The function of allergens may determine allergenicity.
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Allergens↗
Biomedical subjects
M Schlaak
Publications and source records attributed to M Schlaak.
The major birch pollen allergen, Bet v 1, shows ribonuclease activity.
The major birch (Betula alba L.) pollen allergen, Bet v 1, has been shown to be homologous to pathogenesis-related proteins in a number of plants. Recently, it was demonstrated that a ginseng protein with high homology to an intracellular pathogenesis-related protein of parsley and to Bet v 1 is a ribonuclease (RNase). Birch pollen extract was separated in an RNase activity gel. Four major RNase bands were excised from the gel, reseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting with a specific Bet v 1 monoclonal antibody and patient's serum. Thus the monomer and the dimer of Bet v 1 showed RNase activity. Purified recombinant Bet v 1 was shown to degrade plant RNA. The RNase activity of recombinant Bet v 1 was 180 units.mg-1.
Allergens↗
Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens.
The simultaneous presence of IgE reactivity to tomato fruit and grass pollen allergens is evident in many patients with allergy and may be caused by cross-reactivity. Using sera from polysensitized patients with a positive enzyme allergosorbent test (EAST) result (score > 2), we tested reactivity to both allergen sources. IgE reactivity against both extracts was demonstrated in eight serum samples, and cross-reactivity was confirmed by the EAST inhibition assay. The structures responsible for this cross-reactivity were identified by Western blotting: five of the eight sera demonstrated a 16 kd protein in both extracts, which was identified as profilin. Additionally, seven of the eight sera showed IgE binding to epitopes on carbohydrate moieties, which contained alpha 1, 3 fucosylations. To determine the allergens of tomato fruit extract, we performed two-dimensional polyacrylamide gel electrophoresis blotting. We were able to demonstrate one highly concentrated and about 20 weaker proteins possessing terminal fucose residues. These are similarly found in grass pollen extracts. It is therefore postulated that the cross-reactivity is affected by profilins and similar carbohydrate determinants. If carbohydrate structures can provoke IgE cross-reactivity between phylogenetically distant species, such structures may play an important role in sensitization and mediator release. The ubiquitous nature of the IgE-binding determinants was studied by additional EAST inhibition tests with tomato allergen disks and extract from birch pollen, mugwort pollen, apple, and celery, leading to significant inhibitions among all these allergen sources. Epitopes exclusive to grass pollen and tomato have not been detected.
Allergens↗
Farmer's lung: patients' IgG2 antibodies specifically recognize Saccharopolyspora rectivirgula proteins and carbohydrate structures.
Farmer's lung is a frequent form of extrinsic allergic alveolitis. In Europe and Northern America the main source of the antigenic components that induce farmer's lung is the bacterium Saccharopolyspora rectivirgula (Micropolyspora faeni). It remains unclear, however, which S. rectivirgula components are responsible for the disease. We approached these problems by investigating the serologic reaction of patients with farmer's lung and demonstrated specific binding of patients' IgG2 to S. rectivirgula antigens. No such antibodies were found in exposed, unaffected subjects. Thus IgG2 antibodies reacting with S. rectivirgula antigens are useful for the serologic diagnosis of patients with farmer's lung and for the isolation of disease-causing antigens. After separation of S. rectivirgula extract on concanavalin A-Sepharose (Pharmacia, Uppsala, Sweden), we found that approximately one third of the patients' IgG2 reactivity occurred with nonglycosylated proteins. Among these, we characterized two major acidic proteins with molecular weights of 12 and 30 kd, respectively, and with identical N-terminal sequences. Approximately two thirds of the patients' IgG2 reactivity was observed against concanavalin A-binding glycoproteins that contained mainly glucose, mannose, and galactose residues. Deglycosylation of the concanavalin A-bound fraction indicated that most of the IgG2 reactivity occurred with the carbohydrate components.
Amino Acid Sequence↗
Cloning and expression of a murine Fab fragment recognizing a defined linear epitope of Chironomus thummi thummi major allergen Chi t 1-9.
