Targeting proliferating CLL cells with a novel synthetic low density lipoprotein drug delivery system

Chronic lymphocytic leukaemia (CLL) currently remains incurable without stem cell transplantation, an option for only the minority of patients. Despite advances in chemotherapy, most patients relapse owing to the persistence of minimal residual disease (MRD). Substantial evidence has accrued to suggest that the tumour microenvironment is central to disease progression in CLL, with the bone marrow (BM) and lymph nodes (LN) acting as sanctuary sites for MRD. Whilst peripheral blood CLL cells are cell cycle arrested, significant rates of clonal proliferation occur in the BM/LN wherein acquisition of deleterious cytogenetic abnormalities such as 17p deletion may arise. Further, CLL cells co-cultured in vitro on stroma with CD154/IL-4 to give a proliferative signal, are chemoresistant to first line therapies. As proliferating cells require lipids for membrane synthesis, we hypothesise that proliferating CLL cells will have greater requirement for low density lipoprotein (LDL) compared to circulating CLL cells, and also that of normal resting lymphocytes providing a potentially differential cellular property to attack. Proof of concept of drug-loaded synthetic (s)LDL nanoparticles has been provided in glioblastoma and CML. We propose that drug loading into sLDL nanoparticles will allow selective targeting of proliferating CLL cells within the BM/LN proliferation centre, will protect drugs from plasma binding proteins, and will ultimately raise intracellular drug concentrations in the protective microenvironmental niche, to overcome chemoresistance. Aims. To determine (a) the extent of sLDL uptake by CLL cells compared to normal; and (b) whether sLDL uptake by CLL cells changes under proliferative conditions mimicking the proliferation centre. This will determine whether proliferating CLL cells have increased sLDL uptake compared to non-cycling CLL cells or normal B lymphocytes. Methods. sLDL uptake was assessed by flow cytometry, measuring the mean fluorescence intensity in the FITC channel owing to the stable incorporation of dioctadecyloxacarbocyanine (DiO) into the formulation. Internalisation was confirmed by deconvolution fluorescence microscopy. Primary CLL and normal donor samples were enriched for CD19+ B-lineage cells by magnetically activated cell sorting. Cells were cultured in media on tissue culture plastic or NT-L mouse fibroblasts with or without CD154/IL4. Lymphoid cells were stained with CellTrace VioletR to track cell division in response to proliferative signals (CD154/IL4 stroma). Results. HG3, a human lymphoblastoid cell line, avidly took up sLDL nanoparticles in a concentration (0-50 ng/mL cholesterol) and time (0.5-24h) dependent manner. Normal donor peripheral blood B-cells and CLL cells cultured on plastic did not actively take up sLDL but maintained their viability even in the highest concen- tration sLDL tested. Actively proliferating CLL cells on CD154/IL4 stroma could be targeted with sLDL unlike their non-cycling counterparts; interestingly even the minor population of cells that had remained undivided on stroma were also found to be sLDL positive. Summary. CLL cells can be selectively targeted by sLDL nanoparticles with respect to their non-cycling counterparts. We next will investigate the in vivo targeting of sLDL which we hypothesise, by virtue of their size, will home to lymphoreticular organs, sanctuary sites for CLL MRD

Uptake of synthetic low density lipoprotein by leukemic stem cells — a potential stem cell targeted drug delivery strategy

Chronic Myeloid Leukemia (CML) stem/progenitor cells, which over-express Bcr-Abl, respond to imatinib by a reversible block in proliferation without significant apoptosis. As a result, patients are unlikely to be cured owing to the persistence of leukemic quiescent stem cells (QSC) capable of initiating relapse. Previously, we have reported that intracellular levels of imatinib in primary primitive CML cells (CD34<sup>+</sup>38<sup>lo/−</sup>), are significantly lower than in CML progenitor cells (total CD34<sup>+</sup>) and leukemic cell lines. The aim of this study was to determine if potentially sub-therapeutic intracellular drug concentrations in persistent leukemic QSC may be overcome by targeted drug delivery using synthetic Low Density Lipoprotein (sLDL) particles. As a first step towards this goal, however, the extent of uptake of sLDL by leukemic cell lines and CML patient stem/progenitor cells was investigated. Results with non-drug loaded particles have shown an increased and preferential uptake of sLDL by Bcr-Abl positive cell lines in comparison to Bcr-Abl negative. Furthermore, CML CD34<sup>+</sup> and primitive CD34<sup>+</sup>38<sup>lo/−</sup> cells accumulated significantly higher levels of sLDL when compared with non-CML CD34<sup>+</sup> cells. Thus, drug-loading the sLDL nanoparticles could potentially enhance intracellular drug concentrations in primitive CML cells and thus aid their eradication

