Cellular microparticles are vesicular plasma membrane fragments with a diameter of 100-1,000 nanometres that are shed by cells in response to various physiological and artificial stimuli. Here we demonstrate that tumour cell-derived microparticles can be used as vectors to deliver chemotherapeutic drugs. We show that tumour cells incubated with chemotherapeutic drugs package these drugs into microparticles, which can be collected and used to effectively kill tumour cells in murine tumour models without typical side effects. We describe several mechanisms involved in this process, including uptake of drug-containing microparticles by tumour cells, synthesis of additional drug-packaging microparticles by these cells that contribute to the cytotoxic effect and the inhibition of drug efflux from tumour cells. This study highlights a novel drug delivery strategy with potential clinical application.

Ca2+ currents, especially those activated at low voltages (LVA), influence burst generation in thalamocortical circuitry and enhance the abnormal rhythmicity associated with absence epilepsy. Mutations in several genes for high-voltage-activated (HVA) Ca2+ channel subunits are linked to spike-wave seizure phenotypes in mice; however, none of these mutations are predicted to increase intrinsic membrane excitability or directly enhance LVA currents. We examined biophysical properties of both LVA and HVA Ca2+ currents in thalamic cells of tottering (tg; Cav2.1/alpha1A subunit), lethargic (lh; beta4 subunit), and stargazer (stg; gamma2 subunit) brain slices. We observed 46, 51, and 45% increases in peak current densities of LVA Ca2+ currents evoked at -50 mV from -110 mV in tg, lh, and stg mice, respectively, compared with wild type. The half-maximal voltages for steady-state inactivation of LVA currents were shifted in a depolarized direction by 7.5-13.5 mV in all three mutants, although no alterations in the time-constant for recovery from inactivation of LVA currents were found. HVA peak current densities in tg and stg were increased by 22 and 45%, respectively, and a 5 mV depolarizing shift of the activation curve was observed in lh. Despite elevated LVA amplitudes, no alterations in mRNA expression of the genes mediating T-type subunits, Cav3.1/alpha1G, Cav3.2/alpha1H, or Cav3.3/alpha1I, were detected in the three mutants. Our data demonstrate that mutation of Cav2.1 or regulatory subunit genes increases intrinsic membrane excitability in thalamic neurons by potentiating LVA Ca2+ currents. These alterations increase the probability for abnormal thalamocortical synchronization and absence epilepsy in tg, lh, and stg mice.

The binding of Ni(II) and Cu(II) to histidine, to the tripeptides GlyGlyHis and HisGlyHis, and to the protein bovine serum albumin has been studied by isothermal titration calorimetry (ITC) to determine the experimental conditions and data analysis necessary to reproduce literature values for the binding constants and thermodynamic parameters. From analysis of the ITC data, we find that there are two major considerations for the use of this method to accurately quantify metal ion interaction with biological macromolecules. First, to determine true pH-independent binding constants, ITC data must be corrected for metal ion competition with protons by accounting for the experimental pH and pKa values of the metal-binding residues. Second, metal interaction with the buffer (stability and enthalpy of formation of metal-buffer complex(es)) must be included in the analysis of the ITC data to determine the binding constants and the change in enthalpy. While it may be possible to use a buffer that forms only weak, and therefore negligible, complexes with the metal, a buffer that has a strong and well-characterized interaction has the benefit of suppressing metal ion hydrolysis and precipitation, and of allowing the quantification of high-affinity metal-binding sites on biological macromolecules. This study has also quantified the contribution of the N-terminal imidazole of HisGlyHis to the stability of the Cu(II) and Ni(II) complexes of this protein sequence and has provided new insight about Cu(II) binding to albumin.

Diabetes mellitus (DM) is a high risk factor for stroke and leads to more severe vascular and white-matter injury than stroke in non-DM. We tested the neurorestorative effects of delayed human umbilical cord blood cell (HUCBC) treatment of stroke in type-2 diabetes (T2DM). db/db-T2DM and db/+-non-DM mice were subjected to distal middle cerebral artery occlusion (dMCAo) and were treated 3 days after dMCAo with: 1) non-DM + PBS; 2) T2DM + PBS; 3) T2DM + naïve-HUCBC; 4) T2DM + miR-126−/−HUCBC. Functional evaluation, vascular and white-matter changes, neuroinflammation, and miR-126 effects were measured in vivo and in vitro. T2DM mice exhibited significantly decreased serum and brain tissue miR-126 expression compared with non-DM mice. T2DM+HUCBC mice exhibited increased miR-126 expression, increased tight junction protein expression, axon/myelin, vascular density and M2-macrophage polarization; However, decreased blood-brain barrier leakage, brain hemorrhage and miR-126 targeted gene VCAM-1 and MCP-1 expression in the ischemic brain as well as improved functional outcome were present in HUCBC treated T2DM mice compare with control T2DM mice. MiR-126−/−HUCBC-treatment abolished the benefits of naïve-HUCBC-treatment in T2DM stroke mice. In vitro, knock-in of miR-126 in primary cultured brain endothelial cells (BECs) or treatment of BECs with naïve-HUCBCs significantly increased capillary-like tube formation, and increased axonal outgrowth in primary cultured cortical neurons; whereas treatment of BECs or cortical neurons with miR-126−/− HUCBC attenuated HUCBC-treatment induced capillary tube formation and axonal outgrowth. Our data suggest delayed HUCBC-treatment of stroke increases vascular/white-matter remodeling and anti-inflammatory effects; MiR-126 may contribute to HUCBC-induced neurorestorative effects in T2DM mice.

Background and Purpose Co-morbidity of diabetes mellitus and stroke results in worse functional outcome, poor long term recovery and extensive vascular damage. We investigated the neurorestorative effects and mechanisms of stroke treatment with human bone marrow derived mesenchymal stromal cells (hMSCs) in type two diabetes mellitus (T2DM) rats. Methods Adult male Wistar rats were induced with T2DM, subjected to 2 hours of middle cerebral artery occlusion (MCAo) and treated via tail-vein injection with: 1) PBS (n=8); 2) hMSCs (n=10, 5×106) at 3 days after MCAo. Results In T2DM rats, hMSCs administered at 3 days after MCAo significantly improves neurological function without affecting blood glucose, infarct volume and incidence of brain hemorrhage in comparison to T2DM-MCAo PBS treated rats. Delayed hMSC treatment of T2DM stroke significantly improves blood brain barrier integrity, increases vascular and arterial density and cerebral vascular perfusion, and promotes neuroblast cell migration and white matter remodeling as indicated by increased doublecortin, axon, myelin and neurofilament density, respectively. Delayed hMSC treatment significantly increases platelet-derived growth factor (PDGF) expression in the ischemic brain, decreases pro-inflammatory M1 macrophage and increases anti-inflammatory M2 macrophage compared to PBS treated T2DM-MCAo rats. In vitro data show that hMSCs increase sub-ventricular zone explant cell migration and primary cortical neuron neurite outgrowth while inhibition of PDGF decreases hMSCs induced SVZ cell migration and axonal outgrowth. Conclusion In T2DM stroke rats, delayed hMSC treatment significantly improves neurological functional outcome, and increases neurorestorative effects and M2 macrophage polarization. Increasing brain PDGF expression may contribute to hMSC induced neurorestoration.