We have cloned and expressed in bacteria the genes coding for the Fab fragment of the monoclonal antibody (MoAb) M3. M3 is a murine IgG1 antibody reactive with the major allergen Chi t 1-9 of Chironomus thummi thummi. The major allergen Chi t 1-9 is known to be an aggressive inhalant allergen and causes type-I allergy. The immunoglobulin (Ig) fragment genes were cloned as a synthetic dicistronic operon. In this operon each gene is preceded by a bacterial signal sequence to direct the recombinant protein to the periplasmic space of the bacteria. The cloned genes were expressed in Escherichia coli using the strong T7-RNA-polymerase-based system. Sequence analysis revealed that the light and heavy chains of MoAb M3 belong to the V kappa II and V kappa IIC group of the Ig family, respectively. Genes of the V kappa II and V kappa IIC group are known to be used in response to haptens. The apparent affinity constants of the parent antibody M3 and the recombinant Fab fragment are nearly equivalent.
Allergens↗
Identification and characterization of the major allergens of velvet grass (Holcus lanatus), Hol l 1 and Hol l 5.
Although extract of velvet grass pollen is an important ingredient of commercial allergen extracts for immunotherapy, it is not defined concerning its major allergenic components. We have investigated the extract of velvet grass pollen by Western blotting using the sera of 97 patients and identified two major allergens of 34 and 30 kD. By two-dimensional immunoblotting with monoclonal antibodies and lectins, and by biochemical analysis the 34-kD protein was identified as a group I grass pollen allergen and the 30-kD protein as a group V grass pollen allergen. According to the nomenclature for grass pollen allergens we designated these allergens Hol l 1 and Hol l 5, respectively. Fragmentation of the velvet grass pollen extract by CNBr provided the first information on the localization of IgE binding epitopes on these two allergens. With Hol l 1 and Hol l 5 we identified two important components of commercial allergen extracts.
Allergens↗
Assessment of mycobacterial DNA in cells and tissues of mycobacterial and sarcoid lesions.
In this study we applied a polymerase chain reaction (PCR) assay for the detection and species-specific identification of mycobacteria to samples from patients with sarcoidosis and mycobacterial infections and from control patients. The PCR-technique is based on the amplification of mycobacterial DNA coding for 16S rRNA, which is present in all mycobacterial species, and on the additional sequencing of the PCR fragment to determine the species. Mycobacterial DNA could be detected in lung tissues and bronchoalveolar lavage cells from cases of tuberculosis and infections with atypical mycobacteria. On the other hand, mycobacterial DNA was amplified only in lung tissue from one patient with sarcoidosis. Twenty-three samples from patients with sarcoidosis were negative for mycobacterial DNA. From our results we conclude that the granulomatous lesions in sarcoidosis may not be due to mycobacterial infections.
Base Sequence↗
Obstructive sleep apnea syndrome and circadian rhythms of hormones and cytokines.
Several cytokines exhibit a high degree of temporal regulation as well as somnogenic potency (e.g., interleukin-1 [IL-1], tumor necrosis factor-alpha [TNF-alpha]). Seeking the underlying cause of obstructive sleep apnea syndrome (OSAS), we investigated whether circadian rhythms of cytokine release were altered in 10 patients with OSAS. Ten healthy volunteers served as the control population. Seven of the 10 OSAS patients were reexamined after 3 mo of therapy with nasal continuous positive airway pressure (nCPAP) mask ventilation. Circadian cytokine release (IL-1, IL-6, gamma-interferon [gamma-IFN], TNF-alpha) was investigated ex vivo by short-term culture of blood samples. The circadian rhythm of TNF-alpha release was significantly disturbed in OSAS patients: nocturnal physiologic peaks in this cytokine had almost disappeared and an additional daytime peak had developed. Circadian variations in IL-1, IL-6, and gamma-IFN, and in the immunoregulatory hormones melatonin and cortisol, did not differ from those in the controls. Because TNF-alpha is a known modulator of sleep, and nCPAP therapy did not normalize TNF rhythms, we assume that TNF-alpha could well play a pathophysiologic role in OSAS. Further studies should be directed at whether a physiopathologic and/or pathogenic link exists between TNF-alpha and OSAS.
Adult↗
Anti-inflammatory cytokine release by alveolar macrophages in pulmonary sarcoidosis.
