocols using such peptides.

Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I.

The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide-linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty-one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope-mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I. Two monoclonal antibodies directed against reduced/alkylated chain 1 bound to the overlapping peptides 53-66 and 60-70 of chain 1. The monoclonal antibody directed against reduced/alkylated chain 2 bound to the overlapping peptides 36-49 and 43-56 of chain 2. Binding specificity was demonstrated by inhibition by reduced/alkylated Fel d I for all three monoclonal antibodies. Another monoclonal antibody against reduced/alkylated Fel d I had been found to bind predominantly to reduced/alkylated chain 2 on immunoblot in previous studies. It bound to peptides 1-16 and 60-70 of chain 1 and peptides 1-14 and 50-63 of chain 2; it is therefore probably directed against a conformational epitope formed by these four regions. Possibly because of low affinity of this monoclonal antibody, specificity of its binding could not be verified by inhibition studies. A panel of monoclonal antibodies directed against native Fel d I bound to peptides 1-16 and 60-70 of chain 1 and peptides 1-14 and 43-56 of chain 2.(ABSTRACT TRUNCATED AT 250 WORDS)