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Biomedical subjects

R Urbanek

Publications and source records attributed to R Urbanek.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effects of ambient ozone on lung function in children over a two-summer period.

There is a general consensus that short term exposure to ozone (O3) causes a decrease in lung function parameters such as forced vital capacity (FVC) and forced expiratory volume in one second (FEV1). The objective of this study was to assess the reproducibility of lung function decrements after ambient O3 exposure over a two-summer period. The authors studied 797 children with a mean age of 8.2 yrs (95% confidence interval: 6.9-9.5) from the second and third grades of ten elementary schools in Austria and southwestern Germany. At the outset the various study locations were stratified into three groups with low (L), medium (M) and high (H) O3 exposure (range of mean O3 concentration in the locations April-October 1994: 24-30 (L); 33-38 (M); 44-52 (H) parts per billion (ppb)). Four lung function tests were performed on each child between March 1994 and November 1995. The increases in FVC and FEV1 recorded from one test period to the next were expressed as mL x day(-1). A significantly lower FVC and FEV1 increase was observed in children exposed to high ambient O3 concentration during the summer season. (FVC in summer 1994: 0.83 (L); 0.56 (M); 0.55 (H) mL x day(-1); p=0.004; and summer 1995: 0.80 (L); 0.63 (M); 0.56 (H) mL x day(-1); p=0.011; FEV1 in summer 1994: 0.48 (L); 0.34 (M); 0.18 (H) mL x day(-1); p=0.004 and summer 1995: 0.68 (L); 0.45 (M); 0.41 (H) mL x day(-1), p=0.006). There was no significant difference in FVC or FEV1 increase between the groups during the winter period. Adjusting for sex, age, height and passive smoke exposure, linear regression revealed a statistically significant negative association of average ambient O3 concentration with the FVC and FEV1 increase in both summers. During the winter period no association of O3 with FVC or FEV1 was observed. In conclusion, in two consecutive summer periods the authors found reproducible lung function decrements in children exposed to high levels of ambient ozone. Reoccurrence of ozone associated lung function deficits might increase the likelihood of persisting effects on the childrens' airways.

Child↗
PubMed2000

Direct evidence for transplacental allergen transfer.

Allergies are increasing, and despite deeper insights into the immunologic basis of these diseases, preventive measures are not yet efficient. As the induction of allergic diseases is often triggered in early childhood, perinatal or prenatal preventive strategies would be beneficial. We investigated the transfer of inhalant and nutritive allergens across the human placenta. For this purpose, the maternal side of a placental cotyledon was perfused in vitro with an allergen-containing medium, and a specific ELISA was used to detect the allergens on the fetal side. Both allergens evaluated, birch pollen major allergen Bet v1 and the milk allergen beta-lactoglobulin, could be shown to cross the placenta. The nutritive allergen beta-lactoglobulin was not only transferred across the placenta in all eight experiments, but was also detectable within the first minutes of perfusion. The peak allergen concentration on the fetal side could be increased by addition of human immunoglobulin. For the inhalant allergen Bet v1, transfer was observed in two of 10 placental experiments, and only if human immunoglobulin was added. A pulsatility wave with a frequency of 30-35 min suggested an active transfer mechanism. We conclude that allergens are actively and selectively transferred across the placenta. Therefore, controlled maternal allergen exposure might offer new ways to induce tolerance to specific allergens in the fetus.

Allergens↗
PubMed2000

[Incidence of osteoporosis in patients with rheumatoid arthritis].