Sarcoidosis is a systemic, granulomatous disorder with a high rate of spontaneous remission indicating the presence of antiinflammatory mechanisms. Antiinflammatory mediators such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) should be able to induce spontaneous remission of sarcoidosis. By measuring the release of both mediators in culture supernatants of bronchoalveolar lavage (BAL) cells, we investigated their relevance in the spontaneous remission of sarcoidosis. No spontaneous IL-10 release was observed by BAL cells of sarcoid patients. In supernatants of BAL cells of seven patients found retrospectively to be free of any interstitial lung disease, we found 612 +/- 261.2 pg/ml (mean +/- SEM) TGF-beta. TGF-beta release was recorded in 20 of 39 patients with active disease. Patients with active disease without TGF-beta release in BAL cell culture either required therapy (n = 21; 677 +/- 159 pg/ml) or showed evidence of persisting disease (n = 6; 762 +/- 419 pg/ml). Patients with active disease without indications for therapy and with significantly increased TGF-beta release (n = 12; 1,422 +/- 215 pg/ml; p < 0.004 in all comparisons) had a spontaneous remission within 6 mo. Increased TGF-beta release (1,560 +/- 353 pg/ml) was observed in five of five patients receiving therapy. We conclude that TGF-beta is a regulator of the inflammatory process in sarcoidosis.
Adrenal Cortex Hormones↗
Reinvestigation of the major peanut allergen Ara h 1 on molecular level.
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Allergens↗
Major allergen Phl p Vb in timothy grass is a novel pollen RNase.
A cDNA coding for the major group V allergen Phl p Vb was isolated from a timothy grass pollen cDNA library by immunoscreening with a specific monoclonal antibody. It was discovered for the first time that the recombinant Phl p Vb pollen allergen after expression and purification has ribonuclease activity. High homology of Phl p Vb to other group V allergens in grass pollen indicates similar function. By RNase activity gel of natural pollen extract of timothy grass and consecutive Western blot analysis of the excised proteins, the RNase active bands were shown to be group V allergens. Additionally it was demonstrated that an homologous protein to Phl p Vb in the mother plant could be induced by salicylic acid. This indicates that group Vb allergens may be involved in host-pathogen interactions because in pollen they are quickly exported RNases and in the mother plant they depend on a hormone which is related to expression of plant resistance genes.
Allergens↗
Studies on the carbohydrate moieties of the timothy grass pollen allergen Phl p I.
Timothy grass pollen was investigated in order to determine the carbohydrate moieties of its major grass group I (Phl p I) and to study its impact on allergenicity. Based on computer calculations one N-glycosylation site was deduced from the cDNA data of Phl p I. After two-dimensional polyacrylamide gel electrophoresis, followed by blotting of pollen extract and by use of the monoclonal antibody IG 12 we identified at least six isoallergens of Phl p I with the main spots at a molecular mass of 35-37 kDa and a pI range of 6.5-7.3. Deglycosylation by trifluoromethanesulfonic acid resulted in a decrease of about 2 kDa. Treatment with N-glycosidase A resulted in a partial deglycosylation, while N-glycosidase F and O-glycosidase had no effect. Ten lectins were investigated for their binding to Phl p I components: Aleuria aurantia agglutinin showed strong reactivity (indicating fucose residues), while Galanthus nivalis agglutinin (indicating mannose residues) and concanavalin A (indicating mannose, glucose or N-acetylglucosamine residues) showed weak binding. By neutral sugar analysis we determined similar contents of the monosaccharides in the isoallergens. In order to study the influence of the carbohydrate structures of Phl p I on IgE reactivity we tested some patient sera for their reactivity with intact and deglycosylated Phl p I. Even though most of the IgE antibodies bind at the protein core, we detected one serum that recognized carbohydrate moieties on the Phl p I.
Allergens↗
Common CFTR mutations are not likely to predispose to chronic bronchitis in northern Germany.
The frequency of six common mutations in the cystic fibrosis transmembrane conductance regulator gene was studied in 100 patients hospitalized with chronic bronchitis. Only one patient with chronic bronchitis and diffuse bronchiectasis was heterozygous for the common delta F508 mutation. R553X, G542X, G551D, N1303K and 621 + 1G-->T were not detected. This result is not significantly different from the frequency of cystic fibrosis carriers in Northern Europe. Predisposition of heterozygotes for chronic bronchitis is therefore unlikely.
Aged↗
In vitro synthesis of human IgE is suppressed by human IgG.
