Allergenic epitopes of bovine αS1-casein recognized by human IgE and IgG

B-cell epitopes of bovine α_S1-casein, one of the major allergens of cow's milk, were identified by a screening method based on synthetic peptides. According to the known amino acid sequence of α_S1-casein, a set of 188 overlapping sequential decapeptides shifted by one amino acid was manually synthesized on polyethylene pins by the 9-fluorenyl-methoxycarbonyl (Fmoc) method. Peptides were screened by an enzyme-linked immunosorbent assay (ELISA) specific for human IgE and IgG. Bound antibodies were detected by successive incubation with up to three polyclonal antibodies, the last one conjugated to horseradish peroxidase. Tested sera were from 15 patients with acute clinical reactions to cow's milk and IgE-specific reactions to bovine α-casein in the ELISA and immunoblot. Sera from 10 healthy subjects without remarkable reactions to cow's milk proteins were used as controls. All sera from allergic subjects showed reactions with three regions of α_S1-casein, corresponding to amino acids 19–30, 93–98, and 141–150. Furthermore, individual sera showed reactions with other parts of the protein. No essential differences in the epitope specificity of IgE and IgG were found. Inhibition of IgE binding to α_S1-casein with soluble synthetic peptides confirmed the results and revealed peptide CN-2 as the most inhibiting one.     

Characterization of T cell epitopes in αs1-casein in cow''s milk allergic, atopic and non-atopic children

BACKGROUND: One to two percent of infants suffer from IgE-mediated allergic reactions against cow's milk proteins. Most children develop clinical tolerance, but approximately 15% are still allergic by the age of 10 years. Little is known about the T cell epitopes in individual cow's milk protein in relation to allergy and tolerance. OBJECTIVE: To identify T cell epitopes in alphas1-casein, the most abundant milk protein, and to investigate T cell responses toward these epitopes in allergic, atopic and non-atopic children. METHODS: Allergen-specific T cell lines (TCLs) were derived from peripheral blood mononuclear cells of 11 cow's milk allergic, nine atopic and nine non-atopic children. T cell responses were measured to alphas1-casein and to overlapping peptides (18-mers), spanning the alphas1-casein molecule. Proliferation was determined by incorporation of (3)H-thymidine, and cytokine production (IL-10, IL-13 and IFN-gamma) was measured by ELISA. RESULTS: Four main regions (amino acid (AA) residues 43-66, 73-96, 91-114 and 127-180) in the alphas1-casein molecule were immunogenic to T cells, among which the AA residues 133-156 spanned the immunodominant part. Only subtle differences were found in peptide recognition between the subject groups. Some of the peptides induced slightly Th1- or Th2-skewed cytokine responses. The increased levels of IL-10 in response to alphas1-casein observed in TCLs from atopic children appeared not to be linked to recognition of specific IL-10-inducing epitopes. CONCLUSIONS: The immunodominant sequence in alphas1-casein is spanned by AA residues 133-156. Tolerance towards alphas1-casein in atopic children may be mediated by an overall induction of IL-10 and not by recognition of certain T cell epitopes. The identified T cell epitopes in children with cow's milk allergy may be useful targets in developing peptide immunotherapy.     

Identification of IgE and IgG binding epitopes on β- and κ-casein in cow's milk allergic patients

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of children aged less than 2 years of age. Although beta- and kappa-casein are considered among the major allergens responsible for CMA, no data are available on their allergenic epitopes in humans. OBJECTIVE: The aim of the study was to identify IgE- and IgG-binding epitopes on beta- and kappa-casein and to determine whether the pattern of epitope recognition is associated with the natural history of CMA. METHODS: Overlapping decapeptides representing the entire length of beta- and kappa-casein, respectively, were synthesized on a cellulose-derivatized membrane. Sera from 15 milk-allergic children, 4-18 years of age, with high levels of specific IgE antibodies to cow's milk were used to identify IgE- and IgG-binding epitopes. In addition, IgE epitopes were screened with pooled or individual sera from younger patients aged less than 3 years and who had low levels of specific serum IgE, who are likely to outgrow CMA. RESULTS: Six major and three minor IgE-binding epitopes, as well as eight major and one minor IgG binding regions, were identified on beta-casein. Eight major IgE-binding epitopes, as well as two major and two minor IgG-binding epitopes, were detected on kappa-casein. Three of the IgE binding regions on beta-casein and six on kappa-casein were recognized by the majority of patients in the older age group, but not by the younger patients. CONCLUSION: Information regarding the immunodominant epitopes in beta- and kappa-casein may be important for understanding the pathophysiology and natural history of CMA. Differences in epitope recognition may be useful in identifying children who will have persistent milk hypersensitivity.     