Juxta-articular osteoporosis in rheumatoid arthritis (RA) is an early and specific pathology in this disease, however in RA patients there are also factors predisposing for the development of the generalised bone atrophy. 103 female patients with the diagnosis of RA established according to ACR criteria were included in the study. Its aim was to evaluate the generalised osteoporosis incidence in women suffering from RA as well as to find out how osteoporosis was related to certain clinical symptoms and laboratory findings, the disease process activity, and the treatment used. The mean age of the patients was 57.6 +/- 12 years, and the disease duration was 11.4 +/- 9 years. The bone mineral density (BMD) was measured by DEXA method with a LUNAR device and included: the radial bone in its 1/3 of the shaft's distal part; L1-L4 vertebral region; and the proximal part of the femoral bone. The division into subgroups was done basing on the 1994 WHO criteria. In RA female patients generalised osteoporosis was observed more frequently than in the control group. The study discovered a negative, statistically significant, correlation between BMD values and: the disease duration, the time of joint involvement acc. to Steinbrocker, the degree of functional impairment, the presence of extra-articular lesions, periods of immobilisation. There was also a dependence found upon the presence of positive Waaler-Rose reaction, and upon the therapy with Methotrexate and D-penicillamine, and when the patients were treated with glicocorticosteroids--upon the cumulative dose.

Absorptiometry, Photon↗
PubMed1999

Latex sensitization in spina bifida appears disease-associated.

OBJECTIVE: The high prevalence of latex sensitization (up to 80%) in patients with spina bifida (SB) has been attributed to repeated exposure to latex products, whereas disease-associated factors have not been considered. METHODS: We compared children with SB (n = 21) and children with posthemorrhagic or congenital hydrocephalus (PH, n = 32), all of whom had a ventriculoperitoneal shunt since young age. Latex sensitization, number of operations, atopic history, and total IgE levels were evaluated. RESULTS: The following characteristics were recorded: age (SB: 52 months, range 1 to 264 months; PH: 71 months, range 1 to 192 months) and mean number of operations (SB: 2. 09; PH: 2.53). Of the SB group, 43% (9 of 21) showed elevated latex-specific IgE antibodies in contrast to 6% (2 of 32) in the PH group (P <.01). Latex-specific IgE antibodies were detected by 1 year of age, and one surgical operation was sufficient to induce latex-specific IgE-antibody production in patients with SB. CONCLUSIONS: The results suggest that the SB population bears a disease-associated propensity for latex sensitization. Sensitization to latex antigens may occur after the very first contact, arguing for latex avoidance measures from the very beginning of life.

Adolescent↗
PubMed1999

Assessment of serum myeloperoxidase in children with bronchial asthma.

BACKGROUND: The role of neutrophils and myeloperoxidase (MPO) - assumed to be a marker of neutrophil activation - in bronchial asthma is still unclear, and the literature is controversial. METHODS: To investigate the participation of neutrophils and their products in childhood asthma, we assessed neutrophil counts and serum MPO in 175 children with bronchial asthma. Ninety of them were asymptomatic, and 85 of them were symptomatic within the last 2 weeks before examination. Bacterial infection of the lower respiratory tract (LRTI) was present in 34 and viral infection in 49 patients. As controls, 45 patients with cystic fibrosis, 23 patients with bacterial LRTI, and 87 healthy children were recruited. RESULTS: Median neutrophil counts (3135 cells/microl) and serum MPO levels (352 microg/l) were not different in children with bronchial asthma from healthy controls (2220 cells/microl and 401 microg/l, respectively), whereas in patients with cystic fibrosis and bacterial LRTI, neutrophil counts and MPO levels were increased. Asthmatic children with bacterial infection had significantly higher serum MPO and neutrophil counts then asthmatic children with viral infection or without infection. In addition, a significant correlation was found between serum MPO and neutrophil counts and C-reactive protein (CRP), and between neutrophil counts and CRP, but no relationship was detected for serum MPO and disease activity or lung function. CONCLUSIONS: Our data indicate that serum MPO - a marker of neutrophil activation - does not contribute to the assessment of the inflammatory process in childhood asthma. In addition, measurement of serum MPO appears not to be useful in assessing the participation of the neutrophil in asthmatic children. However, assessment of MPO may be useful to distinguish between bacterial and viral infection.

