Toxocara Seropositivity, Atopy and Wheezing in Children Living in Poor Neighbourhoods in Urban Latin American

Background

Toxocara canis and T. cati are parasites of dogs and cats, respectively, that infect humans and cause human toxocariasis. Infection may cause asthma-like symptoms but is often asymptomatic and is associated with a marked eosinophilia. Previous epidemiological studies indicate that T. canis infection may be associated with the development of atopy and asthma.

Objectives

To investigate possible associations between Toxocara spp. seropositivity and atopy and childhood wheezing in a population of children living in non-affluent areas of a large Latin American city.

Methods

The study was conducted in the city of Salvador, Brazil. Data on wheezing symptoms were collected by questionnaire, and atopy was measured by the presence of aeroallergen-specific IgE (sIgE). Skin prick test (SPT), total IgE and peripheral eosinophilia were measured. Toxocara seropositivity was determined by the presence of anti-Toxocara IgG antibodies, and intestinal helminth infections were determined by stool microscopy.

Findings

Children aged 4 to 11 years were studied, of whom 47% were seropositive for anti-Toxocara IgG; eosinophilia >4% occurred in 74.2% and >10% in 25.4%; 59.6% had elevated levels of total IgE; 36.8% had sIgE≥0.70 kU/L and 30.4% had SPT for at least one aeroallergen; 22.4% had current wheezing symptoms. Anti-Toxocara IgG was positively associated with elevated eosinophils counts, total IgE and the presence of specific IgE to aeroallergens but was inversely associated with skin prick test reactivity.

Conclusion

The prevalence of Toxocara seropositivity was high in the studied population of children living in conditions of poverty in urban Brazil. Toxocara infection, although associated with total IgE, sIgE and eosinophilia, may prevent the development of skin hypersensitivity to aeroallergens, possibly through increased polyclonal IgE and the induction of a modified Th2 immune reaction.     

A Protein Allergen Microarray Detects Specific IgE to Pollen Surface,Cytoplasmic, and Commercial Allergen Extracts

Background

Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens.

Methodology/Principal Findings

We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences.

Conclusions/Significance

These results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy.     

High Serum β-Lactams Specific/Total IgE Ratio Is Associated with Immediate Reactions to β-Lactams Antibiotics

Total serum IgE result from the combination of specific and non-specific pools. High specific/total IgE ratio may reflect high level of allergen-specific IgE on mast cells. No data regarding its applications to drug allergies is available. One hundred seventy-one patients with a history of immediate reactions to β-lactams, confirmed by positive skin testing, and 122 control subjects tolerant to β-lactams, were studied. CAP System was used for the detection of serum total and specific IgE antibodies. The specific/total IgE ratio was tested for diagnostic accuracy compared with conventional criteria. We finally performed a simulation study to expand our investigation of the performance of the specific/total IgE ratio index in a scenario in which the new CAP detection threshold is lowered further. Specific/total IgE ratio values ≥0.002 were observed more frequently in reactive than in controls. Seventy-four of 80 subjects with values ≥0.002 were allergic to β-lactams, yielding a positive predictive value of 92.5%. The application of specific/total IgE ratio significantly improves the positive likelihood ratio and the overall diagnostic performance. In addition, we showed the capability of this new criterion to identify true reactive patients even among subjects with high levels of total IgE (>200 kU/L). Significant increase in both receiver operator characteristic (ROC) curve and sensitivity were observed in imputed case of the simulation study. The β-lactams-specific/total IgE ratio may be an additional index compared to the common criterion of positivity to a single hapten in the allergological work-up of patients with β-lactams immediate adverse reactions.     

Diagnosis of <Emphasis Type="Italic">Chenopodium album</Emphasis> allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.     

The determination of allergen-specific IgE and IgG antibodies in patients sensitized to the house dust mite Dermatophagoides pteronyssinus

45 patients, hypersensitive to house-dust mites, were examined by the method of skin tests to D. pteronyssinus allergen. Besides, in their blood sera the levels of allergen-specific IgE antibodies were determined in the radioallergosorbent test and allergen-specific IgG antibodies, in the enzyme immunoassay. These tests revealed that in 91% of the patients the results of skin tests were positive, in 68% an elevated level of specific IgE antibodies and in 93% of the patients an elevated level of specific IgG antibodies were detected. All patients showed the positive result in one of the above-mentioned tests. The largest group of the patients (55%) included persons showing the positive result of the skin test and having elevated levels of allergen-specific IgG and IgE antibodies. Thus, in cases of hypersensitivity to house-dust mites the levels of allergen-specific IgG and IgE antibodies in the patients' blood sera should be determined.     

