Immunodominant semen proteins II: contribution of seminal proteins to female immune infertility.

Seminal fluid is a protective medium for sperm, but it also represents potential immunogenic structures for the female immune system. Anti-seminal antibodies may threaten early fertilization. The aim of our work is to detect and identify seminal proteins that are related to female isoimmunization. In this report, we quantified serum anti-seminal IgG antibodies. Seminal proteins were analysed by two-dimensional gel electrophoresis followed by immunoblotting. To identify IgG-binding proteins of interest, a proteomic approach was selected. The dominant seminal antigens were detected within the relative molecular mass ranging from 25 to 85 kDa and the isoelectric point from 5 to 7. The detected proteins were further identified as prostate-specific antigen, prostatic acid phosphatase, zinc-α-2-glycoprotein and zinc finger protein 778. Since these proteins were recognized by IgGs produced by infertile women and not by fertile women, we presume that major seminal antigens may play an important role in the pathogenesis of female immune infertility. Our study suggests the pattern of seminal proteins for further therapeutic attempts in the diagnosis of female immune infertility.

Heterogeneity of antibody responses among clinical responders during grass pollen sublingual immunotherapy.

BACKGROUND: During allergen-specific sublingual immunotherapy (SLIT), the relevance of changes in specific IgE and IgG antibody titres to treatment efficacy remains to be evaluated at an individual patient level. OBJECTIVE: To investigate whether antibody responses can be used as biomarkers for SLIT efficacy. METHODS: Comprehensive quantitative, qualitative and functional analyses of allergen-specific IgA, IgE, IgG1-4 and IgM responses were performed using purified Phl p 1 to 12 allergens in sera, saliva and nasal secretions from 82 grass pollen allergic patients. These patients were enrolled in a randomized, double-blind placebo-controlled study and assessed in an allergen challenge chamber (ClinicalTrials.gov NCT00619827). Antibody responses were monitored in parallel to clinical responses before and after daily sublingual treatment for 4 months with either a grass pollen or a placebo tablet. RESULTS: A significant mean improvement (i.e. 33-40.6%) in rhinoconjunctivitis total symptom scores was observed in SLIT recipients, irrespective of their baseline patterns of IgE sensitization (i.e. narrow, intermediate, broad) to grass pollen allergens. SLIT did not induce any de novo IgE sensitization. Clinical responders encompassed both immunoreactive patients who exhibited strong increases in titres, affinity and/or blocking activity of grass-pollen-specific IgGs (representing 17% of treated patients), as well as patients with no detectable antibody responses distinguishing them from the placebo group. No significant changes were detected in antibody titres in saliva and nasal washes, even in clinical responders. CONCLUSIONS AND CLINICAL RELEVANCE: Sublingual immunotherapy with a grass pollen tablet is efficacious irrespective of the patients' baseline sensitization to either single or multiple grass pollen allergens. Seric IgG responses may contribute to SLIT-induced clinical tolerance in a fraction (i.e. 17%) of patients, but additional immune mechanisms are involved in most patients. Consequently, antibody responses cannot be used as a marker of SLIT efficacy at an individual patient level.

Female serum immunoglobulins G, A, E and their immunological reactions to seminal fluid antigens.

One in five couples of reproductive age has been diagnosed with infertility. Some diagnoses indicate an immunological basis for this disorder. Female immune infertility may be caused by iso-immunization by seminal components. We focused on the characterization of seminal proteins to illustrate the IgG, IgA and IgE immune responses of 31 infertile women. The biochemical characterization was performed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing, both of which were followed by immunoblotting analyses. IgG mainly recognized the antigens with relative molecular masses (Mr) 95 and 183 kDa and isoelectric points ranging from 6.9 to 7.0. The immunodominant antigens recognized by IgA had the Mr of 35 kDa and isoelectric points ranging from 6.2 to 7.2. The reactivity of IgE was not confirmed within our group of patients. The seminal IgG- and IgA -binding patterns were analysed immunochemically to determine the characteristics of possible seminal proteins associated with female immune infertility.

