B cell signalling in mechanisms of central and peripheral tolerance

Tolerance against self is a necessary feature of the immune system to prevent autoimmunity. Hence during B cell development, a number of central and peripheral developmental checkpoints ensure the deletion of self-reactive B cells and selection of tolerant B cells. For example, antigen-driven ligation of the B cell receptor (BCR) on immature B cells results, by default, in receptor editing, anergy and/or apoptosis, whereas it provides survival, proliferative and effector differentiation signals to mature B cells. Moreover, various factors can influence the functional outcome downstream of BCR ligation. Thus, T helper cell-derived signals, such as those following ligation of the CD40 receptor, can rescue pathogen-specific immature B cells from growth arrest and apoptosis, thereby providing a mechanism in which cells programmed to die because of their immature status can survive after receiving appropriate T cell help. On the other hand, mitogenic BCR-mediated signalling of mature B cells can be suppressed by co-ligation of the inhibitory receptor FcγRIIb. This allows the maintenance of B cell homeostasis in the periphery, as cross-linking of the BCR and FcγRIIb by immune complexes enables the system to terminate ongoing immune responses following clearance of pathogens. The precise signalling mechanisms involved in dictating these differential functional outcomes remain to be elucidated but it is becoming increasingly clear that the developmental stage as well as the integration of various extracellular signals decide the cell’s fate. The core aim of this study has therefore been to characterise the signalling pathways coupling BCR ligation to survival, proliferation and apoptosis during development. In particular, it was planned to focus on the differential signalling mechanisms involved in the negative selection of immature B cells and FcγRIIb-mediated homeostatic regulation of mature B cells. Negative selection of self-reactive immature B cells constitutes a major mechanism of sustaining central tolerance. The WEHI-231 cell line provides a well-established model system for dissecting the signalling mechanism underlying such clonal deletion of immature B cells as these cells, which have the phenotype of immature B cells, undergo growth arrest and apoptosis following stimulation of their BCR. Previously, this laboratory has identified ERK signalling as a key regulator of immature B cell fate. Thus, spontaneously proliferating WEHI-231 B cells exhibit a sustained yet cyclic pattern of ERK activation that is necessary for their survival and proliferation. By contrast, BCR-ligation induces a strong transient activation of ERK followed by sustained downregulation of the cyclic activation pattern observed in spontaneously proliferating cells. However, the pathways linking BCR ligation with suppression of ERK signalling and consequent growth arrest have not been delineated in full. For example, in B cells, the Ras/Raf-1/MEK cascade is considered to be a major pathway regulating ERK activation and consistent with this, this laboratory has previously shown that overexpression of Ras can rescue WEHI-231 B cells from BCR-mediated growth arrest. However, although MEK activation was found to be compromised under conditions of BCR-driven growth arrest, such BCR-signalling did not suppress Ras activation indicating that other pathways normally contributed to the MEK-dependent activation of ERK in spontaneously proliferating WEHI-231 cells. The data presented in this thesis now provide evidence for the existence of a second pathway controlling ERK activation in immature B cells, the Rap/B-Raf/MEK cascade, which has now been implicated in the maintenance of cycling ERK activation observed in spontaneously proliferating WEHI-231 B cells. BCR signalling was found to reduce the levels of active Rap, hence providing a mechanism for the BCR-mediated uncoupling of the Ras-independent, MEK-dependent ERK activation contributing to the cycling ERK signalling responsible for survival and proliferation of WEHI-231 cells. Consistent with the role of Rap in promoting such ERK signalling, additionally, it has now been demonstrated that levels of SPA-1, a negative