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Ulrich Blank

Publications and source records attributed to Ulrich Blank.

12 recordsLinked to original sources
PubMed2006

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens.

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.

Allergens↗
PubMed2006

IgA Fc receptor I signals apoptosis through the FcRgamma ITAM and affects tumor growth.

The IgA Fc receptor (FcalphaRI) has dual proinflammatory and anti-inflammatory functions that are transmitted through the immunoreceptor tyrosine-based activation motifs (ITAMs) of the associated FcRgamma subunit. Whereas the involvement of FcalphaRI in inflammation is well documented, little is known of its anti-inflammatory mechanisms. Here we show that monomeric targeting of FcalphaRI by anti-FcalphaRI Fab or serum IgA triggers apoptosis in human monocytes, monocytic cell lines, and FcalphaRI+ transfectants. However, the physiologic ligand IgA induced apoptosis only when cells were cultured in low serum conditions, indicating differences with induction of anti-inflammatory signaling. Apoptosis signaling required the FcRgamma ITAM, as cells transfected with FcalphaRI or with a chimeric FcalphaRI-FcRgamma responded to death-activating signals, whereas cells expressing a mutated FcalphaRI(R209L) unable to associate with FcRgamma, or an ITAM-mutated chimeric FcalphaRI-FcRgamma, did not respond. FcalphaRI-mediated apoptosis signals were blocked by treatment with the pan-caspase inhibitor zVAD-fmk, involved proteolysis of procaspase-3, and correlated negatively with SHP-1 concentration. Anti-FcalphaRI Fab treatment of nude mice injected subcutaneously with FcalphaRI+ mast-cell transfectants prevented tumor development and halted the growth of established tumors. These findings demonstrate that, on monomeric targeting, FcalphaRI functions as an FcRgamma ITAM-dependent apoptotic module that may be fundamental for controlling inflammation and tumor growth.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Mast cell-mediated remodeling and fibrinolytic activity protect against fatal glomerulonephritis.

Mast cells are detrimental in several inflammatory diseases; however, their physiological roles are also increasingly recognized. Recent data suggest that mast cells may also be involved in renal diseases. We therefore used congenitally mast cell-deficient W/W(v) mice and normal +/+ littermates to assess their role in anti-glomerular basement membrane-induced glomerulonephritis. Following administration of anti-glomerular basement membrane Abs, W/W(v) mice exhibited increased mortality as compared with +/+ mice owing to rapid deterioration of renal function. Reconstitution of the mast cell population in W/W(v) mice restored protection. This was independent of activating FcgammaR, as protection was also obtained using mast cells deficient in FcRgamma. Comparative histological analysis of kidneys showed that deterioration of renal function was caused by the presence of thick layers of subendothelial glomerular deposits in W/W(v) mice, while +/+ mice or mast cell-reconstituted W/W(v) mice showed significantly less. Deposits appeared during the early phase of disease and persisted thereafter, and were accompanied by enhanced macrophage recruitment. Immunohistochemical analysis revealed increased amounts of fibrin and type I collagen in W/W(v) mice, which were also unable to maintain high tissue plasminogen activator and urinary-type plasminogen activator activity in urine in the heterologous phase of disease. Our results indicate that mast cells by their ability to mediate remodeling and repair functions are protective in immune complex-mediated glomerulonephritis.

Animals↗
PubMed2005

Identification of FcalphaRI as an inhibitory receptor that controls inflammation: dual role of FcRgamma ITAM.