Adolescent↗
PubMed1999

In vivo histamine release during the first minutes after deliberate sting challenges correlates with the severity of allergic symptoms.

In this study, deliberate sting challenge was investigated as a method for estimating the severity of anaphylactic reactions in bee venom-sensitized subjects. Twenty-one patients with previous anaphylactic reactions to field bee sting were subjected to a deliberate sting challenge (n = 32). To document anaphylactic reactions, plasma histamine levels were measured before, and then 1 and 2 min after, bee sting challenge. Eleven patients were re-challenged after 3-5 weeks. On 18 occasions, sting challenges caused no systemic reactions, in seven cases reactions were mild, in five moderate and in two severe. In all children showing systemic reactions, significant increases of plasma histamine were measured after 2 min. The results correlated significantly with clinical scores but not with skin prick test or with specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibodies against bee venom. In patients developing local reactions only, no increase of plasma histamine was detected. The relative amount of released histamine correlated significantly with the severity of clinical symptoms. Significant histamine release occured during the first 2 min after sting challenge in children with subsequent systemic reactions and the severity of these subsequent anaphylactic reactions correlated with plasma histamine concentrations. The measurement of plasma histamine levels in the first minutes after challenge test may therefore be used as an objective marker of a potential systemic reaction.

Adolescent↗
PubMed1999

Cord blood leucocytes/basophils produce and release sulfidoleucotrienes in response to allergen stimulation.

BACKGROUND: Leucocyte-derived sulfidoleucotrienes (SLT) from children and adults can be detected in vitro in response to specific allergen stimulation, a mechanism thought to require the presence of allergen-specific immunoglobulin (Ig)E antibodies on the surface of basophils. It is unknown whether this mechanism is functional in cord blood basophils. OBJECTIVE: We studied the in vitro SLT-release of leucocytes in response to allergen and anti-IgE stimulation in term newborns and children with allergic diseases. METHODS: Cord blood from randomly selected term newborns were analysed for total IgE-antibodies and in vitro SLT-release in response to allergen and anti-IgE stimulation. Children from an allergy outpatient clinic were used as the control group. The Cellular Allergen Stimulation Test (CAST) was used as read-out system. Allergen stimulation was performed with an allergen-mix containing 21 nutritive and inhalant allergens. RESULTS: Peripheral blood leucocytes/basophils derived from allergic children (n = 56; median SLT release 1049 pg/mL) were more responsive to anti-IgE stimulation as cord blood leucocytes/basophils (n = 104; median 419 pg/mL P < 0.0001). In response to stimulation with an allergen-mix, the two groups did not differ significantly from each other. Only SLT-releasability in response to anti-IgE showed a correlation with cord blood IgE. CONCLUSIONS: Sulfidoleucotriene-release of cord blood basophils is functional in response to allergens. It appears possible that cord blood basophils are armed with allergen-specific IgE-antibodies though not detectable in serum. Therefore, cord blood SLT-release may indirectly reflect prenatal priming with allergens with subsequent production of allergen-specific IgE.

Allergens↗
PubMed1999

Lung function growth and ambient ozone: a three-year population study in school children.

We followed a cohort of 1,150 children for 3 yr to investigate long-term effects of ambient ozone. Nine study sites were selected on the basis of air-quality data to represent a broad range of ozone exposure. In 1994, 1995, and 1996 lung function was recorded biannually, always before and after summertime. The effect of ozone was analyzed with regression analyses and study-site, a child's sex, atopy, passive smoking, baseline lung function, and increase in height were considered as confounding variables. A negative effect of summertime ozone on the pre- to post-summer-time change in FEV(1) (ml/d) was present in 1994 (beta = -0.019 ml/d/ppb; p < 0.01) and in 1995 (beta = -0.017 ml/d/ ppb; p < 0.05), but not in 1996 (beta = 0. 004 ml/d/ppb; p = 0.6); corresponding estimates for FVC were in 1994: beta = -0.022 ml/d/ppb, p < 0.005; 1995: beta = -0.018 ml/d/ppb, p < 0.05; and 1996: beta = 0.006 ml/d/ppb, p = 0.46. When all three study years were considered simultaneously, i.e., the changes in lung function between each of two subsequent surveys being the dependent variable, summertime ozone was associated with a lesser increase in FEV(1) (beta = -0.029 ml/d/ppb; p < 0.001), FVC (beta = -0.018 ml/d/ppb; p < 0.001), and MEF(50) (beta = -0.076 ml/s/d; p = 0.001). No consistent associations were observed for lung function and NO(2), SO(2) and PM(10). Long-term ambient ozone exposure might negatively influence lung function growth.

