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Biomedical subjects

H Fiebig

Publications and source records attributed to H Fiebig.

At least 19 recordsLinked to original sources
PubMed2000

Complementary DNA cloning and expression of a newly recognized high molecular mass allergen phl p 13 from timothy grass pollen (Phleum pratense).

BACKGROUND: Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.

Allergens↗
PubMed1999

Rapid and efficient purification of Phleum pratense major allergens Phl p 1 and group Phl p 2/3 using a two-step procedure.

The standardization of natural allergenic extracts and the characterization of recombinant allergens ensures a continuing requirement for highly purified natural allergens. The extraction and purification methods have to be reproducible and also preserve the biological and immunological activity of the allergen. A simple two-step purification system has been established in order to provide milligram amounts of purified natural Phl p 1 and Phl p 2/3. Both major allergens were separated from other proteins of timothy grass pollen extract in one step by hydrophobic interaction chromatography (HIC) under mild conditions. The allergens elute in the flow-through fraction while the rest of the proteins remain bound to the column. The very different molecular weights of Phl p 1 and Phl p 2/3 permitted separation of the allergens by a second step using gel filtration.

Allergens↗
PubMed1999

"Allergen engineering": variants of the timothy grass pollen allergen Phl p 5b with reduced IgE-binding capacity but conserved T cell reactivity.

One problem of conventional allergen-specific immunotherapy is the risk of anaphylactic reactions. A new approach to make immunotherapy safer and more efficient might be the application of engineered allergens with reduced IgE-binding capacity but retained T cell reactivity. Using overlapping dodeca-peptides, the dominant T cell epitopes of the timothy grass pollen allergen Phl p 5b were identified. By site-directed mutagenesis outside these regions, point and deletion mutants were generated. Allergen variants were analyzed for IgE-binding capacity with sera of different grass pollen allergic patients by Western blotting, Dot blotting, and EAST inhibition test, and for histamine releasing capacity with peripheral blood basophils from different patients. The deletion mutants revealed significantly reduced IgE reactivity and histamine releasing capacity, compared with the wild-type Phl p 5b. Furthermore, in vivo skin prick tests showed that the deletion mutants had a significantly lower potency to induce cutaneous reactions than the wild-type Phl p 5b. On the other hand, T cell clones and T cell lines from different allergic patients showed comparable proliferation after stimulation with allergen variants and wild-type Phl p 5b. Considering their reduced anaphylactogenic potential together with their conserved T cell reactivity, the engineered allergens could be important tools for efficient and safe allergen-specific immunotherapy.

Allergens↗
PubMed1999

Standardization of Alternaria alternata: extraction and quantification of alt a 1 by using an mAb-based 2-site binding assay.

BACKGROUND: Alternaria alternata is recognized as an important cause of allergic disease. As with other molds, the extracts of A alternata used for diagnosis and therapy are highly heterogeneous, and there is a need for improved standardization. The major allergen Alt a 1 is well characterized and has been produced as a recombinant protein, but very few data are available on the Alt a 1 content in extracts. OBJECTIVE: An assay for the quantification of Alt a 1 was developed and used for monitoring batch-to-batch consistency of A alternata extracts, and the correlation between skin prick test responses and Alt a 1 concentrations was studied. METHODS: A 2-site binding assay based on an Alt a 1-specific mAb was developed and used for the quantification of Alt a 1 in allergen extracts. Quantitative skin prick tests were performed on 16 A alternata-sensitive patients and correlated with the Alt a 1 concentration. RESULTS: The Alt a 1-specific mAb was found to be suitable for affinity purification, as well as for a 2-site binding quantification assay. In allergen extracts the Alt a 1 content was estimated as 2% to 4.7% of total protein. Quantitative skin prick tests showed an Alt a 1 concentration-dependent response. CONCLUSION: Quantification of Alt a 1 in A alternata extracts reflects their batch-to-batch consistency. Skin prick test responses to a standardized A alternata extract correlate with the Alt a 1 contents. An extract containing 3.7 microgram/mL Alt a 1 caused a response equal to that of a 1% histamine dihydrochloride solution.

Allergens↗
PubMed1999

Reactivity of T cells with grass pollen allergen extract and allergoid.

