A Human Islet Cell-Culture System for High-Throuput screening.

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds

Roles of AP-2 in clathrin-mediated endocytosis.

The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway

Quantum Dots for Tracking Dendritic Cells and Priming an Immune Response In Vitro and In Vivo

Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here, we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs, and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal, two-photon, and electron microscopies to visualize QD uptake into DCs and compared CD69 expression, T cell proliferation, and IFN-γ production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c+ DCs avidly and preferentially endocytosed QDs, initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size, motion, and brightness distributed throughout the cytoplasm. At later times, endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot, DCs that had endocytosed QDs were visualized up to 400 µm deep within draining lymph nodes. When antigen-conjugated QDs were used, T cells formed stable clusters in contact with DCs. Antigen-conjugated QDs induced CD69 expression, T cell proliferation, and IFN-γ production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response

Calcium Homeostasis and Cone Signaling Are Regulated by Interactions between Calcium Stores and Plasma Membrane Ion Channels

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca2+ entry (SOCE) to Ca2+ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn2+ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca2+ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca2+ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca2+ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca2+ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca2+ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca2+ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca2+ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse

Single channel properties and regulated expression of Ca(2+) release-activated Ca(2+) (CRAC) channels in human T cells.

Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling

Intercellular communication via exosomes

Exosomes are small membrane bound vesicles between 30-100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. They play a role in intercellular communication by transferring proteins, lipids and RNA to recipient cells. The overall aim of this work has been to further investigate the mechanisms by which cells communicate with each other via exosomes. In Paper I we hypothesized that exosomes from human cells could be used as vectors to provide cells with therapeutic RNA. Herein, exogenous short interfering RNAs were successfully introduced into various kinds of human exosomes using electroporation. Flow cytometry, confocal microscopy and northern blot confirmed the presence of siRNA inside the exosomes. The results showed that exosomes from blood plasma could deliver the siRNA to human monocytes and lymphocytes. The siRNA delivered to the target cells was shown to be functional causing selective gene silencing of mitogen activated protein kinase 1. Our results imply that exosomes from human cells could be used as vectors for delivery of therapeutic exogenous nucleic acids to cells. In paper II we investigated if exosomes from activated CD3+ T cells could play a role in an immunological response by conveying signals from their secreting cells to recipient resting T cells in an in vitro autologous setting. The role of these exosomes was explored in IL-2 mediated T cell proliferation. The results showed that neither exosomes nor IL-2 alone could stimulate proliferation in resting T cells. However, exosomes from stimulated T cells together with IL-2 were able to induce proliferation. T cell cultures stimulated with exosomes and IL-2 showed a higher proportion of CD8+ T cells than cultures without exosomes. Moreover, a cytokine array showed significant changes in the levels of cytokines and chemokines when exosomes were present. The results indicate that activated CD3+ cells communicate with resting autologous T cells via exosomes. The main focus in paper III was to study the cellular mechanism by which esRNA is selectively packaged into exosome vesicles during their biosynthesis. Using RNA gel mobility shift assay, we showed the presence of RNA-binding proteins (RBPs) in exosomes. Moreover, we developed a method for the identification of exosomal RBPs able to bind to the esRNA and cellular microRNA. Using this method, we could identify 31 different RBPs in exosomes and 78 in cells. To evaluate the possible role of the identified RBPs in the transfer mechanism of RNA into intraluminal vesicles, five gene transcripts from the identified RBPs were silenced. The results revealed that a selective gene silencing of hnRNPA2B1 caused a reduction of RNA present in the extracellular vesicles. Thus, a novel transport mechanism was suggested for the packaging of esRNA into the exosomes. In conclusion, the studies presented in this thesis have implications for better understanding the RNA and protein transfer mechanism that occurs between cells via exosomes. The described ability of exosomes to deliver exogenous nucleic acids to cells may be of interest in clinical applications e.g. in gene therapy