The pre-B cell receptor; selecting for or against autoreactivity.

Antibodies represent a crucial component of humoral immunity as protection against invading pathogens, to which they bind and thereby trigger mechanisms that lead to the disposal of the pathogen. Antibodies are assembled from Ig heavy chains (HCs) and light chains (LCs) and are found in both a secreted and a membrane-bound form, termed B cell receptors (BCRs), where the latter allows the 'right' B cell to respond upon recognition of its cognate antigen. The antibody repertoire is almost unlimited because of a process in which germ line V(D)J gene segments, encoding the variable (antigen-binding) region of the antibody HCs and LCs, are recombined. As this process is random, it is apparent that it results in a vast variety of antibodies, those that recognize foreign but also those that recognize self- (auto-) antigens. Control mechanisms are, therefore, in place to ensure that as few autoreactive B cells as possible are allowed to proceed in development. This counter-selection takes place through various mechanisms and at several stages as the cells develop from pre-B cells to antibody-secreting plasma cells. At the first major checkpoint, at the pre-BI to pre-BII cell transition, antibody HCs assemble with the invariant surrogate LC (SLC) forming a pre-BCR. Herein, we will discuss the role of the pre-BCR in the selection at this stage, how a dysfunctional pre-BCR affects selection and its effects on later stages, and whether the pre-BCR selects for or against autoreactivity.

Exosomes - nanovesicles with possible roles in allergic inflammation.

Exosomes are nano-sized membrane vesicles which are released extracellularly after fusion of multivesicular endosomes with the cell membrane. Despite their characteristic composition of proteins compared to the cell membrane, no exosome-specific molecule has so far been characterized. Exosomes are found in bronchoalveolar lavage (BAL), urine, serum and breast milk, and are released from several cells implicated in allergy including mast cells, dendritic cells (DC), T cells and epithelial cells. Antigen-loaded exosomes have been shown to be highly immunogenic and we propose that exosomes could be a modulating factor in allergic responses. Allergen-presenting exosomes could transport allergen and stimulate allergen-specific T cells, and possibly also biasing T cell responses depending on the molecules present on the exosome surface. Furthermore, exosomes from mast cells, highly active in allergic reactions, have been found to induce DC maturation and also to be able to transport functional RNA to recipient cells, suggesting a new pathway for cell communication. Reversely, tolerizing exosomes e.g. tolerosomes, from gut or breast milk, could block an allergic response or prevent allergy development. A better understanding of the role of exosomes in allergies could make us understand how allergy can be prevented or lead to the development of more efficient treatments.

Imaging human erythrocyte spectrin with atomic force microscopy.

Isolated spectrin covalently attached to a surface in a liquid environment as well as dried on mica has been studied with a contact-mode atomic force microscope. Both pyramidal and conical-type cantilever tip facets were used in the AFM. Our images show structures and give dimensions that correlate well with previous structural studies using transmission electron microscopy.

Human small intestinal epithelial cells constitutively express the key elements for antigen processing and the production of exosomes.

In humans, the small intestinal epithelial cells (IEC) have a high constitutive expression of MHC class II (MHC II), and contains lysosomes. The IEC also contains MHC II rich multivesicular compartments and has been shown to produce exosomes. This suggests a role for the IEC in antigen processing and presentation either directly or indirectly by the production of exosomes. However, the presence and localisation in the IEC of other key molecules involved in this process has not been studied previously. In the present work, we have investigated small intestinal biopsies from healthy adults and the HT29 IEC cell line with monoclonal antibodies against molecules involved in the antigen processing/presenting systems and molecules typically found on exosomes derived from professional APCs and IECs. Immunohistology was performed to study the expression and localisation of MHC II (HLA-DR), HLA-DM, MHC I (HLA-ABC), CD1d, Invariant chain, Lamp-1, CD68, CD63, B7.1, B7.2, ICAM-1, Cathepsin D/S/L and the IEC specific marker A33 in the IECs. We found that the IECs from the biopsies constitutively express MHC II, HLA-DM, MHC I, Invariant chain, Lamp-1, CD 68, CD63 and A33, and these markers were also found in the IFN-g treated HT-29 cells. All these molecules were found apically in the IECs of the biopsies, localised mainly in vesicular structures. Interestingly, in the baso-latereral area of the IEC, only MHC II, MHC I, Lamp 1, CD68, CD63 and A33 were found and also here with vesicular staining pattern which matches the molecules previously found on exosomes derived professional APCs and human IEC lines. CD1d, B7, ICAM-1, CD9 and cathepsin D and L were absent in the IEC compartment, but cathepsin S showed a relatively weak staining in the apical part of the IEC. The staining pattern and the morphological localisation of these markers suggest a prominent antigen processing/loading and trafficking compartment, and a possible baso-lateral release of exosomes in the normal human IEC.

Elasticity and adhesion force mapping reveals real-time clustering of growth factor receptors and associated changes in local cellular rheological properties.

Cell surface macromolecules such as receptors and ion channels serve as the interface link between the cytoplasm and the extracellular region. Their density, distribution, and clustering are key spatial features influencing effective and proper physical and biochemical cellular responses to many regulatory signals. In this study, the effect of plasma-membrane receptor clustering on local cell mechanics was obtained from maps of interaction forces between antibody-conjugated atomic force microscope tips and a specific receptor, a vascular endothelial growth factor (VEGF) receptor. The technique allows simultaneous measurement of the real-time motion of specific macromolecules and their effect on local rheological properties like elasticity. The clustering was stimulated by online additions of VEGF, or antibody against VEGF receptors. VEGF receptors are found to concentrate toward the cell boundaries and cluster rapidly after the online additions commence. Elasticity of regions under the clusters is found to change remarkably, with order-of-magnitude stiffness reductions and fluidity increases. The local stiffness reductions are nearly proportional to receptor density and, being concentrated near the cell edges, provide a mechanism for cell growth and angiogenesis.

Oriented, active Escherichia coli RNA polymerase: an atomic force microscope study.

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.

Micromechanical and structural properties of a pennate diatom investigated by atomic force microscopy.

The mechanisms behind natural nanofabrication of highly structured silicas are increasingly being investigated. We have explored the use of a standard Nanoscope III Multimode atomic force microscope (AFM) to study the silica shell of diatoms. The delicate structures of the shell surface of the diatom Navicula pelliculosa (Bréb.) Hilse were imaged and the shell's micromechanical properties were measured semi-quantitatively with a resolution down to approximately 10 nm. The technique to measure elasticity and hardness with the AFM was demonstrated to be useable even on these hard glass-like surfaces. Different experimental configurations and evaluation methods were tested. They gave a consistent result of the shell micromechanical properties. The first results showed that the diatom shell's overall hardness and elasticity was similar to that of known silicas. However, regions with different mechanical properties were distinguished. The elastic modulus varied from 7 to 20 GPa, from 20 to 100 GPa and from 30 to hundreds of GPa depending on the location. In general, the hardness measurements showed similar spatial differences. The hardness values ranged from 1 to 12 GPa but one specific part of the shell was even harder. Hence, certain localized regions of the shell were significantly harder or more elastic. These regions coincide with known characteristic features and mechanisms appearing at the different stages of the shell's growth. These results show that this method serves as a complementary tool in the study of silica biomineralization, and can detect eventual crystalline phases.