Provocation testing with recombinant allergens

In the past few decades, DNA technology has enabled the production of defined recombinant allergen molecules for diagnostic and therapeutic purposes. Recombinant allergens containing most of the relevant IgE epitopes present in natural allergen sources are now available and allergen proteins can be produced that are identical, without biological or batch-to-batch variation. A great advantage of recombinant allergens is that they can be used for component-resolved diagnostics, which makes it possible to establish the patient's individual IgE reactivity profile before therapy is selected. However, before recombinant allergens can be applied in clinical practice their biological activity has to be carefully investigated in vivo. We here describe the most commonly used provocation methods (skin tests (prick and intradermal), nasal, bronchial, and conjunctival provocations) and how they can be performed. We also discuss the results so far obtained with in vivo testing using recombinant allergens and envisage their future use for immunotherapy.     

Seasonal variation in food allergy to apple

The aim of the study was to investigate the possibility of a seasonal variation in reactivity to apples in 27 birch pollen allergic patients. Before and during the birch pollen season 1998, the patients were subjected to double-blind, placebo-controlled food challenges (DBPCFCs) with grated fresh Golden Delicious apple followed by an open food challenge with whole fresh apple. The clinical reactions elicited during the challenges were evaluated both by the patients and the investigators. Moreover, the skin reactivity and the in vitro reactivity to apple were evaluated by skin prick test (SPT), leukocyte histamine release (HR), measurement of specific IgE, and immunoblotting experiments. The sensitivity of the DBPCFC, when compared with the result of the open challenge, was 0.74 (14/19) before the season and 0.80 (16/20) during the season. None of the patients reacted to the blinded challenge without a subsequent reaction to the open challenge. One placebo reaction was registered both before and in season, but not in the same patient. The patient scores of the first positive challenges, and the maximal scores of each combined blinded and open challenge session, were significantly increased during the pollen season (P<0.05). The scores of the open challenge were significantly higher than the scores of the DBPCFC both before the season and during the in-season challenges (P<0.05). Specific IgE against Golden Delicious increased during season (P<0.05), while neither SPT, HR, nor immunoblotting experiments could confirm an increase in reactivity. In conclusion, the results of the oral challenge tests indicated an increase in clinical reactivity to apples during the birch pollen season in birch pollen allergic individuals.     

Risk Factors Associated with the Development of Atopic Sensitization in Indonesia

Background

The prevalence of allergic diseases has increased not only in high income but also in low-to-middle income countries. However, risk factors for their development are still not well established, particularly in the latter.

Objective

To assess prevalence and identify risk factors for sensitization to two major inhalant allergens among children from semi-urban and rural areas in Indonesia.

Method

A cross-sectional survey was performed among 1,674 school children aged 5–15 years old. Information on potential risk factors and reported allergic symptoms were obtained by questionnaire. Helminth infections were assessed. Skin prick tests (SPT) were performed, total IgE as well as allergen-specific IgE for house dust mite (HDM) and cockroach were measured.

Result

The prevalence of allergic skin sensitization to both aeroallergens was significantly higher in the semi-urban than in the rural area. However, serum IgE against HDM and cockroach as well as total IgE were significantly lower in semi-urban than in rural children. In the semi-urban area, there was a significant positive association between SPT to HDM and higher paternal education but a negative one with hookworm infection. The risk factors linked to cockroach sensitization were different: being of a farmer offspring and lacking access to piped water were associated with an increased risk for a positive SPT to cockroach. No significant associations between measured risk factors and having a positive SPT were found in the rural area.

Conclusion

Sensitization to HDM and cockroach is common in Indonesia, more often translating into a positive SPT in the semi-urban than in the rural setting. Whereas high paternal education and low hookworm infection were associated with increased risk of SPT to HDM, we were surprised to find parameters of lower SES were identified as risk factor for cockroach SPT.     

