Investigation of dual Src/c-Abl tyrosine kinase inhibition as a novel therapeutic approach for chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the western world, and remains incurable with current chemotherapy. Although CLL was long-regarded as an autonomous accumulation of functionally incompetent lymphocytes that escape apoptosis, significant rates of clonal proliferation and death have now been elegantly demonstrated in CLL patients in vivo. This, coupled with the high rates of spontaneous apoptosis observed on ex-vivo culture, confirms that CLL is a dynamic disorder, in which the tumour microenvironment is central to leukemic cell survival. Recent advances in CLL cell biology implicate signalling through the B cell antigen receptor (BCR) in the pathogenesis and progression of the disease. The absence of significant somatic hypermutation of the immunoglobulin heavy chain variable region (IgVH), which largely correlates with the expression of ZAP-70, in CLL cells is a significant adverse prognostic marker. CLL cases expressing an unmutated IgVH gene generally retain the ability to signal through the BCR. Components of the BCR signalling pathway are therefore attractive novel therapeutic targets, with potential selectivity for adverse prognostic groups. The non-receptor tyrosine kinases Lyn (a Src kinase) and c-Abl are both required for effective BCR signalling. Both are over-expressed, and constitutively active in CLL, and inhibition of either induces a degree of apoptosis. Dasatinib is a Src/c-Abl tyrosine kinase inhibitor in clinical use in chronic myeloid leukaemia. The main objective of this project was to conduct translational studies to determine the anti-leukaemic effects of dasatinib on CLL cells in vitro. While the Src kinase inhibitor PP2, and the c-Abl inhibitor imatinib induced apoptosis of CLL cells at micromolar concentrations, dasatinib induced apoptosis of CLL cells at clinically achievable nanomolar concentrations, with an EC50 in the region of 10-30 nM, and plateau in effect above 100 nM. CLL cell treatment with 100 nM dasatinib for 48 hr led to a mean reduction in viability of 33.7%, but with significant inter-sample variability. No correlation was observed between dasatinib sensitivity and the established prognostic factors clinical stage, ZAP-70 status, or cytogenetic subgroup. Notably, CLL cells known to contain the 17p deletion, resulting in p53 dysfunction, responded similarly to other samples. Apoptosis induced by dasatinib involved loss of mitochondrial membrane potential and was caspase-dependent. Although dasatinib treatment alone rarely induced apoptosis of over 50% of CLL cells, synergy was observed between dasatinib and the current first-line chemotherapeutic agents fludarabine and chlorambucil. Moreover, dasatinib exhibited synergy with a novel Bcl-2 inhibitor, and the HSP90 inhibitor 17-DMAG. Recently, antigen-independent ‘tonic’ BCR signalling has been linked to the pathogenesis of B cell lymphomas. Tonic signalling is proposed to be mediated by basal activity of Lyn and Syk kinases recruited to the BCR. As Syk is also over-expressed in CLL, we hypothesised that dasatinib sensitivity may correlate with inhibition of components of tonic BCR signal transduction. Indeed, CLL cells contained constitutively phosphorylated SykY348. Furthermore, a significant inverse correlation was observed between basal SykY348 phosphorylation and dasatinib sensitivity in individual samples, suggesting its’ utility as a biomarker of response. Dasatinib consistently inhibited an increase in SykY348 phosphorylation on BCR crosslinking. In addition, dasatinib inhibited calcium flux, and prevented Akt and MAPK phosphorylation on BCR stimulation. Moreover, dasatinib prevented up-regulation of Mcl-1 and blocked the increase in CLL cell survival observed on prolonged BCR stimulation, confirming inhibition of BCR signalling as a functionally relevant treatment strategy in CLL. Although dasatinib prevented CXCR4 down-regulation following BCR stimulation of CLL cells, dasatinib also specifically inhibited PI-3K/Akt activation upon CXCR4 stimulation by SDF-1, resulting in reduced actin polymerization and migration following SDF-1 stimulation. While the full translational implications of these observations remain to be determined, these data demonstrate that the anti-leukaemic effects of dasatinib extend beyond direct inhibition of BCR signal transduction. It is well recognised that bone marrow (BM) stromal cells and blood-derived ‘nurse-like’ cells protect CLL cells from spontaneous apoptosis in vitro. More recently, proliferating CLL cells have been identified within specialised ‘proliferation centres’, admixed with appreciable numbers of T lymphocytes, predominantly activated CD4+ T cells expressing CD40 ligand (CD154), and interleukin 4 (IL-4). CD40/IL-4 stimulation of CLL cells in vitro leads to up-regulation of the anti-apoptotic Bcl-2 family proteins Bcl-xL and Mcl-1, and the pro-proliferative protein survivin, mimicking the expression profile of CLL cells isolated from patient lymph nodes (LNs). We were interested to determine whether the effects of dasatinib on CLL cells were modulated by these microenvironmental factors. To achieve this, CLL cells were co-cultured with either the murine BM stromal cell line NT-L, or NT-L cells stably transfected to express CD154, the latter with IL-4 added to the culture medium (154L/IL-4 system). The pro-apoptotic effect of dasatinib in CLL cells was abrogated by stromal cell co-culture, with or without CD154 and IL-4. Stromal cell-mediated resistance to dasatinib involved Akt and MAPK signalling, as evidenced by the ability of both the PI-3K inhibitor LY294002, and the MEK inhibitor PD98059 to restore dasatinib sensitivity of cells in NT-L co-culture. 154L/IL-4 co-culture activated multiple MAPK, associated with up-regulation of Bcl-xL, Mcl-1, and survivin, which was not inhibited by dasatinib. Dasatinib also failed to inhibit CLL cell proliferation in the 154L/IL-4 system. While dasatinib retained the ability to sensitise CLL cells to both fludarabine and chlorambucil in NT-L co-culture, the addition of CD154 and IL-4 rendered cells resistant to all drug combinations. Dasatinib did however retain the ability to sensitise CLL cells to the HSP90 inhibitor 17-DMAG in both NT-L and 154L/IL-4 co-culture. In conclusion, these studies demonstrate that dasatinib offers much as a novel therapeutic strategy for CLL, overcoming pro-survival signalling through the BCR. However, our data suggest that dasatinib may be best utilised in combination treatment strategies with agents that can target antigen-independent signalling networks within the microenvironment. Collectively, this work provides valuable information that will inform future clinical trials of Src/c-Abl inhibitors in CLL

