Recombinant PreS-fusion protein vaccine for birch pollen and apple allergy.

BACKGROUND: IgE cross-sensitization to major birch pollen allergen Bet v 1 and pathogenesis-related (PR10) plant food allergens is responsible for the pollen-food allergy syndrome. METHODS: We designed a recombinant protein, AB-PreS, consisting of non-allergenic peptides derived from the IgE-binding sites of Bet v 1 and the cross-reactive apple allergen, Mal d 1, fused to the PreS domain of HBV surface protein as immunological carrier. AB-PreS was expressed in E. coli and purified by chromatography. The allergenic and inflammatory activity of AB-PreS was tested using basophils and PBMCs from birch pollen allergic patients. The ability of antibodies induced by immunization of rabbits with AB-PreS and birch pollen extract-based vaccines to inhibit allergic patients IgE binding to Bet v 1 and Mal d 1 was assessed by ELISA. RESULTS: IgE-binding experiments and basophil activation test revealed the hypoallergenic nature of AB-PreS. AB-PreS induced lower T-cell activation and inflammatory cytokine production in cultured PBMCs from allergic patients. IgG antibodies induced by five injections with AB-PreS inhibited allergic patients' IgE binding to Bet v 1 and Mal d 1 better than did IgG induced by up to 30 injections of six licensed birch pollen allergen extract-based vaccines. Additionally, immunization with AB-PreS induced HBV-specific antibodies potentially protecting from infection with HBV. CONCLUSION: The recombinant AB-PreS-based vaccine is hypoallergenic and superior over currently registered allergen extract-based vaccines regarding the induction of blocking antibodies to Bet v 1 and Mal d 1 in animals.

Murine model of steroid-resistant neutrophilic bronchial asthma as an attempt to simulate human pathology.

Bronchial asthma (BA) is a heterogeneous chronic inflammatory disease of the airways. The majority of patients with mild to moderate BA develop Th2-biased eosinophilic pulmonary inflammation and respond well to corticosteroid treatment. However up to 10% of BA patients develop severe pathology, which is associated with neutrophilic inflammation and resistant to conventional corticosteroid therapy. Contrary to eosinophil-predominant airway inflammation neutrophilic BA is developed through Th1- and Th17-immune responses. However, the etiology of corticoid insensitive neutrophilic BA is still remains unclear. Therefore, in the current study we developed a mouse model of BA with predominant neutrophilic rather than eosinophilic pulmonary inflammation. BALB/c mice were immunized with the mixture of the ovalbumin allergen and Freund's adjuvant, followed by aerosol challenge with the same allergen mixed with E. coli lipopolysaccharide. As a result, mice developed the main BA manifestations: production of allergen specific IgE, development of airway hyperreactivity, airway remodeling and pulmonary neutrophilic inflammation. Moreover, this pathology developed through Th1- and Th17-dependent mechanisms and mice with induced neutrophilic BA phenotype responded poorly to dexamethasone treatment, that coincide to clinical observations. The established mouse model could be useful both for studying the pathogenesis and for testing novel approaches to control neutrophilic BA.

A pan-metazoan concept for adult stem cells: the wobbling Penrose landscape.

Adult stem cells (ASCs) in vertebrates and model invertebrates (e.g. Drosophila melanogaster) are typically long-lived, lineage-restricted, clonogenic and quiescent cells with somatic descendants and tissue/organ-restricted activities. Such ASCs are mostly rare, morphologically undifferentiated, and undergo asymmetric cell division. Characterized by 'stemness' gene expression, they can regulate tissue/organ homeostasis, repair and regeneration. By contrast, analysis of other animal phyla shows that ASCs emerge at different life stages, present both differentiated and undifferentiated phenotypes, and may possess amoeboid movement. Usually pluri/totipotent, they may express germ-cell markers, but often lack germ-line sequestering, and typically do not reside in discrete niches. ASCs may constitute up to 40% of animal cells, and participate in a range of biological phenomena, from whole-body regeneration, dormancy, and agametic asexual reproduction, to indeterminate growth. They are considered legitimate units of selection. Conceptualizing this divergence, we present an alternative stemness metaphor to the Waddington landscape: the 'wobbling Penrose' landscape. Here, totipotent ASCs adopt ascending/descending courses of an 'Escherian stairwell', in a lifelong totipotency pathway. ASCs may also travel along lower stemness echelons to reach fully differentiated states. However, from any starting state, cells can change their stemness status, underscoring their dynamic cellular potencies. Thus, vertebrate ASCs may reflect just one metazoan ASC archetype.