Disruption of allergenic activity of the major grass pollen allergen Phl p 2 by reassembly as a mosaic protein

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.     

Latex sensitivity in a macaque (Macaca mulatta)

BACKGROUND AND HISTORY: An adult Macaca mulatta was examined because of a history of multiple episodes of conjunctivitis and an acute, pruritic, dermatitic eruption that affected the axillary and inguinal regions, forearms, thorax, and neck. METHODS AND RESULTS: Results of corneal staining, examination of skin scrapings and feces, fungal culture, CBC, and a thyroid profile (thyroxine/triiodothyronine concentrations) were negative or normal, with the exception of eosinophilia (1,040/mm3). Examination of a punch biopsy specimen of the skin indicated chronic, nonsuppurative eosinophilic dermatitis. Skin patch testing against 25 contact allergens was negative for a delayed-type hypersensitivity reaction. Allergen-specific IgE testing, using six monkey chow additives, also yielded negative results, but testing against latex revealed a strong positive result (0.74 KU/L) consistent with a latex allergy. A skin prick test performed by use of a latex supernatant revealed significant inflammation at the latex site at 72 h and one week. Vinyl gloves were substituted for latex gloves, and that resulted in a marked decrease in erythema, pruritus, and lichenification with no flares of dermatitis for four years. Repeat skin biopsy fourteen weeks after the original biopsy revealed normal epidermis; however, mild chronic active nonsuppurative, perifolliculitis persisted. CONCLUSION: Latex can induce allergic dermatitis in nonhuman primates and should be included in the differen tial diagnosis for atopic dermatitis.     

Improving diagnostic tests for food allergy with recombinant allergens

Food allergy is one important manifestation of atopic allergy. Primary food allergy mainly affects young children (class I), whereas adults frequently develop food allergy as a consequence of an inhalant sensitization (class II). At present, the diagnostic instrument for proving class II food allergy is not satisfactory. Skin tests as well as serological tests are in general neither very specific nor highly sensitive because they depend on food extracts, which differ in their content of individual allergens, vary between manufacturers, and even between different batches. Since the presence of food allergen-specific IgE antibodies does not always correlate with clinical symptoms against the respective food, oral provocation tests (ideally double-blind placebo-controlled food challenges) have to be performed to validate serological diagnosis or skin tests. However, oral provocation tests are connected to several practical problems and include a specific risk for the allergic patient. Applying DNA technology, up to 40 food allergens have been produced in recombinant form, which implies standardized quality and unlimited quantity of the respective proteins. Hence, such molecules might be used to solve problems of clinical and molecular allergology in diagnosis, research, and therapy of class II food allergies. First experiments with recombinant food allergens in this respect appear very promising.     

Correlation between airborne pollen incidence,skin prick tests and serum immunoglobulins in allergic people in cracowm,poland

Fifty patients suffering from pollen allergy were observed during the period 1988–1989. At the same airborne pollen content was examined using volumetric and gravimetric sampling methods.

The mean daily symptom scores and percentages of patients with symptoms of allergy during the pollen season correlated positively with the airborne pollen content of wild Gramincae and Cerealia (Secale cereale).

Skin prick test results and elevated levels of specific serum IgE confirmed the presence of allergy to these taxa in the patients observed. Allergy to tree pollen and herb pollen was confirmed in only 3 cases.

Elevated levels of specific serum IgE for Dactylis glomerata, Pltleum pratense, and Secale cereale appeared in 70–80% of patients. Specific serum IgE levels correlated more with skin prick reactions (especially with late phase reactions) than with total serum IgE levels. Airborne pollen concentrations evaluated by volumetric sampling (Burckard trap) ad by a gravimetric method differ in so many details, that their results are not comparable. However, both methods provide largely similar information about the presence of airborne pollen presence.     

