Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

The Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, and the activation of lambda L chain gene rearrangements. In mature B cells, Btk is essential for BCR-mediated proliferation and survival. Upon BCR stimulation, Btk is transphosphorylated at position Y551, which promotes its catalytic activity and subsequently results in autophosphorylation at position Y223 in the Src homology 3 domain. To address the significance of Y223 autophosphorylation and the requirement of enzymatic activity for Btk function in vivo, we generated transgenic mice that express the autophosphorylation site mutant Y223F and the kinase-inactive mutant K430R, respectively. We found that Y223 autophosphorylation was not required for the regulation of IL-7 responsiveness and cell surface phenotype changes in differentiating pre-B cells, or for peripheral B cell differentiation. However, expression of the Y223F-Btk transgene could not fully rescue the reduction of lambda L chain usage in Btk-deficient mice. In contrast, transgenic expression of kinase-inactive K430R-Btk completely reconstituted lambda usage in Btk-deficient mice, but the defective modulation of pre-B cell surface markers, peripheral B cell survival, and BCR-mediated NF-kappaB induction were partially corrected. From these findings, we conclude that: 1) autophosphorylation at position Y223 is not essential for Btk function in vivo, except for regulation of lambda L chain usage, and 2) during B cell development, Btk partially acts as an adapter molecule, independent of its catalytic activity

Acoustical sensing of cardiomyocyte cluster beating

Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cell quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to mu m). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66-168 bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity

Immunoglobulin production induced by CD57+ GC-derived helper T cells in vitro requires addition of exogenous IL-2

Germinal centers (GC) are well-defined areas in lymphoid organs were B cells proliferate and differentiate in response to T-cell-dependent antigens. The GC comprises B cells, follicular dendritic cells, tangible body macrophages, and a low number of CD4+ T cells. A large portion of these T cells expresses CD57. We have examined the ability of the CD4+CD57+ GC T cells to become activated and to take part in B cell activation processes, These T cells coexpress CD45RO, CD69, CD28, and upon mitogenic stimulation CD25, The cell population was found neither to containe nor to be able to produce any specific mRNA for IL-2, IL-4, and IFN-γ upon activation. Levels of mRNA encoding CD40 ligand was also undetectable under similar conditions. Furthermore, in contrast to ordinary CD4+ T cells, this population expressing CD57 was unable to induce B cells to Ig production in the presence of pokeweed mitogen or SEA unless IL-2 was added to the cultures, However, despite their apparent lack of function CD4+CD57+ GC T cells were found to rescue GC B cells from cell death in vitro to the same extent as CD4+CD57- T(h) cells, The phenotypical and functional differences found between these T cells and regular T(h)-cells suggest that they either represent a T cell subset with distinct properties within the GC yet to be determined or that they represent T cells, Irate in the immune response, having lost most of their original functions and capabilities

Bruton's Tyrosine Kinase Cooperates with the B Cell Linker Protein SLP-65 as a Tumor Suppressor in Pre-B Cells

Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in approximately ~50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence (~75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor

Signals sustaining human immunoglobulin V gene hypermutation in isolated germinal centre B cells

Affinity maturation of antibody responses depends on somatic hypermutation of the immunoglobulin V genes. Hypermutation is initiated specifically in proliferating B cells in lymphoid germinal centres but the signals driving this process remain unknown. This study identifies signals that promote V gene mutation in human germinal centre (GC) B cells in vitro. Single GC B cells were cultured by limiting dilution to allow detection of mutations arising during proliferation in vitro. Cells were first cultured in the presence of CD32L cell transfectants and CD40 antibody (the ‘CD40 system’) supplemented with combinations of cytokines capable of supporting similar levels of CD40-dependent GC B-cell growth [interleukin (IL)-10 + IL-1β + IL-2 and IL-10 + IL-7 + IL-4]. Components of the ‘EL4 system’ were then added to drive differentiation, providing sufficient immunoglobulin mRNA for analysis. Analysis of VH3 genes from cultured cells by reverse transcription–polymerase chain reaction (RT–PCR)-based single-strand conformation polymorphism indicated that the combination IL-10 + IL-1β + IL-2 promoted active V gene mutation whereas IL-10 + IL-7 + IL-4 was ineffective. This was confirmed by sequencing which also revealed that the de novo generated mutations were located in framework and complementarity-determining regions and shared characteristics with those arising in vivo. Somatic mutation in the target GC B-cell population may therefore be actively cytokine driven and not simply a consequence of continued proliferation. The experimental approach we describe should facilitate further studies of the mechanisms underlying V gene hypermutation

Mathematical Modeling and Use of Magnetic Resonance Imaging (MRI) for Oil Migration in Chocolate Confectionery Systems

Oil migration is a common problem in chocolate confectionery products leading to quality defects, particularly fat bloom. Several factors such as contact area, ratio of the two fat phases, type of the fat, solid fat content, presence of non-fat solid particles, particle size, viscosity, structure, concentration gradient of triacylglycerols (TAGs), and storage temperature have all effect on migration rate. Mechanism of oil migration has still not been clearly understood, but possible mechanisms have been suggested and studied in the literature. Diffusion mechanism was demonstrated and modeled in many studies. Although there are so many methods to monitor and quantify migration, magnetic resonance imaging (MRI) is among the most promising techniques as being non-destructive. This review covers the literature related to basics of migration, mechanisms, and monitoring and modeling migration in chocolate through MRI and also includes a brief description about chocolate, chocolate processing, and fundamental concepts in MRI

CD44-stimulated human B cells express transcripts specifically involved in immunomodulation and inflammation as analyzed by DNA microarrays

A number of studies have implicated a role for the cell surface glycoprotein CD44 in several biologic events, such as lymphopoiesis, homing, lymphocyte activation, and apoptosis. We have earlier reported that signaling via CD44 on naive B cells in addition to B-cell receptor (BCR) and CD40 engagement generated a germinal center-like phenotype. To further characterize the global role of CD44 in B differentiation, we examined the expression profile of human B cells cultured in vitro in the presence or absence of CD44 ligation, together with anti-immunoglobulin (anti-Ig) and anti-CD40 antibodies. The data sets derived from DNA microarrays were analyzed using a novel statistical analysis scheme created to retrieve the most likely expression pattern of CD44 ligation. Our results show that genes such as interleukin-6 (IL-6), IL-1alpha , and beta 2-adrenergic receptor (beta 2-AR) were specifically up-regulated by CD44 ligation, suggesting a novel role for CD44 in immunoregulation and inflammation

Non-Invasive Acoustical sensing of Drug-Induced Effects on the Contractile Machinery of Human Cardiomyocyte Clusters.

There is an urgent need for improved models for cardiotoxicity testing. Here we propose acoustic sensing applied to beating human cardiomyocyte clusters for non-invasive, surrogate measuring of the QT interval and other characteristics of the contractile machinery. In experiments with the acoustic method quartz crystal microbalance with dissipation monitoring (QCM-D), the shape of the recorded signals was very similar to the extracellular field potential detected in electrochemical experiments, and the expected changes of the QT interval in response to addition of conventional drugs (E-4031 or nifedipine) were observed. Additionally, changes in the dissipation signal upon addition of cytochalasin D were in good agreement with the known, corresponding shortening of the contraction-relaxation time. These findings suggest that QCM-D has great potential as a tool for cardiotoxicological screening, where effects of compounds on the cardiomyocyte contractile machinery can be detected independently of whether the extracellular field potential is altered or not