regulator of Rap, increase upon BCR-stimulation indicating that this may be the mechanism by which the BCR signals to uncouple the Rap/B-Raf/MEK pathway. Although ERK had previously been identified as the major regulatory element governing survival and proliferation of immature B cells, the pathways linking ERK to the regulation of survival/apoptosis and cell cycle progression have been only poorly characterised. This study has now highlighted the connection between sustained ERK signalling and the stabilisation of c-Myc protein levels. For example, abrogation of ERK activity by BCR-ligation, or pharmacological inhibition, reduced c-Myc levels in a transcription-independent fashion indicating regulation at the post-translational level. This proposal was corroborated by analysis of the phosphorylation status of c-Myc that indicated that ERK signalling promoted the expression of stabilised forms (S62) of c-Myc and reduced the expression of those (T58) targeting c-Myc for proteosomal degradation. The importance of such c-Myc stabilisation was illustrated by Laser Scanning Cytometric analysis that revealed that the increasing levels of c-Myc expressed by individual WEHI-231 B cells correlated with their cell cycle progression, presumably reflecting widely established findings that c-Myc promotes cell cycle progression by increasing the expression and activation of cyclin/Cdk complexes and reducing the levels of Cdk inhibitors such as p27. Consistent with this, negative signalling via the BCR, or suppression of ERK activation by pharmacological inhibition, also increased p27 levels and resulted in the reduction of the hyperphosphorylation of retinoblastoma (Rb) proteins required for transition through the G1-S-phase checkpoint. As Rb is a target of cyclin/Cdks, collectively these data further confirm the links not only between BCR-signalling and c-Myc stability but also the differential cell fate decisions of apoptosis, survival and/or cell cycle progression. By contrast, in mature B cells, BCR-signalling is linked to survival and proliferation. However, to prevent autoimmunity resulting from an expanded pool of potentially weakly autoreactive B cells, such survival and proliferation of mature B cells needs to be homeostatically regulated. Hence, the immune system has evolved inhibitory signalling cascades such as that triggered by cognate immune-complexes co-ligating the BCR and FcγRIIb to terminate ongoing antigen-driven responses. This study has emphasised the crucial role of caspase 8 in the apoptosis of mature B cells resulting from such co-ligation of the BCR and FcγRIIb and has indicated that such caspase 8 activation is likely to be downstream of Fas signalling. Consistent with this, blocking the Fas/FasL death receptor cascade was found to reduce the levels of apoptosis detected and B cells from MRL/MpJ-Faslpr mice, a strain harbouring a mutation causing the abrogation of Fas expression, exhibited defective apototic responses upon such BCR/FcγRIIb co-ligation. Thus, Fas/FasL death receptor signalling might be a major mechanism underpinning the FcγRIIb-mediated apoptosis pathway. Finally, to determine whether dysfunctional regulation of FcγRIIb-mediated signalling plays a role in human autoimmune conditions, B cells from patients with Rheumatoid arthritis and Systemic lupus erythematosus were examined. Overall, the homeostatic regulatory responses between B cells from healthy controls and RA and SLE patients were not found to be significantly different. However, the B cells derived from a small proportion of RA and SLE patients were found to exhibit defective FcγRIIb-mediated inhibitory responses. Moreover, significant differences were found in the ratio of FcγRIIb1/FcγRIIb2 expression between the cohorts of healthy controls and RA and SLE patients. The RA and SLE patients expressed relatively higher levels of the FcγRIIb2 isoform which promotes antigen-processing suggesting that these B cells may play some role in priming autoreactive responses in such individuals. Thus, as these inflammatory disorders constitute spectrum diseases, such defects in the regulation of B cell responses could be one of the contributing factors aggravating autoimmune disease development in some subgroups of patients

