Fabian Kohler

Chronic lymphocytic leukaemia is driven by antigen-independent cell-autonomous signalling

B-cell antigen receptor (BCR) expression is an important feature of chronic lymphocytic leukaemia (CLL), one of the most prevalent B-cell neoplasias in Western countries. The presence of stereotyped and quasi-identical BCRs in different CLL patients suggests that recognition of specific antigens might drive CLL pathogenesis. Here we show that, in contrast to other B-cell neoplasias, CLL-derived BCRs induce antigen-independent cell-autonomous signalling, which is dependent on the heavy-chain complementarity-determining region (HCDR3) and an internal epitope of the BCR. Indeed, transferring the HCDR3 of a CLL-derived BCR provides autonomous signalling capacity to a non-autonomously active BCR, whereas mutations in the internal epitope abolish this capacity. Because BCR expression was required for the binding of secreted CLL-derived BCRs to target cells, and mutations in the internal epitope reduced this binding, our results indicate a new model for CLL pathogenesis, with cell-autonomous antigen-independent signalling as a crucial pathogenic mechanism.

Autoreactive B Cell Receptors Mimic Autonomous Pre-B Cell Receptor Signaling and Induce Proliferation of Early B Cells

The majority of early immature B cells express autoreactive B cell receptors (BCRs) that are, according to the current view, negatively selected to avoid the production of self-reactive antibodies. Here, we show that polyreactive BCRs, which recognize multiple self-antigens, induced autonomous signaling and selective expansion of B cell precursors in a manner comparable to the pre-BCR. We found that the pre-BCR was capable of recognizing multiple self-antigens and that a signaling-deficient pre-BCR lacking the non-Ig region of the surrogate-light-chain component lambda5 was rescued by the complementarity-determining region 3 derived from heavy chains of polyreactive receptors. Importantly, bone marrow B cells from mice carrying Ig transgenes for an autoreactive BCR showed increased cell-cycle activity, which could not be detected in cells lacking the transgenic BCR. Together, the pre-BCR has evolved to ensure self-recognition because autoreactivity is required for positive selection of B cell precursors.

Characterization of the DNA-Binding and Dimerization Properties of the Nuclear Orphan Receptor Germ Cell Nuclear Factor

ABSTRACT The orphan receptor germ cell nuclear factor (GCNF) is a member of the superfamily of nuclear receptors. During development, GCNF exhibits a restricted brain-specific expression pattern, whereas GCNF expression in the adult is germ cell specific. Therefore, the receptor may participate in the regulation of neurogenesis and reproductive functions. No natural GCNF target gene has yet been identified, but recent data demonstrate specific and high-affinity binding of GCNF either to the direct repeat DNA element AGGTCAAGGTCA (DR0) or to extended half-sites, such as TCAAGGTCA. In this study, we show that murine GCNF (mGCNF) can bind as a homodimer to extended half-sites, thus describing a novel property within the nuclear receptor superfamily. Homodimeric binding to extended half-sites requires the presence of a dimerization function within the mGCNF DNA-binding domain (DBD) and a novel dimerization surface encompassing the putative helix 3 and the helix 12 region of the mGCNF ligand-binding domain (LBD). In addition, the mGCNF LBD has the potential to adopt different conformations with distinct dimerization properties. The helix 12 region of the mGCNF LBD not only regulates the switch between these dimerization conformations but also dictates the DNA-binding behavior and transcriptional properties of the different dimerization conformations. In summary, our findings describe unique DNA-binding and dimerization properties of a nuclear receptor and suggest a novel mechanism that allows mGCNF to modulate target gene activity.

A leucine zipper in the N terminus confers membrane association to SLP-65

Membrane recruitment of adaptor proteins is crucial for coupling antigen receptors to downstream signaling events. Despite the essential function of the B cell adaptor SLP-65, the mechanism of its recruitment to the plasma membrane is not yet understood. Here we show that a highly conserved leucine zipper in the SLP-65 N terminus is responsible for membrane association. Alterations in the N terminus abolished SLP-65 membrane localization and activity, both of which were restored by replacement of the N terminus with a myristoylation signal. The N terminus is an autonomous domain that confers specific localization and function when transferred to green fluorescent protein or the adaptor protein SLP-76. Our data elucidate the mechanism of SLP-65 membrane recruitment and suggest that leucine zipper motifs are essential interaction domains of signaling proteins.

FoxO1 induces Ikaros splicing to promote immunoglobulin gene recombination

During murine B cell development, PI3 kinase inhibits Ig gene rearrangement by suppressing FoxO1, which mediates Ikaros mRNA splicing; Ikaros is needed for Ig gene recombination.