BACKGROUND: Successful allergen-specific immunotherapy is achieved with progressively increasing doses of allergen or allergoid. In order to gain further insight into the mechanism of action of allergoids several in vitro investigations were conducted. METHODS: Peripheral blood mononuclear cells (PBMC) from grass pollen allergic and nonallergic subjects were stimulated with either grass pollen extract or allergoid and the proliferation and cytokine production (IL-5, IFN-gamma) were measured. Similar investigations were performed with Phl p 5-specific T cell lines (TCL) and clones (TCC). Dendritic cells and PBMC were compared in terms of their relative efficacies as antigen-presenting cells. RESULTS: Both allergen and allergoid induced proliferation and Th2 and Th1 cytokine synthesis by PBMC of allergic subjects, whereas PBMC of nonallergic subjects did not produce IL-5. The maximum level of IL-5 was obtained with a lower concentration than was necessary for maximal IFN-gamma production. Higher stimulation doses of allergen and allergoid shifted the cytokine profiles towards a Th1 phenotype. TCL and TCC clearly showed reactivity with both allergen and allergoid when using autologous PBMC for antigen presentation, but compared with the native allergen the reactivity of the allergoid was reduced with most of the TCC. Using dendritic cells for antigen presentation a pronounced increase of stimulation of the TCC especially for the allergoids becomes obvious. CONCLUSION: In common with grass pollen allergen the corresponding allergoids possess a strong allergen-specific T cell-stimulating capacity. However, the degree of T cell stimulation by the allergoid seems to be dependent on the type of the antigen-presenting cell. Both, allergen and allergoid, can modulate T cell responses in a dose-dependent manner.

Allergens↗
PubMed1998

Detection and quantification of group 4 allergens in grass pollen extracts using monoclonal antibodies.

BACKGROUND: Grass pollen extracts are complex mixtures consisting of different major allergenic and non-allergenic components. Phl p 4 is an important allergen, because more than 75% of grass pollen allergic patients produce specific IgE antibodies against group 4 allergens. OBJECTIVE: This study was designed to investigate the specificity of monoclonal antibodies (MoAbs) produced against Phl p 4 and to verify the presence of group 4-like proteins in different grass pollen. Furthermore the usefulness of MoAbs for quantification of group 4 allergens was studied. METHODS: Group 4 analogues were investigated by immunoblotting and ELISA inhibition using three MoAbs. The specificity of antibodies was studied using isolated group 1 and group 5 allergens. Quantification of group 4 allergen was achieved by a two-site solid-phase ELISA. Phl p 4 was purified from whole pollen extract by chromatographic or electrophoretic techniques and used as standard. RESULTS: The MoAbs studied bound strongly to proteins from timothy grass pollen extract at a mw of 55 kDa and a pI of 9.0-9.3. Phl p 4 homologes with similar mw were detected in Dactylis glomerata, Festuca pratensis, Holcus lanatus, Poa pratensis, Lolium perenne. Epitope mapping showed that all three MoAb recognized unrelated regions on Phl p 4. A two-site binding ELISA using MoAbs was developed for determination of Phl p 4 in Phleum pratense extracts. The method was able to evaluate group 4 in mass units with a working range between 150 and 2000 ng/mL. The absolute amounts of group 4 in extracts of several grasses varied considerably but was always-less than 1% of the total protein. CONCLUSION: Group 4 homologes are present in the various grass extracts but to different extents. The group 4 ELISA could be very useful as a additional tool for providing information concerning the composition of grass pollen extracts.

Allergens↗
PubMed1998

Mapping of T-cell epitopes of Phl p 5: evidence for crossreacting and non-crossreacting T-cell epitopes within Phl p 5 isoallergens.

BACKGROUND: Group 5 allergens represent major grass pollen allergens because of their high sensitization indices. The identification of T-cell epitopes of these allergens is a prerequisite for the design of immunotherapeutic strategies based on peptide vaccination or modified allergens with conserved T-cell epitopes. OBJECTIVE: This study was undertaken to determine T-cell epitopes on Phl p 5 major pollen allergen of timothy grass (Phleumn pratense). METHODS: T-cell lines (TCLs) and T-cell clones (TCCs), specific to Phl p 5, were established from the peripheral blood of 18 patients allergic to grass pollen. All TCCs were mapped for epitope specificities using 178 overlapping dodecapeptides representing the primary structures of two isoforms of Phl p 5 (Phl p 5a and Phl p 5b). Phenotype and cytokine production profiles of TCCs were tested. Selected TCCs were analysed for HLA class II restriction. RESULTS: A total of 82 TCCs were isolated. All TCCs displayed the helper cell (TH) phenotype. Their reactivity with two recombinant expressed isoforms of Phl p 5a and Phl p 5b was heterogeneous. The epitope specificity of the TCCs was then revealed. Nineteen T-cell epitopes could be identified on Phl p 5. Eighty-one percent of mapped TCCs recognized three T-cell reactive regions on the Phl p 5 allergen. Some TCCs were reactive with isoepitopes presenting on Phl p 5a as well as Phl p 5b. Allergen-specific stimulation induced a TH0-like type of cytokine production in 25 of 50 TCCs. Almost all TCCs secreted high concentrations of interleukin-13. CONCLUSION: Phl p 5, a major grass pollen allergen, contains several T-cell epitopes. Some epitope regions were recognized by several patients. Epitope recognition pattern could not be correlated with special HLA class II haplotypes. T-cell stimulating isoepitopes were found at corresponding regions of Phl p 5a and Phl p 5b isoforms.