Analyzing Ductile Shear Zone Network Geometries in the Grassy Portage Sill, Rainy Lake Region, Northwestern Ontario, Canada

The Grassy Portage Sill (GPS) is a ~2.7 Ga metagabbroic sill located in the Rainy Lake region of northwestern Ontario. The Rainy Lake region is located in the Superior Province between the metavolcanic Wabigoon subprovince to the north and the metasedimentary Quetico subprovince to the south. Two regional faults bound the region and intersect to the east, forming a wedge which defines the Rainy Lake zone. This area was regionally deformed due to oblique transpression, resulting from the Kenoran Orogeny (~2.7 Ga). The GPS is approximately 20 km long and 1-2 km wide, and has undergone heterogeneous strain along its length. This strain variation is a function of the competence contrast between the GPS, the gneissic Rice Bay Dome to the west, and the metavolcanic and metasedimentary units between the two. The GPS has a higher competence than the adjacent metavolcanic and metasedimentary units, but all have a lower competence than the Rice Bay Dome. Within the GPS, anastomosing ductile shear zone networks accommodated the bulk of the deformation within the largely competent sill. The orientations of the networks vary along the length of the sill, apparently related to strain variations, as the inferred shortening directions of the shear zone networks matches those assumed from regional foliations and calculated from previous work on deformed dikes and veins. At all locations, both steeply dipping dextral and sinistral sets of shear zones formed, presumably simultaneously as evidenced by mutual cross-cutting relationships. The shear zones are curviplanar and dip more shallowly near some of their intersections. The style of deformation varies within the gabbro based on grain size. Fine grained lithologies are deformed via a pervasive foliation. Coarser grained lithologies generally contain discrete (mm-cm scale) shear zones, often without a pervasive foliation. There is no relationship between mineralogy and shear zone or foliation formation. Microstructural analysis of shear zone bearing samples indicated that dislocation creep served as the primary deformation mechanism throughout the GPS, but showed no pattern regionally, or with grain size. At the lowest strain sites, the gabbro has a pervasive foliation, but few, if any, shear zones. At low-medium strain sites, the sinistral and dextral shear zone sets have fairly consistent orientations, approximately 65-75° apart from one another. As strain increases, the orientations of both sets become increasingly more variable and the average angle between the two sets decreases. We hypothesize that the shear zone sets formed at relatively high angle to one another and rotated to a lower relative angle with increasing strain. The newer strands in the higher strained sites formed at high angle, causing the orientations of each shear zone set to become more diffuse at higher strain

Inertia controlled instability and small scale structures of sheet and cloud cavitation

The present investigation focuses on the numerical simulation of inertia driven dynamics of 3-D sheet and cloud cavitation on a 2-D NACA 0015 hydrofoil. Special emphasis is put on the numerical analysis of the re-entrant flow, the break-up of the sheet cavity and the formation of clouds. We demonstrate that our CFD-Tool CATUM (CAvitation Technische Universität Mu?nchen) is able to predict even delicate 3-D flow features such as irregular break-up patterns, cavitating hairpin and horseshoe vortices, 3-D instabilities in spanwise direction and the formation and propagation of shocks due to collapsing clouds close to the trailing edge of the hydrofoil. The numerically predicted flow features agree well with the experimental observations of Kawanami et al [1].http://deepblue.lib.umich.edu/bitstream/2027.42/84219/1/CAV2009-final17.pd

Strukturelle Analysen geschleuster Fragesätze

In dieser Arbeit versuche ich einen Überblick zu geben über Schleusenkonstruktionen (engl. Sluicing), über die mit ihnen assoziierten Problemstellungen sowie über die zwei strukturellen Erklärungsansätze dieser Konstruktion – Tilgungsansätze und interpretative Ansätze. Zur Illustration dieser Konstruktion dienen mir vor allem Daten aus dem Deutschen, dem Englischen und dem Serbokroatischen. Nach einer kurzen Einführung in den theoretischen Rahmen der Arbeit in Kapitel 1, lege ich in Kapitel 2 die Probleme dar, auf die jede adäquate Theorie geschleuster Sätze eine Antwort finden muss. In Kapitel 3 diskutiere ich Tilgungsansätze und interpretative Ansätze und versuche zu zeigen, welche Strategien diese beiden Ansätze entwickeln (müssen), um die im vorangegangenen Kapitel formulierten Fragestellungen zu beantworten. Kapitel 4 bespricht drei zentrale Phänomene, die in der neueren Forschungsliteratur ausführlich diskutiert wurden. Ziel dieses Kapitel ist es, einen Teil dieser Diskussionen zusammenzufassen. Ich komme in der vorliegenden Arbeit zu dem Schluss, dass es nicht möglich ist eine eindeutige Antwort zu geben auf die Frage, welche der beiden strukturellen Theorien (Tilgungsansatz oder interpretativer Ansatz) zu favorisieren ist. Für Tilgungsanalysen stellt die Tatsache, dass geschleuste Fragewörter nicht sensitiv sind auf Inseln das größte Problem dar. Dies war bereits in der ersten generativen Analyse dieser Konstruktion der Fall (Ross 1969) und ist nach wie vor ungelöst, obwohl eine Reihe von Vorschlägen existiert, dieses Problem zu lösen (z.B. Merchant 2001; 2008). Tilgungsanalysen haben aber keine Probleme Konnektivitätseffekte zu erklären. Da sie für Ellipsen eine gewöhnliche syntaktische Derivation annehmen, folgen diese Effekte aus ihrer Theorie. Auf der anderen Seite stellen Konnektivitätseffekte das Hauptproblem dar für interpretative Theorien. Sie können diese Effekte nur unter Zuhilfenahme von Stipuationen erklären (z.B. Kasuszuweisung in [Spec,CP]; doppelte Formulierung der Präpositionsstrandungsgeneralisierung). Interpretative Theorien haben hingegen kein Problem die Inselinsensitivität geschleuster Sätze zu erklären. Dies war auch eine der Hauptmotivationen für diese Art von Theorien. Wenn auch keine eindeutige Antwort auf die Frage, welche der beiden Theorien zu favorisieren sei, möglich ist, so scheint mir vorläufig doch der Tilgungsansatz aus mehreren Gründen einem interpretativen Ansatz vorzuziehen zu sein: - Die Parallelität der elliptischen und der nicht-elliptischen Sätze folgt aus der Theorie (Konnektivitätseffekte). - Es müssen keine gesonderten Mechanismen für die Erklärung des größten Teils der Phänomene stipuliert werden. (Ausnahme: Inseln