Air Pollutants↗
PubMed1998

Cord blood mononuclear cells and milk-specific T-cell clones are tools to evaluate the residual immunogenicity of hydrolyzed milk formulas.

BACKGROUND: Hydrolyzed milk formulas (HFs) are given to infants allergic to cow's milk proteins and, for preventive reasons, to atopy-prone newborns for which breast feeding is not feasible. The ultimate properties of HFs are not only a reduced allergenicity but also decreased immunogenic capacity combined with good taste and caloric value. No information is available concerning the capacity of HFs to induce immune responses. OBJECTIVE: We sought to determine the residual immunogenic capacity of partially (pHF) and extensively hydrolyzed milk formula (eHF), and we studied the cellular reactivity of cord blood-derived (n = 71) mononuclear cells induced by 10 different HFs. METHODS: To test the effect of HF on T-helper cells, beta-casein-specific T-cell clones (TCCs, n = 21) from individuals allergic to milk were established, and T-cell proliferation and cytokine profiles (interferon-gamma and IL-4) were determined on stimulation with HF. RESULTS: We found significantly reduced proliferative responses of eHF compared with milk proteins. Whey-based pHF displayed the same proliferative capacity as unmodified milk proteins. As expected, extensively processed whey products displayed lower cellular responses compared with partially hydrolyzed products (pHF whey vs eHF whey, p < 0.0001). No difference in cellular response was found between casein-based pHF and casein-based eHF. Beta-casein-specific TCCs (n = 21) proliferated in response to casein-derived hydrolysates (14% with casein/whey-based pHF, 4% with casein-based pHF, and 0% with casein-based eHF). Whey-based pHF was also found to induce proliferation in beta-casein-specific TCCs, indicating the presence or the generation of peptides displaying cross-reactivity with these whey-derived hydrolysates. TCCs stimulated with whey- or casein-based pHF or eHF produced the same amount of cytokines (IL-4, interferon-gamma) as the same clones stimulated with unmodified products. CONCLUSION: Our data indicate that whey- and casein-derived eHFs display highly reduced immunogenic properties at the T-cell level. In contrast, pHFs display residual immunogenic properties detectable at the T-cell level, reflecting a potential for the induction of pathogenetically important T-cell responses.

Adult↗
PubMed1997

IgE-mediated allergic reaction to hyaluronidase in paediatric oncological patients.