Recently it has been shown that intravenous immunoglobulin (IVIG) preparations suppress the in vitro synthesis of IgG, IgA, and IgM. In this paper we demonstrate that IVIG and IgG purified from a single donor's serum also suppress the in vitro synthesis of IgE. We had noticed this effect when we added human serum (HS) to in vitro cultures for IgE synthesis. The interleukin-4 (IL-4)-induced IgE synthesis from human peripheral blood mononuclear cells (PBMC) in the presence of fetal calf serum (FCS) was suppressed by HS in a dose-dependent fashion. The following results indicate that this suppression is mediated by IgG: (1) IVIG preparations, which consist mainly of IgG, suppressed the IgE synthesis from IL-4-stimulated PBMC in a dose-dependent way; (2) when HS was fractionated by protein G sepharose or anti-IgG sepharose, the eluate fractions (containing IgG), but not the effluent fractions (void of IgG) suppressed IgE synthesis, whereas the opposite was found when HS was fractionated by FCS-coupled sepharose. We conclude from these data that human IgG preparations suppress the in vitro synthesis not only of the IgG, IgA, and IgM isotypes, but also of the IgE isotype.
Adult↗
Structural investigations of the major allergen Phl p I on the complementary DNA and protein level.
Until now investigations of group I grass allergens have mainly been performed on ryegrass allergen (Lol p I). We studied this major allergen grass group with timothy grass pollen (Phl p I), a very common and important cause of type I allergy, to determine intraspecific and interspecific variations among different grass species. By immunoscreening a timothy grass pollen complementary DNA library we obtained three full-length clones. They revealed identical nucleotide sequences in the coding regions consisting of 262 amino acids, including a leader sequence of 23 amino acid residues. The comparison of our data with the amino acid sequences deduced from Lol p I and Hol 1 I clones showed sequence identities of greater than 85% and homologies of greater than 90%, indicating a high degree of sequence conservation. Despite the high degree of homology, amino acid differences were in immunodominant positions, which may be responsible for the differing immune response to group I allergens of different grass species.
Allergens↗
Characterization of the allergen group VI in timothy grass pollen (Phl p 6). I. Immunological and biochemical studies.
For the identification and characterization of allergen grass group VI we performed two-dimensional immunoblotting of timothy grass pollen (Phleum pratense). Two intense 13-kD protein spots of pI 5.2 and 5.5 were found to be IgE reactive. By N-terminal microsequencing and amino acid analysis we identified them as Phl p 6 isoallergens. An antiserum was raised against Phl p 6 by immunizing BALB/c mice with allergen bearing nitrocellulose particles of one isoform. The antiserum revealed an intense reactivity to Phl p 6 isoforms, but also showed a weak cross-reactivity with Phl p 5 allergens. After immunoabsorption of patients' serum to Phl p 6 spots on the blotting membrane, we were able to demonstrate that the eluted human IgE antibodies cross-react with the grass group V allergens as well. Therefore, Phl p 5 and Phl p 6 possess one or more common IgE binding epitopes.
Allergens↗
Characterization of the allergen group VI in timothy grass pollen (Phl p 6). II. cDNA cloning of Phl p 6 and structural comparison to grass group V.
The complete primary structure of the major allergen Phl p 6 was determined by cDNA cloning. A cDNA library of timothy grass pollen was screened by a Phl p 6 directed mouse antiserum. Six clones were obtained. From the cDNA data we deduced a protein sequence of 110 amino acids. While the size of the leader sequences varied considerably between the different clones, only one amino acid exchange affected the mature proteins. Sequence comparison of group VI and V allergens showed a high degree of homology in the N- and C-terminal regions. The structural data strongly indicate that group VI and V are independent groups of allergens, which have derived from a common precursor gene.
Allergens↗
Frequency of common cystic fibrosis gene mutations in chronic bronchitis patients.
It has been suggested that the delta F508 deletion, the most common mutation in the cystic fibrosis (CF) gene, might be linked to chronic bronchial hypersecretion. We investigated whether such an association could be found in chronic bronchitis, since chronic bronchial hypersecretion is an important and specific element of chronic bronchitis. We screened 100 patients hospitalized for chronic bronchitis with six of the most frequently occurring CF gene mutations: delta F508, R553X, G542X, G551D, N1303K, and 621-1G-->T. Only one patient affected by chronic bronchitis and diffuse bronchiectasis was heterozygous for the deletion delta F508; no other mutations were found. This is not significantly different from the expected frequency of CF carriers in northern Europe, which is 1 in 25. Thus, no association between the most commonly occurring cystic fibrosis genes and chronic bronchitis is likely to exist and routine screening of patients without further signs of cystic fibrosis would seem to be of no benefit in northern Europe.
Aged↗