Human α-lactalbumin and bovine β-lactoglobulin absorption in infants

We investigated gut permeability to human α-lactalbumin (ALA) and bovine β-lactoglobulin (BLG) in 20 infants from birth to 8 months or until weaning, before which they were on a strictly cow's-milk-free diet. We measured the proteins with a sensitive, solid-phase, double-sandwich immunofluorometric assay. Median (range) levels of serum ALA on days 3-4 after birth, and at 1 and 2 months of age were 31 (12-225), 6 (0–55), and 2 (0–16) μg/1 serum per g ALA given per kg body weight, respectively. At 3, 5, and 8 months of age, only trace amounts of ALA were found. One week after weaning, serum BLG was found in 5/13 infants (38%) and at 2 weeks in 3/14 infants (21°0), with median concentrations of 7 and 4 μg/1 serum per g BLG given per kg body weight, respectively. No ALA could be detected in any of these samples. In absorption of ALA, the four infants who had allergic symptoms did not differ from those without symptoms. Thus, systemic absorption of ALA and BLG does occur in infants. Absorption of ALA is greatest after birth, when 3 × 10⁴ (median) of the given antigens are absorbed, but absorption decreases rapidly. The gut may often be transiently permeable to BLG when cow's-milk-based formula is started.     

Role of conformational and linear epitopes in the achievement of tolerance in cow's milk allergy

BACKGROUND: Cow's milk (CM) is one of the leading causes of food allergy in children. However, approximately 85% of milk-allergic children become clinically tolerant to CM within the first 3 years of life. The mechanisms involved in the achievement of tolerance remain unknown. OBJECTIVE: To study whether IgE antibodies from children with persistent cow's milk allergy (CMA) differ from children who become clinically tolerant in their ability to recognize linear and conformational epitopes of alpha(s1)- and beta-casein. METHODS: Thirty-six milk-allergic children were included in the study: 11 of the children became clinically tolerant, and 25 had persistent CMA. Blood was obtained from all patients during the time they showed clinical reactions to milk challenge. Six non-milk-allergic children served as controls. Specific IgE antibodies against linear (denatured) as well as conformational (native) milk proteins were determined by probing dot-blots with patients' sera. In addition, selected decapeptides from alpha(s1)- and beta-casein, previously found to be suggestive of persistent CMA, were synthesized on a cellulose-derivatized membrane and probed with individual sera from 10 patients who outgrew CMA and from 10 patients with persistent CMA. RESULTS: Analysis of immunodot-blots showed that, in comparison to tolerant patients, milk-allergic children with persistent symptoms had a significantly higher ratio of specific IgE antibodies to linearized than to native alpha- and beta-casein (P < 0.005 and P < 0.02, respectively). Comparing the selected decapeptides, six of the 10 patients with persistent allergy recognized the peptide corresponding to amino acids 69-78 from alpha(s1)-casein while none of the patients who outgrew CMA had IgE binding to this epitope. CONCLUSION: Patients with persistent milk allergy possess higher detectable levels of IgE antibodies to linear epitopes from alpha(s1)- and beta-casein than children who have achieved tolerance. Specific IgE binding to particular linear epitopes in alpha(s1)-casein may be a predictive factor for persistence of CMA.     