Adult↗
PubMed1996

Conformational changes in CD45 upon monoclonal antibody crosslinking.

The CD45 protein tyrosine phosphatase is expressed in different isoforms that result from alternative splicing of three exons (A, B, and C) encoding regions near the N-terminus of the extracellular part of the molecule. We describe here a novel epitope on the N-terminal end of CD45 that is recognized by the MAb BL-TSub/2. Crossblocking studies showed that BL-TSub/2 and UCHL1 (CD45RO) binding sites are partially overlapping. However, in marked contrast to the CD45RO epitope, protease treatment of cells strongly diminished BL-TSub/2 binding. Similar to the UCHL1 epitope, the BL-TSub/2 binding site involves carbohydrate moieties, since neuraminidase treatment abrogated the reactivity of the MAb. Markedly, preincubation of cells with both CD45 common and CD45RA MAb induced a pronounced increase of BL-TSub/2 binding. This latter finding suggests that crosslinking of the CD45 molecule leads to conformational changes that could influence association of the molecule with putative ligands.

Animals↗
PubMed1996

Characterization of major allergens of Parietaria officinalis.

The major allergens of Parietaria officinalis were characterized with a panel of nine monoclonal antibodies (mAbs). The binding of mAbs and patients' IgE in Western blots revealed two proteins with similar molecular weights in the range of 8-10 kD. Analysis of the mAb-binding patterns in Western blots of P. officinalis extract under reducing and nonreducing conditions allows the mAbs to be divided into three different groups. mAbs of group I recognize the higher-molecular-weight component (9.4 kD), mAbs of group II recognize the lower component (8.8 kD) and mAbs of group III recognize both proteins. A comparable mAb-binding pattern was observed with Western blots of Parietaria judaica. The mAbs were used for affinity purification of the corresponding proteins from a P. officinalis extract. The purified proteins obtained with mAbs of group I-III inhibit the binding of patients' IgE (serum pool) to a high degree, indicating that they posses the major IgE-reactive epitopes. The affinity-purified proteins were subjected to SDS-PAGE, blotted and immunologically stained by mAb binding. The results confirmed those obtained with the complete extracts. The N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of all the proteins contained highly conserved regions: GGVV (positions 4-7) and MPPLL (positions 11-15), alternating with highly variable regions (positions 1-3 for group II and 8-10 for group I). A specific group I sequence appears to be at position 1-3 with the amino acids APA and a specific group II sequence appears to be at position 8-10 with the amino acids GAL. It is possible that the two similar proteins are isoforms of Par o 1.

Allergens↗
PubMed1995

In vitro production of monoclonal antibodies in high concentration in a new and easy to handle modular minifermenter.

This paper describes a new and easy to handle reusable minifermenter for high-density culture of hybridoma and other cells. The culture apparatus is composed of two modules: a 40 ml disposable cell culture and antibody production chamber (the 'production module') and a 550 ml medium reservoir (the 'supply module'). The two modules are separated from each other by a dialysis membrane allowing passage of low molecular mass nutrients and metabolites. The monoclonal antibodies are produced and enriched in the production module. The outer part of this module is made from a thin gas-permeable silicone rubber membrane allowing exchange of gases (oxygen and carbon dioxide). To start the culture, the cells are injected into the production module through ports in the silicone rubber which are equipped with Luer Lock connectors. Samples can be removed in the same way. For culturing, the minifermenter is rolled on a roller apparatus in a carbon dioxide-supplied incubator. Depending on the individual properties of the hybridoma cells cultured, cell densities of more than 10 x 10(6) (in some cases up to 35 x 10(6)) cells per ml and monoclonal antibody concentrations of several mg per ml can be obtained in the new minifermenter. On average, 61 mg (range: 9-159 mg) could be produced within 1-4 weeks. In terms of their properties the monoclonal antibodies produced in the new modular minifermenter were indistinguishable from antibodies prepared from ascitic fluid or from the supernatant of conventional stationary culture. The culture method is a useful alternative to the in vivo production method in mice. In addition, it represents a completely new, inexpensive and easy to handle general solution to the problem of culturing cells in high density and obtaining cellular products in high concentrations.

Antibodies, Monoclonal↗
PubMed1993

Expression cloning of the early activation antigen CD69, a type II integral membrane protein with a C-type lectin domain.