Molecular and immunological characterization of profilin from mugwort pollen

In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No information about the complete molecular structure of any mugwort allergen has been published so far. Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin. Thirtysix percent of the mugwort allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgEbinding properties of the isoforms. One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions. Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions. Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollenallergic patients. However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgEmediated histamine release. Profilin isolated from mugwort pollen also formed multimers. Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen. Inhibition experiments showed extensive IgE crossreactivity of recombinant mugwort profilin and profilin from various pollen and food extracts

Journal of Immunology Research / Prophylactic mRNA vaccination against allergy confers long-term memory responses and persistent protection in mice

Recently, mRNA vaccines have been introduced as a safety-optimized alternative to plasmid DNA-based vaccines for protection against allergy. However, it remained unclear whether the short persistence of this vaccine type would limit memory responses and whether the protective immune response type would be maintained during recurrent exposure to allergen. We tested the duration of protective memory responses in mice vaccinated with mRNA encoding the grass pollen allergen Phl p 5 by challenging them with recombinant allergen, 3.5, 6, and 9 months after vaccination. In a second experiment, vaccinated mice were repeatedly challenged monthly with aerosolized allergen over a period of 7 months. Antibody and cytokine responses as well as lung inflammation and airway hyperresponsiveness were assessed. mRNA vaccination induced robust TH1 memory responses for at least 9 months. Vaccination efficiently suppressed TH2 cytokines, IgE responses, and lung eosinophilia. Protection was maintained after repeated exposure to aerosolized allergen and no TH1 associated pathology was observed. Lung function remained improved compared to nonvaccinated controls. Our data clearly indicate that mRNA vaccination against Phl p 5 induces robust, long-lived memory responses, which can be recalled by allergen exposure without side effects. mRNA vaccines fulfill the requirements for safe prophylactic vaccination without the need for booster immunizations.(VLID)179427

Induction of pathogenic Th17 cells by inducible salt sensing kinase SGK1

Th17 cells are highly proinflammatory cells critical for clearing extracellular pathogens and for induction of multiple autoimmune diseases1. IL-23 plays a critical role in stabilizing and reinforcing the Th17 phenotype by increasing expression of IL-23 receptor (IL-23R) and endowing Th17 cells with pathogenic effector functions2, 3. However, the precise molecular mechanism by which IL-23 sustains the Th17 response and induces pathogenic effector functions has not been elucidated. Here, we used transcriptional profiling of developing Th17 cells to construct a model of their signaling network and nominate major nodes that regulate Th17 development. We identified serum glucocorticoid kinase-1 (SGK1), a serine-threonine kinase4, as an essential node downstream of IL-23 signaling. SGK1 is critical for regulating IL-23R expression and stabilizing the Th17 cell phenotype by deactivation of Foxo1, a direct repressor of IL-23R expression. SGK1 has been shown to govern Na+ transport and salt (NaCl) homeostasis in other cells5, 6, 7, 8. We here show that a modest increase in salt concentration induces SGK1 expression, promotes IL-23R expression and enhances Th17 cell differentiation in vitro and in vivo, accelerating the development of autoimmunity. Loss of SGK1 abrogated Na+-mediated Th17 differentiation in an IL-23-dependent manner. These data demonstrate that SGK1 plays a critical role in the induction of pathogenic Th17 cells and provides a molecular insight into a mechanism by which an environmental factor such as a high salt diet triggers Th17 development and promotes tissue inflammation