Allergy to goat and sheep milk without allergy to cow's milk

BACKGROUND: Cow's milk (CM) allergy is the most frequent cause of food allergy in infants. Most children who are allergic to CM are also sensitized to whey proteins and/or to the casein fraction and many of them cannot tolerate goat's or sheep's milk (GSM) either. Conversely, the GSM allergies that are not associated with allergic cross-reactivity to CM are rare. METHODS: Twenty-eight children who had severe allergic reactions, including anaphylaxis, after consumption of GSM products but tolerated CM products were recruited in a retrospective study. Whole casein and whey proteins were fractionated from CM and GSM. beta-Lactoglobulin and the different caseins were isolated, purified and used to perform enzyme allergosorbent tests (EAST) and EAST inhibition studies with the sera of the allergic children. RESULTS: Clinical observations, skin prick testing and immunoglobulin (Ig)E-binding studies confirmed the diagnosis of GSM allergy without associated CM allergy. EAST determinations demonstrated that GSM allergy involves the casein fraction and not whey proteins. Cow's milk caseins were not at all or poorly recognized by the patient's IgE, while alphaS(1)-, alphaS(2)- and beta-caseins from GSM were recognized with a high specificity and affinity. In all cases, increasing concentrations of CM caseins failed to inhibit the binding of patient's IgE to sheep or goat milk caseins, whereas this binding was completely inhibited by GSM caseins. CONCLUSIONS: The characteristics of GSM allergy differ from those of the CM allergy because it affects older children and appears later. CM products do not elicit any clinical manifestation in GSM allergic patients, whereas CM allergic patients, usually cross-react to GSM. In all the GSM allergic children, the IgE antibodies recognized the caseins but not the whey proteins. Moreover, IgE specificity and affinity was high to GSM and lower to CM caseins despite their marked sequence homology. Doctors and allergic individuals should be aware that GSM allergy requires a strict avoidance of GSM and milk-derived products because reactions could be severe after ingestion of minimal doses of the offending food.     

Effect of terfenadine on TNFα release from peripheral blood mononuclear cells during cow's milk allergy

Background In infants with cow's milk allergy and intestinal symptotns, peripheral blood mononuclear cells stimulated in vitro with cow's milk proteins, secrete large amounts of the proinflammatory cytokine TNFα thus altering intestinal barrier capacity. Terfenadine, an antihistaminic drug, inhibits the release of several inflammatory mediators, including histamine, prostaglandins and leukotrienes. Objectives To test the potential ability of terfenadine to inhibit TNFα secretion by mononuclear cells from infants with cow's milk allergy. Methods Mononuclear cells from infants allergic to cow's milk proteins were stimulated in vitro for 6 days by a mixture of milk proteins (β-lactoglobulin, α-lactalbumin and casein) with or without terfenadine (0.1 –1 μM) and culture supernatants were assayed for TNFα by enzyme immunoassay. The effect of culture supernatants on intestinal barrier capacity was evaluated by measuring the electrical resistance (index of integrity) of filter-grown HT29–19 A intestinal cells in Ussing chambers. Results During active cow's milk allergy, mononuclear cells stimulated with cow's milk proteins secreted large amounts of TNFα which significantly reduced the electrical resistance of HT29–19 A intestinal cells. There was a dose-dependent decrease in TNFα secretion in the presence of terfenadine, with a maximal inhibition of 62% of this secretion at 1 μM. Accordingly, terfenadine-treated mononuclear cells supernatants did not alter the electrical resistance of intestinal HT29. 19 A cells. Conclusion These results indicate that in infants with intestinal dysfunction due to cow's milk allergy, terfenadine is a potent inhibitor of the TNFα secretion induced by sensitizing milk protein antigens. This inhibition prevents the degradation of intestinal function as measured in an intestinal cell line, in vitro.     