CD69 is a very early activation Ag of T lymphocytes. It is a cell surface glycoprotein that can only be detected after stimulation of lymphocytes. Despite extensive studies on its biologic activities, little is known about its molecular function. To investigate the latter in more detail, we have cloned a cDNA encoding CD69 on the basis of its expression in COS cells. The nucleotide sequence of clone CD69.13 is 1676 bp in length and contains a single open reading frame of 600 bp encoding a protein of 199 amino acids. The predicted molecular mass of 22,559 Da could be confirmed by in vitro translation. The protein contains a hydrophobic transmembrane region between amino acids 41 and 61 but no N-terminal signal peptide, which suggests that it is a type II membrane protein. It has one potential N-glycosylation site at amino acid 166. Two glycosylated forms of 26 to 28 kDa and 32 to 34 kDa were detected both in transfected COS cells and in in vitro translation in the presence of canine microsomes. Proteinase K degradation of the N-terminal part after in vitro protein synthesis supports the view of CD69 being a type II integral membrane protein with the N-terminal 40 amino acids in the cytoplasm, a transmembrane domain of 21 amino acids, and C-terminal 138 amino acids as the extracellular domain. Homology searches revealed sequence similarity with members of a supergene family of type II integral membrane proteins with a C-type lectin domain, indicating that CD69 is involved in signal transduction.

Amino Acid Sequence↗
PubMed1993

Regional sublocalization of the human CD69 gene to chromosome bands 12p12.3-p13.2, the predicted region of the human natural killer cell gene complex.

The early activation antigen CD69 is a member of a supergene family of type II integral membrane proteins with a C-type lectin domain. In recent reports the genes encoding the natural killer (NK) cell-related molecules of this supergene family, NKR-P1, NK1.1 and Ly-49, were shown to be clustered in a chromosomal region in mouse, termed the NK gene complex. The human homologue of this complex is likely to reside on chromosome 12 near the PRP locus (12p13.2). By analyzing T cell hybrids, the CD69 gene was previously mapped to human chromosome 12. Here we report the regional sublocalization of the human CD69 gene to chromosome bands 12p12.3-p13.2, suggesting that CD69 belongs to one linkage group together with different cell surface molecules on NK cells.

Animals↗
PubMed1991

[Characterization of a T-cell subpopulation with a new monoclonal antibody BL-TSub/2].

The mab BL-TSub/2 binds to a majority (80%) of thymocytes. The antigen isolated from these cells has an apparent molecular weight of about 170 kDa. The mab BL-TSub/2 recognized antigen is expressed on approximately 50% of mature T cells in peripheral blood. The mab BL-TSub/2 does not bind to monocytes, granulocytes, platelets and CD16+NK cells. The antigen is possibly present on a part of peripheral B cells in very low density. Double labelling experiments showed that about 30% of CD4+ and 60% of CD8+ cells react with the mab BL-TSub/2. This BL-TSub/2+ subset is not identical with T-cell subpopulations till now defined. Within the CD4+ subset the majority of BL-TSub/2+ cells is included in the CD45RO+ subset while only a small proportion is CD45RA+. However, the CD8+ BL-TSub/2+ cells are mostly CD45RA+ with only few cells belonging to the CD45RO+ subset.

Antibodies, Monoclonal↗
PubMed1991

[Epitope analysis of CD68 antigens].

Different epitopes of CD68 antigen are detectable by three new generated monoclonal antibodies (MABs) BL-M68/1-3 (all mouse IgM). The MAB BL-M68/3 reacts with an epitope, which is stable also in formalin-fixed paraffin-embedded tissues. In immunohistochemical blocking experiments using the own biotinylated MABs and six CD68 MABs from the IV. Workshop on Human Leucocyte Differentiation Antigens two new epitopes could be defined. The number of known CD68 epitopes increases at least up to seven.

Antibodies, Monoclonal↗
PubMed1991

[Characterization of a monoclonal antibody against a differentiation antigen of human natural killer cells (NK cells)].

The new murine monoclonal antibody BL-(H5) reacts with a novel surface molecule which is mainly expressed on human NK and B cells. The antigen is not expressed on peripheral T lymphocytes, thymocytes and different human T-cell lines. BL-(H5) does not bind to erythrocytes and platelets. The monoclonal antibody reacts in western blotting experiments with an antigen of 78kDa.

Antibodies, Monoclonal↗
PubMed1991

[Circulating lymphocyte subpopulations in patients with Crohn's disease].

Circulating lymphocytes were enumerated in 25 patients with Crohn's disease and in 43 healthy donors by immunofluorescent staining using monoclonal antibodies for T cell surface phenotypic markers (CD 3, CD 4, CD 8) or for surface immunoglobulin of B cells (mu-chain). Proportions of peripheral T cells and percentages of T cell subsets in Crohn's disease were not significantly different from those in controls. For analyzing the lymphocytes for the expression of activation associated antigens (HLA-DR, CD 25) we found increased numbers of activated lymphocytes during acute exacerbation, whereas, in remission, the population of activated lymphocytes was in the upper normal range. The results leads to the conclusion that the assessment of activated lymphocytes may serve as a parameter in the evaluation of the activity of Crohn's disease.

Adolescent↗