Regulation of B cell function by plasmacytoid dendritic cells

Dendritic cells (DCs) are early sentinels of pathogen exposure and central in the initiation and orchestration of adaptive immune responses. Apart from the prominent role of DCs in the activation of T cells, DCs have been shown to influence humoral B cell mediated responses. DCs are therefore important cells for regulating immune responses to vaccines. Many of the vaccines under development today are against pathogens such as Mycobacterium tuberculosis and HIV-1 that likely require induction of both cellular and humoral responses to cause protection. This generates a need for new safe and effective vaccine adjuvants that can stimulate such responses. One class of adjuvants that has attracted a lot of interest over the last years is TLR (toll-like receptor)-ligands. Selected TLR-ligands can specifically activate subsets of DCs and B cells according to their cognate receptor expression and therefore represent promising candidates to shape vaccine-induced responses. The first aim of this thesis was to investigate the responsiveness of B cells to TLR stimulation to proliferate and differentiate into antibody (Ab) producing cells. Furthermore, the aims were to study, whether the distinctly different myeloid (MDCs) and plasmacytoid (PDCs) can support these responses in a T helper cell-independent or dependent manner. In addition, differences in the responses of B cells and DCs from humans versus non-human primates (NHP) were addressed. In paper I, we established and refined isolation protocols for subsets of primary human PDCs, MDCs and B cells from blood and methods to examine their functions. We found that total B cells responded strongly to engagement of TLR9, less to TLR7/8 and not to engagement of TLR3. Furthermore, PDCs but not MDCs markedly enhanced B cell proliferation and differentiation into Ab producing cells in response to TLR7/8-ligand stimulation and to a lesser extent to TLR9-ligands (CpG ODN classes A, B, and C). PDCs strongly enhanced TLR7/8-ligand-induced proliferation of both memory and naive B cells but were only able to support memory cells to differentiate to CD27high plasmablasts. Type I IFN produced to high levels by PDCs was the principal mediator of the enhanced responses upon TLR7/8 stimulation. This effect may at least in part be explained by the reported upregulation of TLR7 and MyD88 by IFNα. Although MDCs expressed high levels of the known B cell growth factors IL-6, IL-10, and B cell-activating factor (BAFF) in response to TLR7/8 stimulation, they were unable to enhance B cell responses in this system. In paper II, we found that PDCs also had the ability to augment naïve B cell responses induced by BCR engagement and T cell help. The presence of supernatants from TLR-stimulated PDCs increased B cell proliferation, the frequency of B cells that differentiated to CD27high CD38high cells, and secretion of IgM. IFNα produced by PDCs was again instrumental in these processes and increased cell viability or proliferation were not main reasons for the improved B cell function. We found that PDC supernatants or IFNα induced upregulation of the co-stimulatory molecule CD86 on B cells. Further, these B cells showed improved ability to interact with and activate T cells. Thus, increased B cell responsiveness to T cell contact, mediated by PDCs via their production of IFNα, may facilitate B cell proliferation and differentiation into Ab producing cells. In order to further explore the influence of IFNα and PDCs on B cell functions in vivo in humans, models such as NHPs that more closely resemble humans than rodents need to be utilized. NHPs have the advantage that they to a large extent exhibit similar subpopulations of DC and B cell subsets as well as similar TLR expression as humans. However, similarities and potential disparities between the species need to be carefully investigated to facilitate the translation of NHP studies into clinical trials. In paper III, we therefore examined whether the effect of activated PDCs or IFNα enhanced B cell functions was comparable in human and NHP rhesus macaque cells in response to TLR ligands. We found similar responses in human and rhesus cultures to the selected TLR ligands in terms of B cell proliferation. B cell proliferation to the TLR7/8-L and CpG class C showed a significant and comparable increase in presence of IFNα. However, upon stimulation only human B cells acquired high expression of CD27, associated with plasmablast formation, although both human and rhesus B cells produced increased levels of IgM. Instead, rhesus B cell differentiation was associated with a more prominent downregulation of CD20. This validates that rhesus macaques are relevant and appropriate in vivo models to study TLR induced B cell responses although the choice of B cell differentiation markers to measure must be considered. In conclusion, the studies included in this thesis highlight the potential of PDCs and IFNα to shape B cell differentiation to Ab secreting cells. These studies add to the understanding on the role of DCs in modulation of B cell responses, which is crucial information for the design of novel vaccines, adjuvants and immuno-modulatory treatment formulations