Allergy to bovine β-lactoglobulin: specificity of human IgE to tryptic peptides

BACKGROUND: Bovine beta-lactoglobulin (Blg) is a major cow's milk allergen. It is the main whey protein, without any counterpart in human milk. Blg chemical hydrolysates appeared to retain most of the immunoreactivity of the native protein. Allergenicity of Blg has already been shown to be associated with the four peptides derived from cyanogen bromide cleavage of Blg. OBJECTIVES: To map the major allergenic epitopes (e.g. regions of the molecule able to bind IgE) on Blg using specific IgE from sera of 46 milk-allergic patients as a probe. METHODS: Direct and competitive inhibition enzyme immunoassays involving immobilized native protein or purified peptides derived from Blg tryptic cleavage. RESULTS: Several peptides capable of specifically binding human IgEs were identified and were classified according to the intensity and frequency of the responses. The major epitopes appeared to be fragments (41-60), (102-124) and (149-162) recognized by 92, 97 and 89% of sera, respectively, whilst a second group which contained the fragments (1-8) and (25-40) was recognized by 58 and 72% of the population. A third group, comprising peptides (9-14), (84-91) and (92-100), was still detected by more than 40% of sera. CONCLUSION: Three peptides were identified as major epitopes, recognized by a large majority of human IgE antibodies. Numerous other epitopes are scattered all along the Blg sequence.     

Mutational analysis of immunoglobulin E-binding epitopes of β-casein and β-lactoglobulin showed a heterogeneous pattern of critical amino acids between individual patients and pooled sera

BACKGROUND: For immunotherapeutic approaches, 'critical' amino acids (AAs) within allergenic epitopes are replaced with alternate AAs to eliminate IgE antibody binding. OBJECTIVE: To determine the critical AAs for IgE binding in beta-casein and beta-lactoglobulin (BLG). METHODS: Peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with a pool of sera from 15 cow's milk-allergic patients and individual sera from six of the 15 patients. In cases where no decrease in binding occurred with a single AA substitution, peptides with two AA substitutions were generated and labelled. RESULTS: Using pooled patient sera, single AA substitutions led to complete elimination of binding to six of 11 peptides for beta-casein and to all six peptides for BLG. Substituting two AAs led to an elimination of binding to four of the remaining five beta-casein epitopes. However, in three of the 11 modified beta-casein peptides and five of the six BLG peptides, no decrease in IgE binding occurred in at least one individual patient. For these patients, critical AAs other than those defined by the patient serum pool were identified, indicating a heterogeneous pattern of IgE recognition. CONCLUSION: These results indicate that AAs critical for IgE binding are more heterogeneous than initially defined by pooled milk-allergic patient sera. For future immunotherapeutic interventions with mutated peptides, critical AAs should also be identified with individual patient sera to account for heterogeneity of IgE binding between patients.     

Allergy to bovine β-lactoglobulin: specificity of immunoglobulin E generated in the Brown Norway rat to tryptic and synthetic peptides

BACKGROUND: Animal models which reflect the induction and development of food-allergic reactions are important in the identification of allergenic potential of food proteins and peptides. A number of rat strains, including PVG, Hooded Lister and Brown Norway have been shown to produce immunoglobulin (Ig) E antibodies to food proteins as well as to inhaled allergens. Previous work in our laboratory using the Brown Norway (BN) rat has shown that specific IgE is produced following administration of ovalbumin and milk products via both enteral and parenteral route; this has allowed us to rank ovalbumin, lactoferrin and bovine serum albumin in terms of their inherent allergenic potential and has enabled us to demonstrate that milk protein allergens recognized by the systemically-sensitized animal are consistent with those recognized by sera from cow's milk-allergic patients (the most common allergens recognized were beta-lactoglobulin and the alpha, beta and kappa-caseins). OBJECTIVE: To demonstrate that the BN rat model can be used to identify the major IgE-binding peptides from beta-lactoglobulin, a known human food allergen, and that those IgE-binding peptides are similar to those recently identified using sera from cow's milk-allergic patients. METHODS: BN rats were exposed to beta-lactoglobulin or to semiskimmed milk via the intraperitoneal route in the presence of the adjuvant carrageenan. Specific IgE raised against beta-lactoglobulin was determined by a direct enzyme immunoassay using acetyl-cholinesterase substrate; specific IgG responses were also monitored. Overlapping synthetic peptides and tryptic peptides were used within the ELISA to identify the major and minor IgE-binding immunoreactive sequences. RESULTS: In terms of comparative immunogenicity, there appeared to be sequences that were predominantly IgE- or IgG-reactive. IgE-dominant regions were amino acid sequences 21-40, 41-60, 107-117 and 148-168 whereas sequences 1-24, 67-77, 82-92, 85-95 and 117-127 appeared more selective for IgG antibody recognition. An increased capacity to induce specific IgE was observed when the allergen was present in the context of whole food. CONCLUSIONS: These studies provide evidence that the immune system of the BN rat and humans - at least in the case of milk allergens - is recognizing similar protein allergens and indeed, at the molecular level, similar peptide epitopes.     