Focus: Allergic Diseases and Type II Immunity: Development of New White Fish Allergy after Bone Marrow Transplantation from a Non-atopic Donor

Background: Transplant-acquired food allergy has become increasingly recognized in solid organ and bone marrow transplantation. As food allergy has no cure and causes considerable impact on the lives of patients who require strict avoidance of foods to avoid potentially severe or fatal reactions, it is crucial for physicians to better understand the risk factors and mechanisms driving development of food allergy post-transplant. We report a case of new food allergy to whitefish in an elderly patient post-bone marrow transplant in which neither donor nor recipient had a history of atopy. Methods: A 70-year-old man experienced an anaphylactic reaction to Swai whitefish (Pangasius hypophthalmus) 6 months post-transplant that he had previously tolerated on multiple occasions both pre-transplant and in the preceding months post-transplant. This allergy was investigated by commercial serum specific IgE testing and fresh prick-to-prick skin test to Swai whitefish. Results: Fresh prick-to-prick demonstrated large positive reaction to the Swai whitefish with wheal of 10 mm and flare of 22 mm compared to positive histamine control with a wheal/flare of 5x8mm. Serum specific IgE testing to commercial whitefish was negative (specific IgE <0.10kU/L). The patient continues to strictly avoid Swai whitefish but tolerates all other fish and shellfish. Conclusions: The unique development of specific Swai whitefish allergy in an elderly man after bone marrow transplant where both donor and recipient had no prior history of atopy strongly supports transplant-related immunomodulation as a major mechanism for transplant-acquired allergy and suggests that that absence of atopy or advanced age may not necessarily be protective.     

Evaluation of a spontaneous canine model of immunoglobulin E-mediated food hypersensitivity: dynamic changes in serum and fecal allergen-specific immunoglobulin E values relative to dietary change

The purpose of the pilot study reported here was to evaluate serum and fecal total and allergen-specific immunoglobulin E (IgE) responses to dietary change in five Maltese x beagle dogs with suspected food hypersensitivity, compared with those of five clinically normal dogs. Clinical parameters (pruritus, otitis, and diarrhea) improved in the Maltese x beagle dogs during feeding of a novel diet, and signs were exacerbated by oral allergen provocation. Relative concentrations of serum and fecal wheat-, corn-, and milk-specific IgE were determined by use of an ELISA. The onset of clinical signs of disease was accompanied by an increase in serum allergen-specific IgE concentrations. In contrast, changes in clinical signs of disease or allergen-specific IgE values were not seen in the control group undergoing the same regimen. Total serum IgE concentration was measured by use of the ELISA, and comparison with known quantities of a monoclonal IgE allowed absolute values to be reported. Values were high in the Maltese x beagle colony (7 to 34 microg/ml), compared with those in the control dogs (0.7 to 6 microg/ml). Total serum and total fecal IgE concentrations did not change in either group during the study. Although allergen-specific IgE was detected in the feces of both groups, significant interassay variability made interpretation of the results difficult. The authors concluded that these Maltese x beagle dogs satisfied the currently recognized clinical criteria for the diagnosis of canine food hypersensitivity. Furthermore, the clinical and serologic responses seen in these dogs in response to oral allergen provocation suggest that this may be a useful model for the study of spontaneous food hypersensitivity.     

Use of solid-phase immunoenzyme analysis for determining allergen-specific IgE antibodies

An ELISA system for the detection of allergen-specific IgE antibodies to ragweed allergen has been developed. The system is highly sensitive and specific. Ragweed pollen allergen has been obtained by the dialysis of water-soluble extract through a kidney membrane. The high molecular fraction of ragweed allergen, showing the whole of the allergenic activity detected by skin tests in untreated patients, has been used for coating polystyrene assay plates. To detect IgE antibodies to ragweed allergen, the conjugate of sheep anti-IgE antibodies with horse-radish peroxidase has been used. The level of allergen-specific IgE antibodies has been determined on the basis of the data on the optical density of the samples in comparison with that of the normal sera. The correlation factor of the results obtained in the assay of specific IgE antibodies with the newly developed assay system and with the commercial kit Phadezyme RAST manufactured by Pharmacia AB (Sweden) has proved to be 0.82 at n = 39, p less than 0.01, while the variation factor in the reproduction of the assay results has proved to be 12% at n = 40.     

Natural History of Perceived Food Hypersensitivity and IgE Sensitisation to Food Allergens in a Cohort of Adults

Background

No longitudinal studies exist on the natural history of food hypersensitivity and IgE sensitisation to food allergens in adults.

Objective

To examine the natural history of food hypersensitivity, the natural history of IgE sensitisation to food allergens and to investigate the risk factors for new onset food hypersensitivity.