Coelomocyte replenishment in adult Asterias rubens: the possible ways.

The origin of cells involved in regeneration in echinoderms remains an open question. Replenishment of circulatory coelomocytes-cells of the coelomic cavity in starfish-is an example of physiological regeneration. The coelomic epithelium is considered to be the main source of coelomocytes, but many details of this process remain unclear. This study examined the role of coelomocytes outside circulation, named marginal coelomocytes and small undifferentiated cells of the coelomic epithelium in coelomocyte replenishment in Asterias rubens. A qualitative and quantitative comparison of circulatory and marginal coelomocytes, as well as changes of circulatory coelomocyte concentrations in response to injury at different physiological statuses, was analysed. The presence of cells morphologically similar to coelomocytes in the context of coelomic epithelium was evaluated by electron microscopy. The irregular distribution of small cells on the surface and within the coelomic epithelium was demonstrated and the origin of small undifferentiated cells and large agranulocytes from the coelomic epithelium was suggested. Two events have been proposed to mediate the replenishment of coelomocytes in the coelom: migration of mature coelomocytes of the marginal cell pool and migration of small undifferentiated cells of the coelomic epithelium. The proteomic analysis of circulatory coelomocytes, coelomic epithelial cells and a subpopulation of coelomic epithelial cells, enriched in small undifferentiated cells, revealed proteins that were common and specific for each cell pool. Among these molecules were regulatory proteins, potential participants of regenerative processes.

Injury affects coelomic fluid proteome of the common starfish, Asterias rubens.

Echinoderms, possessing outstanding regenerative capabilities, provide a unique model system for the study of response to injury. However, little is known about the proteomic composition of coelomic fluid, an important biofluid circulating throughout the animal's body and reflecting the overall biological status of the organism. In this study, we used LC-MALDI tandem mass spectrometry to characterize the proteome of the cell-free coelomic fluid of the starfish Asterias rubens and to follow the changes occurring in response to puncture wound and blood loss. In total, 91 proteins were identified, of which 61 were extracellular soluble and 16 were bound to the plasma membrane. The most represented functional terms were 'pattern recognition receptor activity' and 'peptidase inhibitor activity'. A series of candidate proteins involved in early response to injury was revealed. Ependymin, ß-microseminoprotein, serum amyloid A and avidin-like proteins, which are known to be involved in intestinal regeneration in the sea cucumber, were also identified as injury-responsive proteins. Our results expand the list of proteins potentially involved in defense and regeneration in echinoderms and demonstrate dramatic effects of injury on the coelomic fluid proteome.

Ultrastructure and functional morphology of dermal glands in the freshwater mite Limnochares aquatica (L., 1758) (Acariformes, Limnocharidae).