Prevalence of sensitisation to oilseed rape and maize pollens in France: a multi-center study carried out by the Allergo-Vigilance Network.

BACKGROUND: Oilseed rape and maize crops represent a large part of agriculture fields in European countries. OBJECTIVE: To establish the actual prevalence of sensitization to oilseed rape and maize pollen, and to determine if this is correlated to the amount of exposure as well as to the patient's history of atopy or asymptomatic atopy. METHODS: The study was conducted by 69 allergists belonging to the Allergo-Vigilance Network, in collaboration with the French Agency for Safety of food, and compiles the results of skin prick-tests using oilseed rape and maize pollens and seeds, as well as common aeroallergens. The patients were classified into 3 groups: nonatopic, asymptomatic atopy, and actual atopic diseases. RESULTS: Among the 5372 subjects studied (2515 children, 2857 adults), 62.3% had an atopic disease, 10.2% had an asymptomatic atopy, and 27.5% were non-atopic. The level of sensitization was higher in the subjects with atopic disease, as compared to those with asymptomatic atopy: oilseed rape pollen: 11.8% vs 8%, maize pollen, 26% vs 19%, oilseed rape seeds, 7.7% vs 6.9%, corn seeds: 8.3% vs 4.8% (p < 0.001). The rate of sensitization was significantly increased in those living in high crop density regions. The association of an atopic disease with a high rate of exposure yielded a higher rate of sensitization of 13.8% and 21.3% for rapeseed pollen, and 22.9% and 30.7% for maize pollen in both children and adults, respectively. CONCLUSIONS: The incidence of sensitisation to rapeseed and maize pollen is positively correlated to the level of exposure. This prevalence is higher in patients with actual atopic disease as compared to those with asymptomatic atopy. The frequency of sensitization confirms the allergenicity of these plants destined for food supply and demonstrates the importance of monitoring for respiratory allergies to these pollens, not only in workers exposed to these types of crops, but also in atopic patients living in regions that contain a high density of rapeseed and maize fields. Cross-reactivities between pollens and seeds could potentially elicit cross-reacting food allergies.

Evaluation of ash pollen sensitization pattern using proteomic approach with individual sera from allergic patients.

BACKGROUND: In Europe, sensitization to ash pollen induces pollinosis with cross-reactivities with other pollen sources. The aim of the study was to identify the repertoire of ash pollen allergens and evaluate the extent of the diversity of the IgE response in ash allergic patients. METHODS: The IgE reactivities of 114 ash pollen- and eight grass pollen-sensitized patients were screened by 1D immunoblot (SDS-PAGE) against ash pollen extract. The IgE reactivities of 13 ash pollen- and two grass pollen-sensitized patients were then evaluated in 2D immunoblots. Some IgE- and non-IgE-reactive proteins were identified by mass spectrometry. RESULTS: In 1D analysis, 86% of sera showed binding to Fra e 1 (18-20 kDa), 23% to Fra e 2 (14 kDa), 3% to Fra e 3 (10 kDa) and 57% to High Molecular Weight allergens (HMW, >30 kDa). Individual analysis of 2D immunoblots showed several IgE-binding protein areas among which three were more often recognized: (i) Fra e 1 comprising, at least, 15 isoforms, (ii) a series of acidic spots (45 kDa), and (iii) Fra e 2, the ash profilin. HMW allergens could be resolved in four areas; two unidentified, one homologous to beta-galactosidase and the other to sugar transport proteins. A malate deshydrogenase and calmodulin were shown to be IgE-binding proteins and 10 non-IgE reactive proteins were identified. CONCLUSIONS: No direct correlation was evidenced between IgE profile and the degree of sensitization even though 2 spectrotypes could be distinguished. Our data contribute to a better delineation of ash pollen allergens and patterns of sensitization.

Characterization of peptidic and carbohydrate cross-reactive determinants in pollen polysensitization.

BACKGROUND: Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. MATERIAL AND METHODS: Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. RESULTS: The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. DISCUSSION: This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.

Modifications of Phleum pratense grass pollen allergens following artificial exposure to gaseous air pollutants (O(3), NO(2), SO(2)).