Occupational asthma and rhinoconjunctivitis from inhalation of dried cow's milk caused by sensitization to α-lactalbumin

A chocolate candy worker was diagnosed as having occupational asthma and rhinoconjunctivitis on the basis of clinical record and methacholine challenge. Positive conjunctival and bronchial challenge tests with lactalbumin showed that this protein was the pathogenetic agent. Type I hypersensitivity mechanism is demonstrated by means of skin prick test and RAST.     

Heterozygous α-antichymotrypsin and PiZ (α1-antitrypsin deficiency

In a case-control study we compared the prevalence of heterozygous deficiency of two closely related anti-neutrophil protease inhibitors, alpha 1-antitrypsin and alpha 1-antichymotrypsin, in 172 consecutive children with asthma. In a cohort study the clinical spectrum and severity were compared. On the basis of family studies 5/172 (2.9%) were classified as heterozygotes for alpha 1-antichymotrypsin deficiency, a high prevalence compared with that of an unselected adult population (prevalence ratio 4.5 (1.7-11.9), P less than 0.005). This finding suggests that the carrier state of this rare allele (prevalence 0.64%) may predispose to asthma in children. Among these heterozygous patients the prevalence of positive RAST tests for foodstuffs was significantly increased (prevalence ratio 4.8 (1.7-13.2), P less than 0.005) and 2/5 manifested food allergy with Quincke oedema. Either the PiMZ or SZ phenotype of alpha 1-antitrypsin deficiency was found in 12 (7.0%) of the 172 patients, a prevalence similar to that of a normal population (prevalence ratio 1.3 (0.67-2.6), P = 0.44). However, the asthma was more severe among the Z allele carriers, judged by the number of hospital admissions, compared with the non-Z asthmatic children (mean 2.92 vs. 1.72, P less than 0.05). The results indicate that heterozygous deficiency of protease inhibitors directed against neutrophil proteases may affect the severity and clinical spectrum of childhood asthma, and to some degree be predisposing.     

A sensitive enzyme-linked immunosorbent assay for determination of bovine β-lactoglobulin in infant feeding formulas and in human milk

We developed a sensitive sandwich-type ELISA for measuring low levels of cow's milk (CM) β-lactoglobulin. Purified anti-β-lactoglobulin was used as coating antibody and also as second antibody conjugated with alkaline phosphatase. Polyethylene glycol 6000 was added to the incubation buffers to improve sensitivity. The detection limit of the assay was 0.002 μg/l, which is much better than sensitivities reported for other β-lactoglobulin assays. The sensitivity was not impaired by the presence of other CM proteins. The recovery from breast milk was 93% and from the diluting buffer 127%. The coefficient of variation within day was 5–15% and between days 10%. One hour after oral intake of milk, P-lactoglobulin could be detected in the breast milk of three mothers at concentrations of about 1–2 μg/l. Widely different concentrations of β-lactoglobulin were measured in two protein hydrolysates based on CM whey and casein proteins; the observed concentrations were 200 and 0.0056 μg P-lactoglobulinμ/g dry weight, respectively.     

Emergence of α5β1 fibronectin- and αvβ3 vitronectin-receptor expression in melanocytic tumour progression

Cell adhesion is crucial in the process of tumour progression. As integrins are important receptor molecules involved in cell adhesion, we studied the distribution of the α1-6, αv, αIIb, β1, β3, and β4 integrin subunits in tissue sections of common naevocellular naevi ( n =22), dysplastic naevi (16), thin (24) and thick primary cutaneous melanomas (28), and melanoma metastases (25). We found correlated expression of α1/α2, of α4/α5/β3, and of α6/β4. Decrease of α6 and β4, and increase of α4 and αv were found to be correlated with melanoma progression. Furthermore, expression of α5 and β3 was detected only in primary melanoma and melanoma metastasis. Our findings indicate that during melanoma progression alterations in integrin expression occur, the most striking being emergence of α5β1 fibronectin and αvβ3 vitonectin receptor.     