UNLABELLED: Hyaluronidase has been gaining increasing interest as a spreading factor for better penetration of chemotherapeutics into CNS tumours. Five out of 16 patients with CNS tumours treated with hyaluronidase in addition to chemotherapeutic agents developed symptoms of immediate type allergic reactions, therefore we sought to characterize the harmful allergenic proteins of the bovine testes hyaluronidase enzyme preparation (Neopermease). The role of specific IgE for the allergic reaction was investigated. Using an immunoblotting technique, we investigated sera from 16 children treated with Neopermease (5 of them having developed anaphylactic reactions), 5 patients with atopy (atopic eczema) with high total IgE levels and 4 healthy children. SDS-PAGE of hyaluronidase preparation Neopermease revealed two major bands at 73 and 41-43 kDa. In all 5 sera from patients with adverse reactions, binding of specific IgE antibodies to have 73 and 41-43 kDa bands was found. Two patients reacted with the 73 kDa band exclusively, two patients reacted with both bands, one patient displayed IgE only to the 41-43 kDa band. A specific inhibition of IgE-binding to both bands was achieved after preincubation of the sera in four out of five patients with partially purified bovine hyaluronidase. Furthermore preincubation with gelatin, a stabilising agent in the commercial extract, led to a partial inhibition in the sera of three patients. No specific IgE binding was detected either in the sera of atopic patients, or in the control group. CONCLUSION: IgE mediated allergic reactions to hyaluronidase may occur in paediatric oncological patients treated with hyaluronidase. Whether these children are sensitized by intravenous hyaluronidase treatment or by cross-reactivity of other preformed IgE antibodies, yet to be specified, remains to be elucidated.

Adolescent↗
PubMed1997

Cytokine concentrations in sputum from patients with cystic fibrosis and their relation to eosinophil activity.

To evaluate whether concentrations of cytokines supposed to be involved in eosinophil recruitment and activation were elevated in cystic fibrosis (CF), we assessed interleukin-3 (IL-3), IL-5, IL-8, regulated on activation, normal T-cell expressed and secreted (RANTES); and granulocyte-macrophage colony stimulating factor (GM-CSF) in sputa from 32 patients with CF, eight patients with atopic bronchial asthma, and six patients with bacterial pneumonia. In addition, eosinophil cationic protein (ECP) and eosinophil protein X (EPX) were measured as markers of eosinophil activation. In patients with CF, sputum levels of IL-8 were elevated (p < 0.01) as compared with asthmatic patients. Concentrations of IL-3, ECP, and EPX were not different in the two groups. However, IL-5 (p < 0.0001), RANTES (p < 0.003), and GM-CSF (p < 0.0001) were significantly lower in the CF group than in subjects with asthma. IL-5 was detected only in sputum samples from CF patients with Aspergillus sensitization. In patients with pneumonia, IL-8 levels only were increased. In CF sputum, ECP levels were significantly correlated with the levels of IL-8 (r = 0.626, p < 0.0001) and IL-3 (r = 0.642; p < 0.0001), whereas in asthmatic patients IL-5, IL-8, and RANTES concentrations were significantly related to ECP in sputum. These findings suggest that different cytokine profiles are responsible for eosinophil activation in patients with CF as compared with asthmatic patients. In CF, IL-8 and IL-3 appear to be responsible for increased degranulation of eosinophils.

Adolescent↗
PubMed1997

Traffic-related NO2 and the prevalence of asthma and respiratory symptoms in seven year olds.

The aim of this study was to determine whether outdoor nitrogen dioxide (NO2) was associated with the prevalence of asthma and respiratory symptoms. In eight nonurban communities, 843 children resident for a minimum of 2 yrs were studied. Since industrial sources of air pollution were at least 20 km away from the study communities, NO2 was considered to primarily indicate traffic-related air pollution. NO2 was recorded at central monitors, and the 3 yr mean exposure was calculated. Asthma and respiratory symptoms were assessed according to the International Study on Asthma and Allergy in Childhood. Prevalence of asthma at some time ("ever asthma") was associated with long-term NO2. In parallel with increasing levels of NO2 (community specific 3 yr mean 6.0-17.0 parts per billion (ppb)), asthma prevalence was 2.5, 1.4, 1.6, 2.3, 3.4, 3.6, 7.6 and 8.5%, respectively (p=0.002 for trend). The prevalence odds ratios (PORs) for "ever asthma", following adjustment for gender, age, parental education, passive smoke exposure, type of indoor heating, and parental asthma, were 1.28 (95% confidence interval (95% CI) 0.20-7.98), 2.14 (95% CI 0.40-11.3) and 5.81 (95% CI 1.27-26.5), when each of two communities with low, regular and high NO2, respectively, were compared with the two communities with very low NO2. For symptoms "wheeze" (adjusted PORs for increased NO2: 1.47, 1.23 and 2.27) and "cough apart from colds" (adjusted PORs for increased NO2: 1.49, 1.93 and 2.07), a similar trend was seen. In this study a significant relationship was observed between traffic-related nitrogen dioxide and the prevalence of asthma and symptoms. Whether this association is causal has to be tested in longitudinal studies.