Philipp von Huttens Tod in der Neuen Welt

Der erste Kriminalfall der Neuen Welt, der die Maschinerie kolonialen Schreibens in Gang setzte: Philipp von Hutten, ein Mitglied des berühmten fränkischen Rittergeschlechts, wird im Jahr 1546 in der Provinz Venezuela von seinem Rivalen Juan de Carvajal enthauptet. Susanne Andrea Gujer-Bertschingers Studie stellt erstmals alle Dokumente im Zusammenhang mit dem »Fall Hutten« in einen Kontext: Briefe des Opfers - sie gelten als die ältesten Briefe in deutscher Sprache, die aus Amerika geschrieben wurden -, Prozessakten und Reiseberichte. Die Analyse der deutschen, spanischen und italienischen Texte liefert einen genuinen Beitrag zur Forschungsdebatte über (post-)koloniale Studien

Development of image analysis techniques as a tool to detect and quantify morphological changes in anaerobic sludge : I. application to a granulation process

Image analysis techniques were developed and applied to quantify the process of anaerobic granulation in an expanded granular sludge blanket reactor (EGSB) fed with a synthetic substrate based on glucose [60-30% COD chemical oxygen demand)] and volatile fatty acids (40-70% COD) over 376 days. In a first operation period that lasted 177 days, the aggregation of dispersed sludge was quantitatively monitored through the recognition and quantification of aggregates and filaments. A parameter defined as the ratio between the filaments' length and the aggregates projected area (LfA) has proven to be sensitive to detect changes in the aggregation status of the anaerobic sludge. The aggregation time-defined as the moment when a balance between filaments' length and aggregates' size was establishe-was recognized through the LfA. The percentage of projected area of aggregates within three size ranges (0.01 -0.1 mm, 0.1 - 1 mm, and >1 mm, equivalent diameter) reflected the granular size spectrum during the aggregation process. When sudden increases on the upflow velocity and on the organic loading rate were applied to the previously formed granules, the developed image analysis techniques revealed to be good indicators of granular sludge stability, since they were sensitive to detected filaments release, fragmentation, and erosion that usually leads to washout. The specific methanogenic activities in the presence of acetate, propionate, butyrate, and H2/CO2 increased along the operation, particularly relevant was the sudden increase in the specific hydrogenophilic activity, immediately after the moment recognized as aggregation time.Instituto Cooperação Científica e Tecnológica Internacional (ICCTI), Ambassade de France in Portugal - Project 203 B4.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/3187/2000, PRAXIS XXI/BD/20325/99, POCTI/1999/CTA736524

Anaerobic biodegradation of oleic and palmitic acids : evidence of mass transfer limitations caused by long chain fatty acid accumulation onto the anaerobic sludge