BACKGROUND: Air pollution is frequently proposed as a potential cause of the increased incidence of allergy in industrialised countries. Our objective was to investigate the impact of the major gaseous air pollutants on grass pollen allergens. METHODS: Timothy grass pollen was exposed to ozone (O(3)), nitrogen dioxide (NO(2)) and sulphur dioxide (SO(2)) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. RESULTS: For O(3)-treated pollen, immunoblotting showed an acidification of allergens Phl p 1b, Phl p 4, Phl p 5 and Phl p 6 and an IgE recognition decrease in Phl p 1, Phl p 2, Phl p 6 and Phl p 13. NO(2) exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO(2) treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO(2)/O(3) or NO(2)/SO(2) showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O(3) acidification was also observed with the NO(2)/O(3) mix. CONCLUSION: Exposure of pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts. This decrease could be due to a mechanical loss of allergens from the altered pollen grains and/or post-translational modifications affecting allergen recognition by IgE.

Traffic-related air pollutants induce the release of allergen-containing cytoplasmic granules from grass pollen.

BACKGROUND/AIM: Pollen cytoplasmic granules (PCG) are loaded with allergens. They are released from grass pollen grains following contact with water and can form a respirable allergenic aerosol. On the other hand, the traffic-related air pollutants NO2 and O3 are known to be involved in the current increase in the prevalence of allergic diseases via their adjuvant effects. Our objective was to determine the effects of air pollutants on the release of PCG from Phleum pratense (timothy grass) pollen. METHODS: P. pratense pollen was exposed to several concentrations of NO2 and O3. The induced morphological damages were observed by environmental scanning electron microscopy, and the amount of PCG released from the pollen upon contact with water was measured. RESULTS: The percentages of damaged grain were 6.4% in air-treated controls, 15% after treatment with the highest NO2 dose (50 ppm) and 13.5% after exposure to 0.5 ppm O3. In treated samples, a fraction of the grains spontaneously released their PCG. Upon subsequent contact with water, the remaining intact grains released more PCG than pollen exposed to air only. CONCLUSIONS: Traffic-related pollutants can trigger the release of allergen-containing granules from grass pollen, and increase the bioavailability of airborne pollen allergens. This is a new mechanism by which air pollution concurs with the current increase in the prevalence of allergic diseases.

Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5.

BACKGROUND: Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein. OBJECTIVE: To clone and characterize timothy grass pollen allergen Phl p 4. METHODS: Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies. RESULTS: Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner. CONCLUSIONS: Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.

Intratracheal instillation of cytoplasmic granules from Phleum pratense pollen induces IgE- and cell-mediated responses in the Brown Norway rat.

BACKGROUND: Release of cytoplasmic granules from grass pollen upon contact with water is thought to be an important source of airborne allergens. OBJECTIVES: To investigate the humoral and cellular responses to intratracheal instillation of Phleum pratense (timothy grass) pollen cytoplasmic granules (PCG) in the Brown Norway rat. METHODS: PCG were purified from timothy grass pollen by filtration through 5-microm-mesh filters. Rats were sensitized (day 0) and challenged (day 21) intratracheally with purified PCG suspended in saline (6 x 10(6) PCG/rat). Rats were then challenged 4 weeks later (1.5 x 10(6) PCG/rat). Blood samples, bronchial lymph nodes and lungs were collected from the rats 4 days after the second challenge. PCG-specific IgE and IgG1 levels and specificity were determined by ELISA and Western blotting. Pollen, pollen extract and PCG-induced proliferation of lymph node cells were monitored by [(3)H]-thymidine incorporation in a lymph node assay. Histopathological examination was carried out on the lungs. RESULTS: Specific IgE and IgG1 were present in the sera. Cultured lymph node cells proliferated in the presence of pollen, pollen extract and PCG. Western blots showed that all major pollen allergens are recognized by IgE and IgG1 from PCG-treated rats. Histopathological examination revealed features of a mild allergic reaction. CONCLUSIONS: In our rat model of allergy, purified timothy grass PCG instillation induced specific antibodies and lymph node cell responses, comparable to those obtained with intact pollen.

Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy.

BACKGROUND: Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. OBJECTIVE: We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. METHODS: Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. RESULTS: An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. CONCLUSIONS: It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.

Lipid transfer protein 1 is a possible allergen in Arabidopsis thaliana.

BACKGROUND: The Arabidopsis thaliana genome was recently fully sequenced, and this plant is now considered as the most useful model to study the effects of genetic engineering. The aim of the present study was to identify A. thaliana IgE-binding molecules and to localize their genes in order to evaluate the potential effect of gene insertion on the expression of IgE-binding molecules. METHODS: A. thaliana flower proteins were separated by two-dimensional gel electrophoresis and transferred onto a nitrocellulose sheet. The nitrocellulose sheet was successively incubated with human sera known to contain IgE that binds to rapeseed proteins, alkaline phosphatase-conjugated goat anti-human IgE and 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. One allergen was further identified by N-terminal amino acid microsequencing. RESULTS: The results showed that some individuals possessed IgE that recognized numerous proteins with high molecular masses and various isoelectric points. This binding pattern strongly suggests that the epitopes recognized by these IgE could be, at least partly, sugar residues. Otherwise, out of the 10 sera that possessed IgE to Arabidopsis flower proteins, one serum strongly recognized a unique basic protein with an apparent molecular mass of around 14 kD. This protein was identified by amino acid microsequencing as the lipid transfer protein 1 (LTP1). CONCLUSION: We have demonstrated that A. Thaliana LTP1 is IgE reactive. The gene encoding this protein is located on chromosome 2, but it has been described that family 1 of A. Thaliana LTPs constitutes a multigenic family with genes located on various chromosomes.

Polygalacturonase (pectinase), a new oilseed rape allergen.

BACKGROUND: Type I hypersensitivity to rapeseed pollen allergens was described as the result of a cross-sensitization with various pollens that could constitute an aggravating factor in birch or grass pollen allergies. Recently, a few rapeseed pollen allergens were described. The aim of the present work was to identify new rapeseed pollen allergens by using two-dimensional gel analysis, microsequencing, and mass spectrometry. METHODS: Water extractable proteins from oilseed rape pollen or stamen were separated by two-dimensional gel electrophoresis. The proteins were then electroblotted onto a nitrocellulose (NC) sheet. The NC sheets were successively incubated with (1) individual human sera pre-selected for their immunoglobulin E (IgE) reactivity to rapeseed pollen proteins, (2) alkaline phosphatase (AP)-conjugated goat anti-human IgE and (3) AP substrate. The allergens localized by this method were then identified by microsequencing and MALDI-TOF mass spectrometry analysis. RESULTS: Of the 18 sera studied, five recognized a wide multispot zone with a molecular mass around 43 kD and pIs between 6.5 and 8.5. The results obtained with two representative sera are shown. From this zone, two isoforms of the polygalacturonase enzyme were identified by microsequencing. Confirmation was obtained through MALDI-TOF mass spectrometry analysis. CONCLUSION: The present results allow the identification of a new rapeseed allergen that can be the main allergen for some patients.

Interest of two-dimensional electrophoretic analysis for the characterization of the individual sensitization to latex allergens.