Methods

Food hypersensitivity was questionnaire-assessed in 2307 individuals (aged 20–45 years) from Iceland and Sweden during the European Community Respiratory Health Survey both at baseline and follow-up 9 years later. IgE food and aeroallergen sensitisation were assessed in a subgroup of these individuals (n = 807). Values of 0.35 kU/L and above were regarded as positive sensitisation.

Results

Food hypersensitivity was reported by 21% of the subjects and this proportion remained unchanged at follow-up (p = 0.58). Fruits, nuts and vegetables were the three most common causes of food hypersensitivity, with a similar prevalence at baseline and follow-up. The prevalence IgE sensitisation to food allergens decreased in general by 56% (p<0.001) and IgE sensitisation to peanut decreased in particular by 67% (p = 0.003). The prevalence of timothy grass IgE sensitisation decreased by 15% (p = 0.003) while cat, mite and birch IgE sensitisation did not decrease significantly. Female sex, rhinitis, eczema and presence of IgE sensitisation to aeroallergens were independently associated with new onset food hypersensitivity.

Conclusion

The prevalence of food hypersensitivity remained unchanged while the prevalence of IgE sensitisation to food allergens decreased in adults over a 9-year follow-up period. The decrease in prevalence of IgE sensitisation to food allergens was considerably larger than the change in prevalence of IgE sensitisation to aeroallergens.     

Airway exposure levels of lipopolysaccharide determine type 1 versus type 2 experimental asthma

Allergic asthma is characterized by airway inflammation initiated by adaptive immune responses to aeroallergens. Recent data suggest that severe asthma may be a different form of asthma rather than an increase in asthma symptoms and that innate immune responses to LPS can modulate adaptive immune responses to allergens. In this study, we evaluated the hypothesis that airway exposure to different doses of LPS induces different form of asthma. Our study showed that neutrophilic inflammation and IFN-gamma expression were higher in induced sputum from severe asthma patients than from mild to moderate asthmatics. Animal experiments indicated that allergen sensitization with low-dose LPS (0.1 microg) induced type 2 asthma phenotypes, i.e., airway hyperresponsiveness, eosinophilic inflammation, and allergen-specific IgE up-regulation. In contrast, allergen sensitization with high-dose LPS (10 microg) induced asthma phenotypes, i.e., airway hyperresponsiveness and noneosinophilic inflammation that were not developed in IFN-gamma-deficient mice, but unaffected in the absence of IL-4. During the allergen sensitization period, TNF-alpha expression was found to be enhanced by both low- and high-dose LPS, whereas IL-12 expression was only enhanced by high-dose LPS. Interestingly, the asthma phenotypes induced by low-dose LPS, but not by high-dose LPS, were completely inhibited in TNF-alpha receptor-deficient mice, whereas the asthma phenotypes induced by high-dose LPS were abolished in the homozygous null mutation of the STAT4 gene. These findings suggest that airway exposure levels of LPS induces different forms of asthma that are type 1 and type 2 asthma phenotypes by high and low LPS levels, respectively.     

Alpha-Actinin Is a New Type of House Dust Mite Allergen

Main indoor allergens for humans are from house dust mites. There are more than 30 allergens in Dermatophagoides farinae but only fourteen allergens have been identified from this mite including Der f 1–3, 6, 7, 10, 11, 13–18, and 22. A native allergen protein (Der f 24, 90 kDa) was purified from D. farinae by gel filtration and anionic exchange liquid chromatography combined with IgE immunodetection. Its primary structure was determined by Edman degradation, mass spectrometry analysis and cDNA cloning. Enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, basophil activation test (BAT) and skin prick test (SPT) were performed to evaluate the allergenicity. It was identified as an alpha (α)-actinin containing a CaM-like domain with EF-hand motifs. Der f 24 reacted to sera from 85.4% (35/41) of patients on western blot analysis. It reduced ∼20% sera IgE reactivity to D. farinae extracts on a competitive ELISA. Eighty percent (8/10) of patients with D. farinae allergy showed positive reactions to Der f 24 in skin prick test. The expression of CD63 on basophils from patients was up-regulated by Der f 24 by ∼5.4-fold. Alpha-actinin was identified as a new type of house dust mite allergen. To the best of our knowledge, this is the first report of α-actinin as an allergen.     

A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients' IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.     

Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10-100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2-3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.