Serum IgA is considered a discrete housekeeper of the immune system with multiple anti-inflammatory functions, whereas IgA-immune complexes mediate inflammatory responses. Here, we identify FcalphaRI as a molecular device that determines the nature of IgA responses. In the absence of sustained aggregation, receptor targeting by serum IgA or anti-FcalphaRI Fab inhibits activating responses of heterologous FcgammaR or FcepsilonRI. The inhibitory mechanism involves recruitment of tyrosine phosphatase SHP-1 to FcalphaRI and impairment of Syk, LAT, and ERK phosphorylation induced by FcepsilonRI engagement. SHP-1 recruitment is dependent on ERK. Conversely, sustained aggregation of FcalphaRI by multimeric ligands stimulates cell activation by recruiting high amounts of Syk and aborting SHP-1 binding. Both types of signals require the FcRgamma-ITAM motif. Anti-FcalphaRI Fab treatment suppresses manifestations of allergic asthma in FcalphaRI transgenic mice. These findings redefine FcalphaRI as a bifunctional inhibitory/activating receptor of the immune system that mediates both anti- and proinflammatory functions of IgA.

Animals↗
PubMed2003

Munc18-2/syntaxin3 complexes are spatially separated from syntaxin3-containing SNARE complexes.

Exocytosis of mast cell granules requires a vesicular- and plasma membrane-associated fusion machinery. We examined the distribution of SNARE membrane fusion and Munc18 accessory proteins in lipid rafts of RBL mast cells. SNAREs were found either excluded (syntaxin2), equally distributed between raft and non-raft fractions (syntaxin4, VAMP-8, VAMP-2), or selectively enriched in rafts (syntaxin3, SNAP-23). Syntaxin4-binding Munc18-3 was absent, whereas small amounts of the syntaxin3-interacting partner Munc18-2 consistently distributed into rafts. Cognate SNARE complexes of syntaxin3 with SNAP-23 and VAMP-8 were enriched in rafts, whereas Munc18-2/syntaxin3 complexes were excluded. This demonstrates a spatial separation between these two types of complexes and suggests that Munc18-2 acts in a step different from SNARE complex formation and fusion.

Animals↗
PubMed2003

Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis.

Compound exocytosis of inflammatory mediators from mast cells requires SNARE and a series of accessory proteins. However, the molecular steps that regulate secretory granule movement and membrane fusion as well as the role of the cytoskeleton are still poorly understood. Here, we report on our investigation of the role of syntaxin-binding Munc18 isoforms and the microtubule network in this process. We found that mast cells express Munc18-2, which interacts with target SNAREs syntaxin 2 or 3, as well as Munc18-3, which interacts with syntaxin 4. Munc18-2 was localised to secretory granules, whereas Munc18-3 was found on the plasma membrane. Increased expression of Munc18-2 and derived peptides containing an interfering effector loop inhibited IgE-triggered exocytosis, while increased expression of Munc18-3 showed no effect. Munc18-2 localisation on granules is polarised; however, upon stimulation Munc18-2 redistributed into forming lamellipodia and persisted on granules that were aligned along microtubules, but was excluded from F-actin ruffles. Disruption of the microtubule network with nocodazole provoked Munc18-2 redistribution and affected mediator release. These findings suggest a role for Munc18-2 and the microtubule network in the regulation of secretory granule dynamics in mast cells.

Actins↗
PubMed2003

[The high-affinity IgE receptor: lessons from structural analysis].

The high affinity receptor for IgE, FcERI, is at the core of the allergic reaction. This receptor is expressed mainly on mast cells and basophils. Interaction of an allergen with its specific IgE bound to FcERI triggers cell activation, which induces the release of numerous mediators that are responsible for allergic manifestations. The recent increase in the prevalence of allergic diseases in developed countries has resulted in renewed efforts towards the development of new drugs. One of these is a humanised antibody directed against the IgE ligand. This antibody recognises specifically free but not FcERI-bound IgE thus preventing ligand binding and subsequent cell activation. This antibody has shown some efficacy in clinical trials involving patients with asthma and allergic rhinitis. The recent elucidation of the tridimensional structure of the complex between IgE and FcERI provides unexpected information regarding the mechanism of assembly of the complex, which now can be used to design small chemical compounds capable of specifically inhibiting this interaction.

Amino Acid Motifs↗
PubMed2002

Vav1 is a component of transcriptionally active complexes.