Asthma↗
PubMed1997

Prenatal contact with inhalant allergens.

Pollen contact in early infancy may enhance the risk for subsequent pollen allergy. In this study likelihood of a prenatal antigen contact, as a result of inhalation of pollen allergens by the mother, was investigated. Due to the seasonal occurrence of allergens studied, the date of priming can be estimated, and this can supply data about the maturation of the fetal immune system. Proliferative responses of umbilical cord blood mononuclear cells (UCB MNCs) to the recombinant major allergens of birch (rBet v 1) and timothy grass (rPhl p 1) were analyzed throughout the whole year. A positive proliferative response was regarded as the criterion for a prenatal contact of the immune system with the allergen. Prenatal priming with both allergens was observed. Timothy grass pollen displayed considerably higher antigenicity than did birch pollen. The susceptibility of the fetal immune system to be primed by these allergens varies during the gestation period. The majority of positive responses to rPhl p 1 and rBet v 1 were found in UCB samples in which antigen contact (the respective pollen season) took place in the first 6 mo of pregnancy. Our results offer indirect evidence that, shortly after migration of T cell precursors to the epithelial thymus, T cells are mature enough for priming with antigens. No relationship was found between the susceptibility of the fetal immune system to be primed by these allergens and the clinical history of the family concerning type I allergy.

Allergens↗
PubMed1997

Prenatal allergen contact with milk proteins.

BACKGROUND: Cellular proliferation to various allergens (Dermatophagoides pteronyssinus, beta-lactoglobulin, bovine serum albumin, ovalbumin) has been found in cord blood cells. Whether this reflects a sensitization during foetal life is uncertain. OBJECTIVE: We studied the cellular reactivity and cytokine production of cord blood cells in response to cow's milk proteins in a randomly selected group of newborns. The delineation of possible in utero allergen contact was attempted. METHODS: Cord blood mononuclear cells from 39 neonates were incubated with cow's milk proteins (alpha-lactalbumin, beta-lactoglobulin, casein, alpha-casein, beta-casein, kappa-casein, bovine serum albumin) for 7 days, and proliferation was assessed by incorporation of [3H]thymidine. Cord blood cell-derived interferon-gamma (IFN gamma) and interleukin-4 (IL-4) secretion was evaluated in response to allergen or phytohaemagglutinin (PHA) stimulation. RESULTS: A pronounced proliferation of cells stimulated with alpha-lactalbumin (ALA: mean stimulation index 8.0, 95% confidence interval 5.2-10.8), beta-lactoglobulin (BLG: mean stimulation index 5.9, 95% confidence interval 3.2-8.6) and alpha-casein (2.6, 95% confidence interval 2.9-9.1), as opposed to unstimulated cells in medium, was found. No correlation was found between cellular proliferation to milk proteins and parental atopy, maternal total IgE or cord blood IgE. IFN gamma production (but not IL-4) was inducible by PHA (range 429-1810 pg/ml), but only in one individual upon stimulation with BLG. Preferentially, reduced IFN gamma levels were found in individuals with positive parental allergic history. CONCLUSION: The recognition of allergen by cord blood cells indicates that allergen priming must occur prenatally. The relevance for subsequent sensitization is unclear.

Allergens↗
PubMed1995

Characterization of antigens and allergens in hypo-allergenic infant formulae.