Identification of amino acids critical for IgE-binding to sequential epitopes of bovine kappa-casein and the similarity of these epitopes to the corresponding human kappa-casein sequence

Background:  The delineation of allergenic (i.e. IgE-binding) epitopes in cow's milk proteins and the amino acids (AAs) critical for IgE-binding is necessary to understand better the structural properties of an allergen and to develop more efficacious immunotherapeutic reagents. Furthermore, this information may enable us to understand better cross-sensitivity between different allergens.
Methods:  Eleven peptides, 10–14 AAs in length, representing the IgE-binding epitopes of κ-casein were synthesized on a derivatized cellulose membrane with single AA substitutions at each position. Membranes were incubated with pooled sera from 15 milk-allergic patients and individual sera from 10 of the patients included in the pool.
Results:  For 10/11 allergenic peptides, one to five different single AA substitutions resulted in elimination of IgE-binding of pooled patient sera. Overall at least one mutated peptide could be found for these 10 IgE-binding sites that resulted in a reduction of IgE-binding in at least 80% of the patients who recognized the native protein. Furthermore, the IgE-binding region at AA104–112 on bovine κ-casein showed a high degree of similarity with the human κ-casein, respectively, including the AAs critical for IgE-binding.
Conclusion:  This finding suggests that critical AAs should be assessed with both pooled and individual patient sera to account for the B-cell epitope heterogeneity between patients, with cow's milk allergy. In addition, we identified two potentially cross-reactive peptides between bovine and human caseins of unknown clinical relevance.     

Leukocyte migration inhibition test in children with cow milk allergy

The usefulness of the leukocyte migration inhibition factor (LIF) test to detect cow milk (CM) hypersensitivity was studied in 40 children with suspected allergy to CM. Hypersensitivity was carefully investigated by oral milk challenges, which gave a final confirmation of cow milk hypersensitivity in 12 subjects, and excluded it in the remaining 28 subjects. Leukocyte migration inhibition was measured using beta-lactoglobulin (BLG), alpha-lactalbumin (ALA), alpha-casein (ACA) and bovine serum albumin (BSA) as antigens. IgA and IgG antibodies to these antigens were measured by an enzyme-linked immunosorbent assay, and IgE antibodies to these antigens and to CM by radioallergosorbent test (RAST). Skin prick test with CM was performed in 38 subjects, and with BLG, ALA, ACA and BSA in 29 subjects. Leukocyte migration was more often inhibited by cow milk antigens in the CM challenge positive (CM+) subjects than in the challenge negative (CM-) subjects. Of the specific milk antigens, ALA was the most potent inhibitor, and gave a positive LIF test result in all CM+ subjects, and significantly (P less than 0.02) less often (15/24) in CM- subjects. Also in the skin prick test and RAST, ALA gave positive results more often than the other milk antigens. BLG, ACA and BSA had an inhibiting effect on leukocyte migration, but the difference between the CM+ and CM- subjects was not statistically significant. Two of the 12 CM+ subjects had no demonstrable IgE antibodies to CM proteins; both of them, however, had a positive LIF test with at least one of the CM antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunostaining for α1-antichymotrypsin and α1-antitrypsin in gliomas

Antisera to α₁-antichymotrypsin, α₁-antitrypsin and lysozyme were reacted with 20 cases of glioblastoma multiforme, seven anaplastic astrocytomas, eight astrocytomas, six oligodendrogliomas, four ependymomas and the cerebral cortex from six normal autopsy brains. In addition, two pleomorphic xantho-astrocytomas and two heavily lipidized malignant gliomas were similarly examined. All astrocytic lesions were confirmed with anti-GFAP antisera. Thirty astrocytic tumours (77%), four oligodendrogliomas (67%) and three ependymomas (75%) reacted positively with anti-α₁-antichymotrypsin; 25 astrocytic tumours (64%), three oligodendrogliomas (50%) and three ependymomas (75%) showed positive staining for α₁-antitrypsin. The pattern of staining with either of these two markers did not correlate with tumour grading. None of the gliomas examined stained positively with anti-lysozyme. Non-neoplastic glial elements did not react with any of the three antisera. The results of this study suggest that staining for α₁-antichymotrypsin and α₁-antitrypsin is of little value in the differential diagnosis of neuroepithelial or mesenchymal lesions in the brain.     