This study is the first attempt to describe the ultrastructure and functional morphology of the dermal glands in Limnochares aquatica (L., 1758). The dermal glands were studied using light-optical, SEM and TEM microscopy methods during different stages of their activity. In contrast to the vast majority of other fresh water mites, dermal glands of the studied species are originally multiplied and scattered freely over the mite body surface. The opening of the glands is saddle-like, formed of several tight cuticular folds and oriented freely to the long axis of the mite body. Either a small cuticular spine or, rarely, a slim sensitive seta is placed on one pole of the opening. On the inside, the central gland portion is provided with a complex cuticular helicoid armature. The glands are composed of prismatic cells situated around the intra-alveolar lumen, variously present, and look like a fig-fruit with the basal surface facing the body cavity. The glands are provided with extremely numerous microtubules, frequently arranged in bundles, and totally devoid of synthetic apparatus such as RER cisterns and Golgi bodies. Three states of the gland morphology depending on their functional activity may be recognized: (i) glands without secretion with highly folded cell walls and numerous microtubules within the cytoplasm, (ii) glands with an electron-dense granular secretion in the expanded vacuoles and (iii) glands with the secretion totally extruded presenting giant empty vacuoles bordered with slim cytoplasmic strips on the periphery. Summer specimens usually show the first gland state, whereas winter specimens, conversely, more often demonstrate the second and the third states. This situation may depend on some factors like changes of the seasonal temperature, pH, or oxygenation of the ambient water. On the assumption of the morphological characters, dermal glands may be classified not as secretory but as a special additional excretory organ system of the body cavity. Despite the glands lack cambial cells, restoration of functions after releasing of 'secretion' looks possible. Organization of dermal glands is discussed in comparison to other water mites studied.

EGF-induced dynamics of NF-κB and F-actin in A431 cells spread on fibronectin.

To evaluate the role of actin cytoskeleton in the regulation of NF-κB transcription factor, we analyzed its involvement in the intracellular transport and nuclear translocation of the NF-κB RelA/p65 subunit in A431 epithelial cells stimulated with fibronectin and EGF. Live cell imaging and confocal microscopy showed that EGF activated the movement of RelA/p65 in the cytoplasm. Upon cell adhesion to fibronectin, RelA/p65 concentrated onto stress fibers, and EGF stimulated its subsequent allocation to membrane ruffles, newly organized stress fibers, and discrete cytoplasmic actin-rich patches. These patches also contained α-actinin-1 and α-actinin-4, vinculin, paxillin, α-tubulin, and PI3-kinase. Cytochalasin D treatment resulted in RelA/p65 redistribution to actin-containing aggregates, with the number of cells with RelA/p65-containing clusters in the cytoplasm increasing under the effect of EGF. Furthermore, EGF proved to induce RelA/p65 accumulation in the nucleus after cell pretreatment with actin-stabilizing and actin-destabilizing agents, which was accompanied by changes in its DNA-binding activity after either EGF stimulation or cytochalasin D treatment. Thus, EGF treatment of A431 cells results in simultaneous nuclear RelA/p65 translocation and cytoplasmic redistribution, with part of RelA/p65 pool forming a very tight association with actin-rich structures. Apparently, nuclear transport is independent on drug stabilization or destabilization of the actin.

Small coelomic epithelial cells of the starfish Asterias rubens L. that are able to proliferate in vivo and in vitro.

Echinoderms, due to their outstanding potential for regeneration, are widely used as experimental models for research in regenerative biology. One of the main problems in this field concerns identification and characterization of cells responsible for the restoration of lost body parts and organs in adult animals. In this study, we analyze the probable candidates for this role in the starfish Asterias rubens L., namely, small coelomic epithelial cells with a high nuclear-cytoplasmic ratio that have the ability to proliferate. These cells are one of several cell types common to the coelomic epithelium (CE) and coelomic fluid (CF). They are analyzed with respect to morphology, proportion in the total cell pool, dynamics after injury and distribution between CE and CF. The results of whole-mount and scanning electron microscopy provide evidence that these small cells occupy a boundary position between CE and CF. Moreover, a novel subpopulation of CE cells is identified that is enriched (up to 50 %) with small epitheliocytes capable of migrating from CE into the CF. As shown in experiments with BrdU incorporation and anti-phospho-histone H3 antibody staining, small epitheliocytes cultured on laminin retain proliferative activity for at least 1 month and can form colony-like aggregates. Two types of small proliferating cells are distinguished by their behavior in culture: some cells remain attached to the substrate and form aggregates, while others detach from the substrate during culturing. The morphology of small epitheliocytes, their proliferative activity in vivo and in vitro and the ability to migrate suggest that they possess certain properties characteristic of stem cells.