Infrared Telescope Facility's Spectrograph Observations of Human-Made Space Objects

No abstract availabl

GAS2 upregulation is a targetable vulnerability in chronic myeloid leukaemia

Tyrosine kinase inhibitors (TKIs), such as imatinib (IM), increase the survival of chronic myeloid leukemia (CML) patients but do not eradicate the disease as leukemia stem cells (LSCs) with primitive and quiescent signatures persist after TKI monotherapy, driving disease relapse. Using single-cell publicly available transcriptomic data, we investigated potentially tractable vulnerabilities in this persistent CML LSC population. GAS2 is significantly upregulated when comparing LSCs from CML patients in remission to normal hematopoietic stem cells (HSCs). A topoisomerase IIβ inhibitor, XK469, was proposed to be repurposed as a candidate small-molecule inhibitor of GAS2, and its effect was investigated in cell line models in combination with IM in vitro. Alone, XK469 could induce cell cycle arrest/differentiation in CML cells and reduce cell viability. In combination with IM, XK469 significantly increased CML cell apoptosis and reduced CML cell clonogenic capacity. These results suggest that GAS2 is a targetable vulnerability in CML LSCs and that using XK469 in combination with TKI potentiates the sensitivity of CML cells to IM

Diversity of the microbiota communities found in the various regions of the intestinal tract in healthy individuals and inflammatory bowel diseases

The severe and chronic inflammatory bowel diseases (IBD), Crohn disease and ulcerative colitis, are characterized by persistent inflammation and gut damage. There is an increasing recognition that the gut microbiota plays a pivotal role in IBD development and progression. However, studies of the complete microbiota composition (bacteria, fungi, viruses) from precise locations within the gut remain limited. In particular, studies have focused primarily on the bacteriome, with available methods limiting evaluation of the mycobiome (fungi) and virome (virus). Furthermore, while the different segments of the small and large intestine display different functions (e.g., digestion, absorption, fermentation) and varying microenvironment features (e.g., pH, metabolites), little is known about the biogeography of the microbiota in different segments of the intestinal tract or how this differs in IBD. Here, we highlight evidence of the differing microbiota communities of the intestinal sub-organs in healthy and IBD, along with method summaries to improve future studies

The Effective Fragment Potential: Small Clusters and Radial Distribution Functions

The effective fragment potential (EFP) method for treating solventeffects provides relative energies and structures that are in excellent agreement with the analogous fully quantum [i.e., Hartree-Fock (HF), density functional theory(DFT), and second order perturbation theory (MP2)] results for small water clusters. The ability of the method to predict bulk water properties with a comparable accuracy is assessed by performing EFP molecular dynamics simulations. The resulting radial distribution functions (RDF) suggest that as the underlying quantum method is improved from HF to DFT to MP2, the agreement with the experimental RDF also improves. The MP2-based EFP method yields a RDF that is in excellent agreement with experiment

Augmented visual feedback of movement performance to enhance walking recovery after stroke : study protocol for a pilot randomised controlled trial

Increasing evidence suggests that use of augmented visual feedback could be a useful approach to stroke rehabilitation. In current clinical practice, visual feedback of movement performance is often limited to the use of mirrors or video. However, neither approach is optimal since cognitive and self-image issues can distract or distress patients and their movement can be obscured by clothing or limited viewpoints. Three-dimensional motion capture has the potential to provide accurate kinematic data required for objective assessment and feedback in the clinical environment. However, such data are currently presented in numerical or graphical format, which is often impractical in a clinical setting. Our hypothesis is that presenting this kinematic data using bespoke visualisation software, which is tailored for gait rehabilitation after stroke, will provide a means whereby feedback of movement performance can be communicated in a more meaningful way to patients. This will result in increased patient understanding of their rehabilitation and will enable progress to be tracked in a more accessible way. The hypothesis will be assessed using an exploratory (phase II) randomised controlled trial. Stroke survivors eligible for this trial will be in the subacute stage of stroke and have impaired walking ability (Functional Ambulation Classification of 1 or more). Participants (n = 45) will be randomised into three groups to compare the use of the visualisation software during overground physical therapy gait training against an intensity-matched and attention-matched placebo group and a usual care control group. The primary outcome measure will be walking speed. Secondary measures will be Functional Ambulation Category, Timed Up and Go, Rivermead Visual Gait Assessment, Stroke Impact Scale-16 and spatiotemporal parameters associated with walking. Additional qualitative measures will be used to assess the participant's experience of the visual feedback provided in the study. Results from the trial will explore whether the early provision of visual feedback of biomechanical movement performance during gait rehabilitation demonstrates improved mobility outcomes after stroke and increased patient understanding of their rehabilitation