Palmitic acid was the main long chain fatty acids (LCFA) that accumulated onto the anaerobic sludge when oleic acid was fed to an EGSB reactor. The conversion between oleic and palmitic acid was linked to the biological activity. When palmitic acid was fed to an EGSB reactor it represented also the main LCFA that accumulated onto the sludge. The way of palmitic acid accumulation was different in the oleic and in the palmitic acid fed reactors.Whenoleic acid was fed, the biomass-associated LCFA (83% as palmitic acid) were mainly adsorbed and entrapped in the sludge that became ‘‘encapsulated’’ by an LCFA layer. However, when palmitic acid was fed, the biomass-associated LCFA (the totality as palmitic acid) was mainly precipitated in white spots like precipitates in between the sludge, which remained ‘‘non-encapsulated.’’ The two sludges were compared in terms of the specific methanogenic activity (SMA) in the presence of acetate, propionate, butyrate, and H2CO2, before and after the mineralization of similar amounts of biomassassociated LCFA (4.6 and 5.2 g COD-LCFA/g of volatile suspended solids (VSS), for the oleic and palmitic acid fed sludge, respectively). The ‘‘non-encapsulated,’’ sludge exhibited a considerable initial methanogenic activity on all the tested substrates, with the single exception of butyrate. However, with the ‘‘encapsulated’’ sludge only methane production from ethanol andH2/CO2 was detected, after a lag phase of about 50 h. After mineralization of the biomass-associated LCFA, both sludges exhibited activities of similar order of magnitude in the presence of the same individual substrates and significantly higher than before. The results evidenced that LCFA accumulation onto the sludge can create a physical barrier and hinder the transfer of substrates and products, inducing a delay on the initial methane production. Whatever the mechanism, metabolic or physical, that is behind this inhibition, it is reversible, being eliminated after the depletion of the biomass-associated LCFA.Fundação para a Ciência e Tecnologia (FCT) Fundo Social Europeu (FSE

Structural Determinants in the Calcitonin Receptor-Like Receptor (Crlr) Important for Cgrp and Adrenomedullin (Am) Receptor Function of Crlr/Receptor-Activity-Modifying Protein (Ramp) 1 and Crlr/Ramp2 Heterodimers

Cell surface protein cross-linking, coimmmunoprecipitation, and confocal microscopy identified CRLR/RAMP1-, CRLR/RAMP2-, and calcitonin receptor isotype 2 (CTR2)/RAMP1 heterodimers as CGRP-, AM-, and CGRP/amylin receptors, linked to cAMP production. Along these lines, effects of structural alterations in the N-terminal extracellular domain of the human CRLR on cell surface expression as well as the association with RAMP and CGRP or AM have been investigated

Mdscs in Infectious diseases: regulation, roles, and readjustment

Many pathogens, ranging from viruses to multicellular parasites, promote expansion of MDSCs, which are myeloid cells that exhibit immunosuppressive features. The roles of MDSCs in infection depend on the class and virulence mechanisms of the pathogen, the stage of the disease, and the pathology associated with the infection. This work compiles evidence supported by functional assays on the roles of different subsets of MDSCs in acute and chronic infections, including pathogen-associated malignancies, and discusses strategies to modulate MDSC dynamics to benefit the host

Validity of the 3D VECTRA photogrammetric surface imaging system for cranio-maxillofacial anthropometric measurements

Purpose: The use of three-dimensional (3D) photography for anthropometric measurements is of increasing interest, especially in the cranio-maxillofacial field. Before standard implementation, accurate determination of the precision and accuracy of each system is mandatory. Methods: A mannequin head was labelled with 52 landmarks, and 28 three-dimensional images were taken using a commercially available five-pod 3D photosystem (3D VECTRA; Canfield, Fairfield, NJ) in different head positions. Distances between the landmarks were measured manually using a conventional calliper and compared with the digitally calculated distances acquired from labelling by two independent observers. The experimental set-up accounted for clinical circumstances by varying the positioning (vertical, horizontal, sagittal) of the phantom. Results: In the entire calliper measurement data set (n = 410), a significant difference (p = 0.02) between the directly measured and corresponding virtually calculated distances was found. The mean aberration between both modalities covering all data was 7.96mm. No differences (p = 0.94) between the two groups were found using a cut-off of 10% (leaving n = 369 distances) due to considerable errors in direct measurements and the necessary manual data translation. The mean diversity of both measurement modalities after cut-off was 1.33mm (maximum, 6.70mm). Inter-observer analysis of all 1,326 distances showed no difference (p = 0.99; maximal difference, 0.58mm) in the digital measurements. Conclusion: The precision and accuracy of this five-pod 3D photosystem suggests its suitability for clinical applications, particularly anthropometric studies. Three-hundred-and-sixty degree surface-contour mapping of the craniofacial region within milliseconds is particularly useful in paediatric patients. Proper patient positioning is essential for high-quality imaging