BACKGROUND/OBJECTIVE: Latex allergy is a type 1 hypersensitivity reaction that mainly affects high-risk populations such as health care workers, spina bifida-affected or multiply-operated children. Ten molecules have so far been identified and registered as latex allergens (Hev b 1 to Hev b 10). The aim of the present investigation was to identify the major latex allergens by an individual analysis of the IgE response of latex-allergic patients to latex proteins separated by two-dimensional (2-D) gel electrophoresis. MATERIALS AND METHODS: Latex proteins from a sap or a glove extract were separated by 2-D electrophoresis and transferred to a nitrocellulose membrane. Each membrane was incubated with the serum of one latex-allergic patient. The most frequently recognized latex allergens were characterized in sap and glove extracts using monoclonal antibodies or amino acid microsequencing. RESULTS: The one-dimensional screening of 54 patient sera revealed 4 major bands recognized by IgE. The 2-D analysis of the sensitization to latex allergens allows the identification of allergen isoforms and the characterization of an individual response diversity. Hev b 6.01 was recognized by 88.9% of the patients. Protein spots around 14 kD were recognized by 48.1% of the patients and corresponded to Hev b 6.03 as well as other proteins. A not yet characterized doublet of acidic proteins with molecular masses of 43 and 94 kD was recognized by 20.4% of the sera. Only 5.5% of the sera did not recognize any of these 4 major allergens. Hev b 1 is the main protein from the glove extract but was not constantly found in sap extracts. CONCLUSIONS: One-dimensional electrophoretic analysis of the allergen is usually not sufficient to characterize the individual specificity of the IgE response to latex allergens. Latex-glove proteins which are allergens can be absent from the sap extracts and the sensitization to these allergens could be underestimated. Individual 2-D analysis of the sensitization to latex allergens is useful to define the best allergen mixture required for diagnosis and needed for individual therapy monitoring.

Effects of birch pollen and traffic particulate matter on Th2 cytokines, immunoglobulin E levels and bronchial hyper-responsiveness in mice.

BACKGROUND: Health effects due to air pollution arising from motor vehicles are a major public and political concern world-wide. Epidemiological studies have shown that the manifestations of asthma are increased by air pollution in already affected individuals. OBJECTIVE: To investigate the potential role of air-polluted tunnel dust (traffic particulate matter, TPM) or pure carbon core particles in the initiation and persistence of experimental allergic inflammation. METHODS: BP2 mice were immunized with birch pollen alone (group B) or pollen together with TPM (group A), or with birch pollen and Al(OH)3 (group C), or with birch pollen and carbon core particles (group D). Before methacholine challenge they were challenged intranasally and thereafter bronchial hyper-reactivity (BHR) was evaluated in a whole-body plethysmograph. Levels of Th2 cytokines, fibronectin and lactate dehydrogenase (LDH) were determined, and differential counts were performed in the bronchoalveolar lavage (BAL) fluid. Sera were collected for determination of antibody titres and cytokine levels. RESULTS: Specific IgE titres, BHR, the number of recruited eosinophils and levels of fibronectin and LDH in BAL were increased in mice immunized and challenged with a mixture of birch pollen and TPM. However, mice immunized with birch pollen alone and challenged intranasally with pollen or a mixture of pollen and TPM demonstrated the highest levels of IL-4 and IL-5. CONCLUSION: This study highlights the importance of the exposure to a combination of particulate matters and pollen allergens, in the induction of allergic disease in the airways, and we have demonstrated that polluted tunnel dust has an effect on both the inflammatory and immunological components of experimental allergy. Immunization and challenge with carbon core particles together with birch pollen increased neither the BHR nor the specific IgE production significantly. Our results therefore strongly suggest that it is most likely to be the organic phase bound to the carbon core of the diesel exhaust particles that might have an important adjuvant effect in the induction of experimental allergy.

Characterization of high-molecular-mass allergens in oilseed rape pollen.

BACKGROUND: Oilseed rape pollen allergies have been previously described as the result of cross-sensitization with various pollens. Recently, several proteins have been identified as oilseed rape allergens. The aim of the present work was the characterization of oilseed rape pollen allergens by two-dimensional (2-D) gel analysis and amino acid microsequencing. METHODS: Water extractable proteins from oilseed rape pollen were separated by isoelectrofocusing and then transferred onto a nitrocellulose sheet. Twenty-one human sera from pollen- or mustard-allergic individuals were screened for their reactivity to oilseed rape proteins. Eleven sera possessed IgE which recognized oilseed rape pollen proteins and one serum was selected for further 2-D characterization and amino acid microsequencing of the allergens. RESULTS: The results showed that three molecules from oilseed rape pollen were identified as oilseed rape allergens which have not yet been described. These three proteins were molecules of 70 kD with a pI >8, 40 kD with a pI around 10 and 80 kD with a pI around 5. These proteins displayed identities with the berberine bridge protein, a receptor-like protein kinase and the cobalamin-independent methionine synthetase from Arabidopsis thaliana, respectively. The genes encoding the putative Arabidopsis molecules are located on chromosome 1 (berberine bridge protein) and chromosomes 3 and 4 (receptor-like protein kinases). CONCLUSION: These results show that certain high-molecular-mass proteins from oilseed rape pollen are allergens.