Functional compartmentalisation of NF-kB-associated proteins in A431 cells.

NF-kB proteins belong to a family of ubiquitous transcription factors involved in a number of cellular responses. While the pathways of NF-kB activation and input into the regulation of gene activity have been comprehensively investigated, its cytoplasmic functions are poorly understood. In this study we addressed effects of the compartmentalisation of NF-kB proteins RelA/p65 and p50 in relation to the inhibitor IkB-a, using fibronectin (FN) and epidermal growth factor (EGF) for environmental stimulation of epidermoid carcinoma A431 cells. We thus assessed the presence of NF-kB family proteins in the cytosol, membrane, nuclear and cytoskeletal fractions with a special attention to the cytoskeletal fraction to define whether NFkB was active or not. Sub-cellular fractionation demonstrated that the proportion of RelA/p65 differed in diverse sub-cellular fractions, and that the cytoskeleton harboured about 7% thereof. Neither the nuclear nor the cytoskeleton fraction did contain IkB-a. The cytoskeleton binding of RelA/p65 and p50 was further confirmed by co-localisation and electron microscopy data. During 30-min EGF stimulation similar dynamics were found for RelA/p65 and IkB-a in the cytosol, RelA/p65 and p50 in the nucleus and p50 and IkB-a in the membrane. Furthermore, EGF stimulation for 30 min resulted in a threefold accumulation of RelA/p65 in cytoskeletal fraction. Our results suggest that nuclear-, membrane- and cytoskeleton-associated NF-kB are dynamic and comprise active pools, whereas the cytoplasmic is more constant and likely non-active due to the presence of IkB-a. Moreover, we discovered the existence of a dynamic, IkB-a-free pool of RelA/p65 associated with cytoskeletal fraction, what argues for a special regulatory role of the cytoskeleton in NF-kB stimulation.

RelA/NF-kappaB transcription factor associates with alpha-actinin-4.

The NF-kappaB/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-kappaB in the cytoplasm and the transport mechanism to the nucleus. We found that NF-kappaB is associated with the actin-binding protein alpha-actinin-4. NF-kappaB and alpha-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-alpha, alpha-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-alpha led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-kappaB co-immunoprecipitated alpha-actinin-4 from A431 cell lysates and nuclear extracts, but alpha-actinin-1 and beta-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-kappaB can bind to matrix-bound chicken gizzard alpha-actinin. We suggest that the alpha-actinin-4 is important for the NF-kappaB nuclear translocation and its functions inside the nucleus.

Extra-cellular matrix proteins induce re-distribution of alpha-actinin-1 and alpha-actinin-4 in A431 cells.

Alpha-actinins are actin-binding proteins of non-muscle cells, which can participate in the regulation of transcription factor activity. We describe the distribution of alpha-actinin-1 and -4 depending on different actin cytoskeleton formed as a result of cell adhesion to extracellular matrix proteins, such as fibronectin and laminin 2/4. Immunofluorescent studies show a difference in the distribution of alpha-actinin and -4. Both isoforms localise along stress-fibres, but alpha-actinin-1 localises in the perinuclear region more abundantly than alpha-actinin-4. Western blot analysis demonstrated existence of truncated forms of both isoforms. Truncated alpha-actinin-1 appears in cells spread on fibronectin or laminin. Cell spreading also correlated with more tight association of alpha-actinin-4 with chromatin. Basing on our previous finding of an interaction of alpha-actinin-4 with p65 subunit of the NF-kappaB, we checked the possible influence of immobilised ligands on its redistribution in nuclear complexes containing p65. alpha-Actinin-4 seems to be present in some but not all nuclear complexes containing p65. Immobilised ligands may affect the interaction of alpha-actinin-4/p65 complexes with chromatin. The data suggest that adhesion to extra-cellular matrix may interfere in cellular reactions mediated by alpha-actinin-1 and -4.