Efficient generation of B lymphocytes by recognition of self‐antigens

Antibody diversity is generated by a random gene recombination process with the inherent risk of the production of autoreactive specificities. The current view suggests that B cells expressing such specificities are negatively selected at an early developmental stage. Using the knock‐in model system of the 3‐83 autoreactive B‐cell antigen receptor (BCR) in combination with precursor‐BCR (pre‐BCR) deficiency, we show here that the 3‐83 BCR mediates efficient generation of B cells in the presence, but not the absence, of a strongly recognized auto‐antigen. Experiments with mixed bone marrow chimeras showed that combining the 3‐83 BCR with the corresponding auto‐antigen resulted in efficient reconstitution of B‐cell development in immune‐deficient mice. These results suggest that B cells are positively selected by recognition of self‐antigens during developmental stages that precede receptor editing. Moreover, the data indicate that the pre‐BCR functions as a specialized autoreactive BCR to initiate positive selection at a stage where the cells express immunoglobulin heavy but not light chains.

Hermetic electronic packaging of an implantable brain-machine-interface with transcutaneous optical data communication

Future brain-computer-interfaces (BCIs) for severely impaired patients are implanted to electrically contact the brain tissue. Avoiding percutaneous cables requires amplifier and telemetry electronics to be implanted too. We developed a hermetic package that protects the electronic circuitry of a BCI from body moisture while permitting infrared communication through the package wall made from alumina ceramic. The ceramic package is casted in medical grade silicone adhesive, for which we identified MED2-4013 as a promising candidate.

Mapping of sheep sensory cortex with a novel microelectrocorticography grid.

Microelectrocorticography (µECoG) provides insights into the cortical organization with high temporal and spatial resolution desirable for better understanding of neural information processing. Here we evaluated the use of µECoG for detailed cortical recording of somatosensory evoked potentials (SEPs) in an ovine model. The approach to the cortex was planned using an MRI‐based 3D model of the sheeps brain. We describe a minimally extended surgical procedure allowing placement of two different µECoG grids on the somatosensory cortex. With this small craniotomy, the frontal sinus was kept intact, thus keeping the surgical site sterile and making this approach suitable for chronic implantations. We evaluated the procedure for chronic implantation of an encapsulated µECoG recording system. During acute and chronic recordings, significant SEP responses in the triangle between the ansate, diagonal, and coronal sulcus were identified in all animals. Stimulation of the nose, upper lip, lower lip, and chin caused a somatotopic lateral‐to‐medial, ipsilateral response pattern. With repetitive recordings of SEPs, this somatotopic pattern was reliably recorded for up to 16 weeks. The findings of this study confirm the previously postulated ipsilateral, somatotopic organization of the sheeps sensory cortex. High gamma band activity was spatially most specific in the comparison of different frequency components of the somatosensory evoked response. This study provides a basis for further acute and chronic investigations of the sheeps sensory cortex by characterizing its exact position, its functional properties, and the surgical approach with respect to macroanatomical landmarks. J. Comp. Neurol. 522:3590–3608, 2014.

Mechanical characterization of neural electrodes based on PDMS-parylene C-PDMS sandwiched system

Manufacturing of neural electrodes based on metal foil and silicone rubber using a laser is a simple and promising method. A handicap of such electrode arrays is the mechanical robustness of the thin metal tracks that connect the electrode sites with the interconnection pads. Embedding of structured parylene C foil in silicone rubber turned out to be an interesting method to increase the robustness. Test samples with 12.5 μm thick platinum tracks and a 15 μm thick embedded and RIE-structured parylene C foil showed more than 800 % higher ultimate strength until breakage of the tracks. Different structured parylene C foil showed increasing robustness with increasing hole-spacing.

Parylene-coated metal tracks for neural electrode arrays - Fabrication approaches and improvements utilizing different laser systems

In the past we developed a method for the fabrication of neural electrodes based on laser-structuring metal foil to form tracks and electrode sites within a silicone rubber substrate. Here, this process was refined by an additional coating of the laser-patterned metal tracks to improve their mechanical properties. Parylene C has been found to be the coating material of choice due to excellent electrical and mechanical characteristics and its well known biocompatibility. An almost ten times increased tensile strength compared to uncoated tracks could be achieved. Investigating the electrical properties of parylene C and silicone rubber attested both materials excellent insulating capabilities by withstanding voltages of more than 400 VDC for layer thicknesses as intended to be used in electrode array fabrication (some 10 μm). This paper outlines the feasibility of the manufacturing process using a 1064 nm Nd:YAG laser in the nanosecond pulse regime. However, an improvement of the whole processing was demonstrated when a 355 nm Nd:YVO4 laser in the picosecond regime is used. Benefits of this short pulse duration range from ablating materials independent of their optical properties to increased manufacturing speed and superior processing quality.