A European study on the genetics of mite sensitization.

BACKGROUND: Sensitization to mite allergens represents a prominent feature of atopy and an important predictor of bronchial asthma. OBJECTIVE: It was the intention of this study to define genetic loci linked to mite sensitization because these could represent the genetic basis of the important atopic component of asthma. METHODS: We studied a multiethnic white population of 99 families, including 224 sib pairs sensitized to Dermatophagoides pteronyssinus. A genome-wide candidate-region search was performed that covered potential asthma and atopy regions. RESULTS: As for nonparametric linkage (NPL) analysis, 14 of the candidate regions showed evidence for linkage (NPL > 2.0), and 4 of them showed prominent linkage (NPL > 3.0). However, there were substantial ethnic differences. Maximizing the LOD score analysis identified candidate regions with suspected dominant and recessive mode of inheritance. Furthermore, genetic imprinting models provided significant evidence for linkage in the 8p23 region and revealed potential maternal imprinting. The regions found are distinct to those in asthma searches that have been found to be linked to asthma, as well to other inflammatory diseases. In addition, we could not find linkage to the HLA region. By different cutoff points of the phenotype definition, the IL cluster showed evidence of being linked to the degree of sensitization rather than to sensitization per se. CONCLUSION: The results indicate that the genetic basis of the atopic component of asthma is different from that of the inflammatory component. Furthermore, it seems reasonable to assume that specific sensitizations are influenced by distinct genetic variants leading to their initiation versus those leading to their enhancement.

Electrotitration curves of human gastric pepsinogens in agarose gels.

Electrotitration curves (ETC) of a marker protein mixture, pH 2.5-5.65, and human pepsinogens were performed in an agarose gel, containing 2% acid carrier ampholytes, forming a pH range of 2.5-5. Although the establishment of the pH gradient by isoelectric focusing was not quite complete and linear, both biochemically and immunochemically different types of pepsinogen C (PGC) and pepsinogen A (PGA) zymogens as well as the acid isoelectric points (pI) marker proteins were separated with good resolution. Three main fractions of PGA (Pg3, Pg4, and Pg5) were detected. To obtain an exact determination of the pepsinogen pIs, a simple and very fast 10 s pressure blot technique was applied. Human pepsinogens were separated alone or mixed with pI marker proteins in the pH range 2.4-5.65. No effect of the markers was observed on the pepsinogen migration. To visualize the different protein samples in the gel and on nitrocellulose membrane, we have used colloidal gold (AuroDye) staining, proteolytic activity, and immunostaining with monoclonal antibodies anti PGA and PGC. The described method shows an ability to separate proteins at acidic conditions with a resolution comparable to isoelectric focusing with immobilized pH gradients, but much faster, easier, and cheaper. In addition, the technique allows us to determine precise and exact pI values, and is suitable for studies of the pepsinogen polymorphism and its role in gastric diseases.

Molecular basis of IgE cross-reactivity between human beta-casein and bovine beta-casein, a major allergen of milk.

Twenty patients allergic to cow's milk proteins and with high levels of specific IgE directed against bovine whole casein were selected to evaluate reactivity of their IgE antibodies with human beta-casein. Highly purified human and bovine beta-caseins were prepared by selective precipitations and FPLC separation. Their identity and purity were assessed by HPLC, analysis of amino acid composition, sequencing of the five N-terminal amino acid residues and immunochemical tests. Direct and indirect ELISAs were performed using human and bovine beta-casein coated into microtiter plates and monoclonal anti-human IgE antibody AChE labelled for revelation. Seven sera contained specific IgE directed against human beta-casein. Inhibition studies using native human and bovine beta-caseins as well as bovine beta-casein-derived peptides demonstrated that, depending on the sera, one or several common epitopes located in different parts of the molecule were shared by the two homologous proteins.