The innate immune repertoire in Cnidaria - ancestral complexity and stochastic gene loss

Background  

Characterization of the innate immune repertoire of extant cnidarians is of both fundamental and applied interest - it not only provides insights into the basic immunological 'tool kit' of the common ancestor of all animals, but is also likely to be important in understanding the global decline of coral reefs that is presently occurring. Recently, whole genome sequences became available for two cnidarians, Hydra magnipapillata and Nematostella vectensis, and large expressed sequence tag (EST) datasets are available for these and for the coral Acropora millepora.     

A physical approach to reduce nonspecific adhesion in molecular recognition atomic force microscopy.

Atomic force microscopy is one of the few techniques that allow analysis of biological recognition processes at the single-molecule level. A major limitation of this approach is the nonspecific interaction between the force sensor and substrate. We have modeled the nonspecific interaction by looking at the interaction potential between a conical Si3N4 tip with a spherical end face and a mica surface in solution, using DLVO (Derjaguin, Landau, Verwey, Overbeek) theory and numerical calculations. Insertion of the tip-sample potential in a simulation of an approach-retract cycle of the cantilever gives the well-known force-distance curve. Simulating a force-distance curve at low salt concentration predicts a discrete hopping of the tip, caused by thermal fluctuations. This hopping behavior was observed experimentally and gave rise to a novel approach to making measurements in adhesion mode that essentially works in the repulsive regime. The distance between tip and sample will still be small enough to allow spacer-involved specific interactions, and the percentage of nonspecific interactions of the bare tip with the mica is minimized. We have validated this physical model by imaging intercellular adhesion molecule 1 (ICAM-1) antigen with a tip functionalized with anti-ICAM-1 antibody. The measurement demonstrated that a significant decrease in the number of nonspecific interactions was realized, and the topographical image quality and the specific bonding capability of the tip were not affected.     

Modulation of phenotypic and functional properties of human peripheral blood monocytes by IL-4

Highly purified peripheral blood monocytes were cultured in the presence of rIL-4. Major changes in the morphology of the monocytes were observed. After day 5 of culturing the cells acquired a macrophage-like appearance, with increased cell size and extensive processes, suggesting that IL-4 may induce monocyte-macrophage differentiation. This notion is supported by the observed increased expression of MHC class II Ag, which is thought to be associated with monocyte differentiation. Exposure of monocytes to IL-4 resulted in a dose-dependent increase of the expression of MHC class II Ag, which became apparent after only 20 h of incubation. Maximal expression was obtained after incubation for 6 days, and persisted throughout the whole culture period. Similarly, IL-4 increased the expression of R for C3bi and p150.95 Ag, two members of the leukocyte function-associated Ag 1 family, whereas the expression of the third member, leukocyte function-associated Ag 1, remained unchanged during culture. Furthermore, it was shown that IL-4 inhibited the secretion of cytostatic and chemotactic compounds. Supernatants of monocytes cultured with IL-4 were, in contrast to control cultures, much less effective in inhibiting the growth of A375 melanoma cells. In addition, these supernatants failed to direct the migration of freshly isolated monocytes in a chemotaxis assay. Further analysis revealed that these supernatants exhibited reduced IL-1 activity, as measured in a mouse thymocyte proliferation assay, which might explain the low cytostatic and chemotactic activity. Taken together these results show that IL-4 modulates monocyte phenotype and function and may induce monocyte-macrophage differentiation in vitro.     

Zur Anatomie des Stammes der Dammarpflanze

Ohne Zusammenfassung     

Spectral analysis of flow cytometric data: design of a special-purpose low-pass digital filter

Spectral decomposition of flow cytometric datafiles of arbitrary dimension reveal information of both the signal and the noise components that constitute the histograms. This spectral information is used to construct a low-pass digital filter, which removes the high-frequency noise from the actual data. It is shown that this procedure guarantees non-trivial smoothing of the flow cytometric data in accordance with the local experimental situation. As a consequence optimal reconstruction of the signal is possible, which facilitates unambiguous interpretation of the data files and mathematical estimation of the statistical parameters.     

Über das Restitutionsvermögen der Blätter von Bryophyllum calycinum Salisb

Ohne ZusammenfassungMit 2 Textabbildungen.     

Proteome Based Construction of the Lymphocyte Function-Associated Antigen 1 (LFA-1) Interactome in Human Dendritic Cells

The β2-integrin lymphocyte function-associated antigen 1 (LFA-1) plays an important role in the migration, adhesion and intercellular communication of dendritic cells (DCs). During the differentiation of human DCs from monocyte precursors, LFA-1 ligand binding capacity is completely lost, even though its expression levels were remained constant. Yet LFA-1-mediated adhesive capacity on DCs can be regained by exposing DCs to the chemokine CCL21, suggesting a high degree of regulation of LFA-1 activity during the course of DC differentiation. The molecular mechanisms underlying this regulation of LFA-1 function in DCs, however, remain elusive. To get more insight we attempted to identify specific LFA-1 binding partners that may play a role in regulating LFA-1 activity in DCs. We used highly sensitive label free quantitative mass-spectrometry to identify proteins co-immunoprecipitated (co-IP) with LFA-1 from ex vivo generated DCs. Among the potential binding partners we identified not only established components of integrin signalling pathways and cytoskeletal proteins, but also several novel LFA-1 binding partners including CD13, galectin-3, thrombospondin-1 and CD44. Further comparison to the LFA-1 interaction partners in monocytes indicated that DC differentiation was accompanied by an overall increase in LFA-1 associated proteins, in particular cytoskeletal, signalling and plasma membrane (PM) proteins. The here presented LFA-1 interactome composed of 78 proteins thus represents a valuable resource of potential regulators of LFA-1 function during the DC lifecycle.     

TRPM7, a novel regulator of actomyosin contractility and cell adhesion

Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, alpha-kinases control actomyosin relaxation, a similar role for mammalian alpha-kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an alpha-kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca(2+)- and kinase-dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca2+ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase-dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase-dependent and -independent pathways on actomyosin contractility.     

Dendritic cell interaction with Candida albicans critically depends on N-linked mannan

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, because a detailed characterization at the structural level is lacking. Antigen-presenting dendritic cells (DCs), strategically located at mucosal surfaces and in the skin, may play an important role in anti-Candida protective immunity. However, the contribution of the various Candida-associated molecular patterns and their counter-receptors to DC function remains unknown. Here, we demonstrate that two C-type lectins, DC-SIGN and the macrophage mannose receptor, specifically mediate C. albicans binding and internalization by human DCs. Moreover, by combining a range of C. albicans glycosylation mutants with receptor-specific blocking and cytokine production assays, we determined that N-linked mannan but not O-linked or phosphomannan is the fungal carbohydrate structure specifically recognized by both C-type lectins on human DCs and directly influences the production of the proinflammatory cytokine IL-6. Better insight in the carbohydrate recognition profile of C-type lectins will ultimately provide relevant information for the development of new drugs targeting specific fungal cell wall antigens.     

In vivo19F MRI for Cell Tracking

In vivo¹⁹F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.