Optimized methods for fungal α-amylase airborne exposure assessment in bakeries and mills

BACKGROUND: In order to enable reproducible and comparable exposure measurements of fungal alpha-amylase (alpha-amylase) in different laboratories and countries, the entire procedure from sampling of airborne dust to measuring extracted samples (including standards and the used enzyme) immunoassays must be standardized. The aim of this study was to establish optimal elution and assay conditions. METHODS: A parallel sampler was used for simultaneous collection of 10 samples of inhalable dust in bakeries and mills in Germany, England, the Netherlands and Spain. Three enzyme-immunoassays (EIAs) for detection of fungal alpha-amylase based on monoclonal antibodies or polyclonal antibodies were used for the measurement of the parallel-sampled filters (n=432) extracted using several methods. The results were analysed by regression analysis of variance. Additional filters (n=54) were extracted and analysed using two EIAs to investigate the storage stability of the extracts. RESULTS: Although alpha-amylase concentrations correlated well (r> or =0.88), differences were found between the EIAs in the sensitivity and nominal values (up to a mean factor 5.8). The best elution medium for airborne filters (phosphate-buffered saline 'PBS' with 0.05% Tween-20) led to 1.2 to two times higher alpha-amylase allergen yields than extraction in PBS only, while higher Tween-20 concentrations decreased the extracted alpha-amylase yield. During storage of frozen dust/filter extracts for 3-4 months at -20 degrees C, a loss of approximately 40% of measurable alpha-amylase was observed, which could be partially prevented by addition of 0.1% casein to the medium directly after extraction. CONCLUSION: Although the effects of only a few of many possible causes of variation were investigated, for these factors a clear choice could be made with regard to optimal elution conditions and the use of validated EIAs with calibrated standards, thus making significant progress towards a completely standardized procedure for airborne alpha-amylase measurements.     

Integrated morphogen signal inputs in γδ versus αβ T-cell differentiation

Summary:  Morphogens, a class of secreted proteins that regulate gene expression in a concentration-dependent manner, are responsible for directing nearly all lineage fate choices during embryogenesis. In the thymus, morphogen signal pathways consisting of WNT, Hedgehog, and the transforming growth factor-β superfamily are active and have been implicated in various developmental processes including proliferation, survival, and differentiation of maturing thymocytes. Intriguingly, it has been inferred that some of these morphogen signal pathways differentially affect γδ and αβ T-cell development or maintenance, but their role in T-cell lineage commitment has not been directly probed. We have recently identified a modulator of morphogen signaling that significantly influences binary γδ versus αβ T-cell lineage diversification. In this review, we summarize functions of morphogens in the thymus and provide a highly speculative model of integrated morphogen signals, potentially directing the γδ versus αβ T-cell fate determination process.     

Pseudodeficiency of α-galactosidase A

Apparent deficiency of alpha-galactosidase A was observed in a 51-year-old, clinically healthy male, with no clinical symptoms of Fabry disease, and without excess urinary excretion of ceramide trihexoside. The deficiency, which was similar to that found in Fabry disease patients, could be demonstrated using both synthetic and natural substrates. This pseudodeficiency was transmitted in his family by classical X-linked inheritance. His wife showed enzyme activity in the normal range, two daughters were heterozygotes for this mutation as demonstrated by hair root assay, and three sons showed normal alpha-galactosidase activity. Kinetic studies in cultured skin fibroblasts indicated a five-fold increase in the apparent Km and a greater heat stability of the residual alpha-galactosidase activity when compared to controls. These data indicate that the residual enzyme activity in this mutation behaves similarly to that observed in Fabry disease patients but does not cause any clinical abnormalities.