An analysis of motor coordination in fit versus less fit young adult males

The purpose of this study was to compare the motor coordination components of movement between young adult males who are physically fit versus those who are less fit

Effect of Concurrent Walking and Interlocutor Distance on Conversational Speech Intensity and Rate in Parkinson\u27s Disease

This study examined the effects of concurrent walking tasks and interlocutor distance on conversational speech production in fifteen individuals with idiopathic Parkinson’s disease (PD) and fourteen age-equivalent controls. Recent studies of speech in PD have demonstrated that changes in the behavioural conditions and the environmental context can have a powerful effect on the severity of speech symptoms in PD. This investigation focused on changes in speech intensity and speech rate in response to changes in walking speed and interlocutor distance. Results suggest that the introduction of a concurrent walking task significantly increased the conversational speech intensity of both controls and individuals with PD. When compared to sitting and talking or standing and talking, current walking and talking appeared to have an enhancing effect on conversational speech intensity. In addition, walking faster was associated with a significant increase in conversational speech intensity relative to normal and slow walking speeds. These results provide important new information about the effect of concurrent walking on speech motor performance and speech symptom severity in PD. The potential energizing effect of concurrent walking conditions on conversational speech intensity may be an important consideration in the assessment and treatment of individuals with low speech intensity in PD

Process for phosphonylating the surface of an organic polymeric preform

A process for phosphonylating the surface of an organic polymeric preform and the surface-phosphonylated preforms produced thereby are provided. Organic polymeric preforms made from various polymers including polyethylene, polyetheretherketone, polypropylene, polymethylmethacrylate, polyamides, and polyester, and formed into blocks, films, and fibers may have their surfaces phosphonylated in a liquid-phase or gas phase reaction. Liquid phase phosphonylation involves the use of a solvent that does not dissolve the organic polymeric preform but does dissolve a phosphorus halide such as phosphorus trichloride. The solvent chosen must also be nonreactive with the phosphorus halide. Such solvents available for use in the present process include the fully-halogenated liquid solvents such as carbon tetrachloride, carbon tetrabromide, and the like. Gas phase phosphonylation involves treating the organic polymeric preform with a gaseous phosphorus halide such as phosphorus trichloride and oxygen. The inventive processes allow for surface phosphonylation of the organic polymeric preform such that up to about 30 percent of the reactive carbon sites in the polymer are phosphonylated. The inventive phosphonylated organic polymers are particularly useful as orthopedic implants because hydroxyapatite-like surfaces which can be subsequently created on the organic implants allow for co-crystallization of hydroxyapatite to form chemically-bound layers between prosthesis and bone tissue

Electrical Stimulation Directs Migration, Enhances and Orients Cell Division and Upregulates the Chemokine Receptors CXCR4 and CXCR2 in Endothelial Cells

© 2019 S. Karger AG, Basel.Peer reviewedPostprin

P 139 Serum dramatically enhances orientation and migration of cultured bovine corneal epithelial cells in an applied electric field (EF)

Digitalitzat per Artypla

Antimicrobial-Drug Prescription in Ambulatory Care Settings, United States, 1992–2000