The antigenicity and allergenicity of so-called hypo-allergenic infant formulae is mainly determined by the degree of hydrolysis and ultrafiltration. Five different formulae were investigated by means of immunoblotting and RAST in order to characterize the antigens and allergens regarding their molecular weights, molecular origin and their ability to bind human IgG and IgE antibodies: A non hydrolysed infant formula (I-F), a mixture of the major cow's milk proteins (PM), a whey-based infant formula (W-H), a whey-based and ultra-filtrated infant formula (U-H), a casein/whey-based infant formula (CW-H). By immunoblotting we demonstrated that all tested formulae still contain antigens with molecular weights from 3 to 67 kD. But when compared with I-F and PM the antigen content of the hydrolysed formulae was considerably lower. The lowest antigen content could be demonstrated in U-H, which contains casein fragments (3-6 kD) and beta-lactoglobulin and its fragments (6-18 kD). W-H and CW-H contain bovine serum albumin, beta-lactoglobulin, casein and their fragments (3-67 kD). All hydrolysed formulae tested showed a reduced IgE-binding capacity. Three out of 12 cow's milk allergic children possessed IgE binding to U-H or W-H, and 5 of them IgE against CW-H. Conclusion. The enzymatic hydrolysis plus ultra-filtration seems to be the most efficient method to reduce the antigen content of so-called hypo-allergenic infant formulae.

Allergens↗
PubMed1995

Subsequent insect stings in children with hypersensitivity to Hymenoptera.

To investigate the risk of life-threatening reactions to future stings, we sequentially challenged 113 children (aged 2 to 17 years) allergic to insect stings with a sting by the relevant insect. The time interval between the challenges varied from 2 to 6 weeks. The history of the index stings was a large local reaction (LR) in 16% and a systemic reaction (SR) in 84% of the test subjects. On the first challenge, 76% had a normal LR, 11% a large LR, and 13% an SR. On the second challenge, 78% of the children had a normal LR, 5% a large LR, and 17% an SR. Thirty-nine of the untreated children were exposed to a field sting during the subsequent 3-year follow-up period. In comparison with other diagnostic evaluations such as skin-prick tests, determinations of specific IgE and IgG antibodies, and single-sting exposure, the dual sting challenge scheme appears to be the best predictor of reactions to subsequent stings. It also appears to be helpful in selecting patients with an uncertain sensitization status for venom immunotherapy.

Adolescent↗
PubMed1995

Natural and recombinant enzymatically active or inactive bee venom phospholipase A2 has the same potency to release histamine from basophils in patients with Hymenoptera allergy.

BACKGROUND: A complementary DNA encoding the major bee venom allergen phospholipase A2 (PLA) has been characterized recently. Recombinant PLA was produced in Escherichia coli and purified to apparent homogeneity. Natural PLA was compared with recombinant PLA in its ability to release histamine from blood basophils. METHODS: A synthetic gene encoding the mature form of PLA was expressed in E. coli, and the polypeptide was purified to homogeneity by affinity chromatography and refolded, yielding fully enzymatically active PLA. In addition, we have produced a genetically engineered enzymatically inactive variant by substitution of a single amino acid residue in the catalytic center. A standard histamine release assay was used to compare the potency of natural PLA with correctly folded enzymatically active and inactive recombinant PLA to release histamine from blood basophils of nine patients with bee venom allergy. RESULTS: Recombinant enzymatically active PLA and purified natural protein were equally effective in releasing histamine from sensitized basophils. By comparing the histamine-releasing capacity of enzymatically active and inactive recombinant allergen, we further demonstrate that catalytic activity is not a requirement for allergenicity in the effector phase. Denaturation of natural PLA or incorrect folding of recombinant protein resulted in a total loss of allergenic potency. CONCLUSION: We demonstrate the feasibility of producing native-like recombinant allergens with or without enzymatic activity. We also provide evidence for the requirement of correct three-dimensional structure of PLA to induce histamine release from basophils and thus evidence for its recognition by IgE.

Bee Venoms↗