Pediatric Allergy and Immunology / DNA and mRNA vaccination against allergies

Allergen-specific immunotherapy, which is performed by subcutaneous injection or sublingual application of allergen extracts, represents an effective treatment against type I allergic diseases. However, due to the long duration and adverse reactions, only a minority of patients decides to undergo this treatment. Alternatively, early prophylactic intervention in young children has been proposed to stop the increase in patient numbers. Plasmid DNA and mRNA vaccines encoding allergens have been shown to induce T helper 1 as well as T regulatory responses, which modulate or counteract allergic T helper 2-biased reactions. With regard to prophylactic immunization, additional safety measurements are required. In contrast to crude extracts, genetic vaccines provide the allergen at high purity. Moreover, by targeting the encoded allergen to subcellular compartments for degradation, release of native allergen can be avoided. Due to inherent safety features, mRNA vaccines could be the candidates of choice for preventive allergy immunizations. The subtle priming of T helper 1 immunity induced by this vaccine type closely resembles responses of non-allergic individuals and-by boosting via natural allergen exposure-could suffice for long-term protection from type I allergy.W 1213(VLID)286547

The therapeutic potential of the filarial nematode-derived immunodulator, ES-62 in inflammatory disease

The dramatic recent rise in the incidence of allergic or autoimmune inflammatory diseases in the West has been proposed to reflect the lack of appropriate priming of the immune response by infectious agents such as parasitic worms during childhood. Consistent with this, there is increasing evidence supporting an inverse relationship between worm infection and T helper type 1/17 (Th1/17)-based inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes and multiple sclerosis. Perhaps more surprisingly, given that such worms often induce strong Th2-type immune responses, there also appears to be an inverse correlation between parasite load and atopy. These findings therefore suggest that the co-evolution of helminths with hosts, which has resulted in the ability of worms to modulate inflammatory responses to promote parasite survival, has also produced the benefit of protecting the host from pathological lesions arising from aggressive proinflammatory responses to infection or, indeed, aberrant inflammatory responses underlying autoimmune and allergic disorders. By focusing upon the properties of the filarial nematode-derived immunomodulatory molecule, ES-62, in this review we shall discuss the potential of exploiting the immunomodulatory products of parasitic worms to identify and develop novel therapeutics for inflammation

Allergy Asthma & Immunol Research / No concentration decrease of house dust mite allergens with rising altitude in alpine regions

Purpose: Several studies over the past 4 decades have indicated a significant reduction in house dust mite (HDM) and HDM allergen concentration in areas higher than 1,500 m above sea level. These have served as basis of allergen avoidance therapies for HDM allergy and asthma. However, modern construction techniques used in the insulation, heating, and glazing of buildings as well as global warming have changed the environmental parameters for HDM living conditions. The present study revisits the paradigm of decreasing HDM allergen concentrations with increasing altitude in the alpine region of Germany and Austria. Methods: A total of 122 dust samples from different abodes (hotels, privates and mountain huts) at different altitudes (400-2,600 m) were taken, and concentrations of HDM allergens were analyzed. Humidity and temperature conditions, and numerous indoor environmental parameters such as fine dust, type of flooring, age of building, and frequency of cleaning were determined. Results: HDM allergen concentrations did not significantly change with increasing altitude or relative humidity. At the level of indoor parameters, correlations could be found for different flooring types and the concentration of HDM allergens. Conclusions: In contrast to the widespread view of the relationship between altitude and HDM allergen concentrations, clinically relevant concentrations of HDM allergens could be detected in high-lying alpine regions in Austria and Germany. These results indicate that improvement in conditions of asthmatic patients sensitized against HDMs during a stay at high altitude can no longer be ascribed to decreased levels of HDM allergens, instead, other mechanisms may trigger the beneficial effect