During the 1990s, as antimicrobial resistance increased among pneumococci, many organizations promoted appropriate antimicrobial use to combat resistance. We analyzed data from the National Ambulatory Medical Care Survey, an annual sample survey of visits to office-based physicians, and the National Hospital Ambulatory Medical Care Survey, an annual sample survey of visits to hospital emergency and outpatient departments, to describe trends in antimicrobial prescribing from 1992 to 2000 in the United States. Approximately 1,100–1,900 physicians reported data from 21,000–37,000 visits; 200–300 outpatient departments reported data for 28,000–35,000 visits; ~400 emergency departments reported data for 21,000–36,000 visits each year. In that period, the population- and visit-based antimicrobial prescribing rates in ambulatory care settings decreased by 23% and 25%, respectively, driven largely by a decrease in prescribing by office-based physicians. Antimicrobial prescribing rates changed as follows: amoxicillin and ampicillin, –43%; cephalosporins, –28%; erythromycin, –76%; azithromycin and clarithromycin, +388%; quinolones, +78%; and amoxicillin/clavulanate, +72%. This increasing use of azithromycin, clarithromycin, and quinolones warrants concern as macrolide- and fluoroquinolone-resistant pneumococci are increasing

The Direction of Neurite Growth in a Weak DC Electric Field Depends on the Substratum: Contributions of Adhesivity and Net Surface Charge

AbstractWe investigated the influence of the growth surface on the direction ofXenopusspinal neurite growth in the presence of a dc electric field of physiological magnitude. The direction of galvanotropism was determined by the substratum; neurites grew toward the negative electrode (cathode) on untreated Falcon tissue culture plastic or on laminin substrata, which are negatively charged, but neurites growing on polylysine, which is positively charged, turned toward the positive electrode (anode). Growth was oriented randomly on all substrata without an electric field. We tested the hypothesis that the charge of the growth surface was responsible for reversed galvanotropism on polylysine by growing neurons on tissue culture dishes with different net surface charges. Although neurites grew cathodally on both Plastek substrata, the frequency of anodal turning was greater on dishes with a net positive charge (Plastek C) than on those with a net negative charge (Plastek M). The charge of the growth surface therefore influenced the frequency of anodal galvanotropism but a reversal in surface charge was insufficient to reverse galvanotropism completely, possibly because of differences in the relative magnitude of the substratum charge densities. The influence of substratum adhesion on galvanotropism was considered by growing neurites on a range of polylysine concentrations. Growth cone to substratum adhesivity was measured using a blasting assay. Adhesivity and the frequency of anodal turning were graded over the range of polylysine concentrations (0 = 0.1 < 1 < 10 = 100 μg/ml). The direction of neurite growth in an electric field is therefore influenced by both substratum charge and growth cone-to-substratum adhesivity. These data are consistent with the idea that spatial or temporal variation in the expression of adhesion molecules in embryos may interact with naturally occurring electric fields to enhance growth cone pathfinding

QUANTITATIVE ANALYSIS OF THE HISTONE LOCUS BODY IN SUMO KNOCKOUT HUMAN CELLS

Small ubiquitin-related modifiers (SUMOs) are proteins that can be reversibly conjugated to many other cellular proteins. Mammalian cells express up to five SUMO paralogs and our lab has recently generated paralog-specific knockout (KO) cells for SUMO1 and SUMO2 using CRISPR-Cas9. Analysis of these cells has exposed unique, paralog-specific phenotypes. In particular, SUMO1 and SUMO2 affected global gene expression patterns and PML nuclear body structure in unique ways. Using RNA-sequencing of poly(A)-selected mRNAs, we detected apparent lower levels of histone transcripts in SUMO1 KO cells but higher levels in SUMO2 KO cells, compared to wild type (WT) cells. Histone genes are not typically polyadenylated in healthy proliferative cells, but they can be polyadenylated in cases of cell differentiation, cancer, or 3’ end processing errors. Our findings suggest that histone mRNA 3’ ends may be misprocessed in SUMO KO cells. Because most core histone genes are both expressed and processed in a membrane-less organelle called the histone locus body (HLB), we assessed the localization of histone locus body factors NPAT and FLASH in WT, SUMO1 KO and SUMO2 KO cells. Using immunofluorescence microscopy, we observed similar colocalization of NPAT and FLASH in the HLBs of WT in SUMO KO cells, indicating no major defects in HLB assembly. We then used NPAT staining to further quantify the number and dimensions of HLBs in WT and SUMO KO cells. The mean HLB focus size was significantly larger in the knockout cells. Moreover, WT cells contained significantly more foci than both SUMO1 KO and SUMO2 KO cells. Based on our findings, we conclude that both SUMO1 and SUMO2 play a role in regulating histone mRNA processing through effects on the structure and function of HLBs