The importance of the hematopoietic protooncogene Vav1 in immune cell function is widely recognized, although its regulatory mechanisms are not completely understood. Here, we examined whether Vav1 has a nuclear function, as past studies have reported its nuclear localization. Our findings provide a definitive demonstration of Vav1 nuclear localization in a receptor stimulation-dependent manner and reveal a critical role for the COOH-terminal Src homology 3 (SH3) domain and a nuclear localization sequence within the pleckstrin homology domain. Analysis of DNA-bound transcription factor complexes revealed nuclear Vav1 as an integral component of transcriptionally active nuclear factor of activated T cells (NFAT)- and nuclear factor (NF)kappaB-like complexes, and the COOH-terminal SH3 domain as being critical in their formation. Thus, we describe a novel nuclear role for Vav1 as a component and facilitator of NFAT and NFkappaB-like transcriptional activity.

Active Transport, Cell Nucleus↗
PubMed2002

The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells.

Rab3 GTPases regulate exocytosis of neurons, endocrine and exocrine cells. In the present paper, we report a system to measure the guanine nucleotide status of Rab3 proteins in living cells. The assay is based on the ability of the Rab3 interacting molecule RIM to extract selectively the GTP-bound form of Rab3. Using this system, we found that approx. 20% of wild-type Rab3A, -B, -C or -D transfected in the insulin-secreting cell line HIT-T15 is in the GTP-bound conformation. The pool of activated Rab3 is decreased under conditions that stimulate exocytosis or by co-expression of the Rab3 GTPase-activating protein. In contrast, co-expression of Mss4 or Rab3-GEP (guanine nucleotide exchange protein) increases by approx. 3-fold the GTP-bound pool of Rab3 isoforms. Rab3-GEP is very similar to MADD, a death domain-containing protein that associates with the type 1 tumour necrosis factor receptor. We observed that the death domain of Rab3-GEP is involved in intramolecular interactions and that deletions or mutations that affect this domain of the protein impair the nucleotide exchange activity towards Rab3. We propose that the death domain of Rab3-GEP acts as a molecular switch and co-ordinates multiple functions of the protein by exchanging its binding partners.

Amino Acid Sequence↗
PubMed2002

SNAREs and associated regulators in the control of exocytosis in the RBL-2H3 mast cell line.

Mast cells participate in inflammation and allergies by releasing biologically active mediators stored in numerous cytoplasmic granules. Degranulation is tightly controlled and requires activation of cell surface receptors, such as the high affinity IgE receptor (FcepsilonRI). Here, we discuss some of the key components of the molecular machinery that regulates the final steps of fusion between the granular and plasma membrane based on results obtained with the rat mast cell line RBL-2H3. We emphasize the role of soluble N-ethylmaleimide attachment protein receptors (SNAREs) proteins such as syntaxin 4 that can promote membrane fusion through formation of a stable complex with SNAP-23. We also highlight the role of a Ser/Thr kinase found to be associated with Rab3D, a negative regulator of degranulation. Associated kinase activity, which diminishes after stimulation as a consequence of intracellular calcium increases, specifically phosphorylates syntaxin 4 thereby affecting its capacity to bind to its t-SNARE partner SNAP-23. Our results suggest a new way of how Rab3 GTPases may intersect with the function of SNAREs thought to be universal mediators of membrane fusion.

Animals↗
PubMedDate not supplied

[The heritage of anaphylaxis].

This text resumes the steps in Immunology that have followed the discovery of anaphylaxis in 1902 by P. Portier and C. Richet. Anaphylaxis was difficult to explain at the beginning of the 20th century where "phylactic" thinking predominated in Immunology. About one hundred years of research were necessary to identify the serum factor, IgE, the cellular actors with their IgE receptors as well as their activation mechanisms and to obtain an atomic view of the interaction between IgE and its receptor. Its also after one century that arrives the first treatment derived from biotechnological research that aims allergies which is the most commonly known expression of an anaphylactic reaction.

Allergy and Immunology↗