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- Lysosomal enzymes may cross the blood-brain-barrier by pinocytosis: Implications for Enzyme Replacement Therapy. [Journal Article]
- Med Hypotheses 2014 Apr; 82(4):478-80.
Here we hypothesized that the water-soluble lysosomal enzymes may cross the blood-brain-barrier and reach the brain using the mechanism of unspecific fluid-phase endocytosis. We also highlight studies that show that, at higher serum concentrations, a fraction of these proteins can reach the brain after intravenous injection, and we suggest some experiments to study this hypothesis. Finally we discuss the implications of this for treatments such as enzyme replacement of lysosomal storage disorders.
- Role of caveolin-1 in the biology of the blood-brain barrier. [JOURNAL ARTICLE]
- Rev Neurosci 2014 Feb 6.
Abstract Caveolin-1 is the principal marker of caveolae in endothelial cells. It plays an important role in physiological and pathological conditions of the blood-brain barrier and serves as a mediator in drug delivery through the blood-brain barrier. Caveolin-1 is related to the diminished expression of tight junction-associated proteins and metabolic pinocytosis vesicles when the blood-brain barrier is destroyed by outside invaders or malignant stimulus. The permeability of the blood-brain barrier, regulated by types of drugs or physical irradiation, is connected with drug transportation with the participation of caveolin-1. Caveolin-1, which serves as a platform or medium for signal transduction, cooperates with several signal molecules by forming a complex. Silencing of caveolin-1 and disruption of caveolae can attenuate or remove pathological damage and even engender the opposite effects in the blood-brain barrier. This review considers the role of caveolin-1 in the blood-brain barrier that may have profound implications for central nervous system disease and drug delivery through the blood-brain barrier.
- The effect of the neonatal Fc receptor on human IgG biodistribution in mice. [Journal Article]
- MAbs 2014 Mar 1; 6(2):502-8.
The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG. Recombinant therapeutic antibodies are typically composed of human or humanized sequences, and their biodistribution, or tissue distribution, is often studied in murine models, although, the effect of FcRn on tissue distribution of human IgG in rodents has not been investigated. In this report, an (125)I-labeled human IgG1 antibody was studied in both wild type C57BL/6 (WT) and FcRn knockout (KO) mice. Total radioactivity in both plasma and tissues (0-96hr post-dose) was measured by gamma-counting. Plasma exposure of human IgG1 were significantly lower in FcRn KO mice, which is consistent with the primary function of FcRn. Differences in biodistribution of human IgG to selected tissues were also observed. Among the tissue examined, the fat, skin and muscle showed a decrease in tissue-to-blood (T/B) exposure ratio of human IgG1 in FcRn KO mice comparing to the WT mice, while the liver, spleen, kidney, and lung showed an increase in the T/B exposure ratio in FcRn KO mice. A time-dependent change in the T/B ratios of human IgG1 was also observed for many tissues in FcRn KO mice. These results suggest that, in addition to its role in IgG elimination, FcRn may also play a role in antibody biodistribution.
- Low dose naltrexone (LDN) enhances maturation of bone marrow dendritic cells (BMDCs). [Journal Article, Research Support, Non-U.S. Gov't]
- Int Immunopharmacol 2013 Dec; 17(4):1084-9.
It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrexone, a non-peptidic δ-opioid receptor selective antagonist and opioid receptors on BMDCs have been detected . However, there is little prior data published on naltrexone and DCs. Therefore, we hypothesized that LDN could exert modulating effect on BMDCs. In present study, we studied influence of LDN on both phenotypic and functional maturation of BMDCs. Changes of BMDC post-treatment with LDN were evaluated using conventional light microscope and transmission electron microscopy (TEM); flow cytometry(FCM); cytochemistry; acid phosphatase activity(ACP) test; FITC-dextran bio-assay; mixed lymphocytes and enzyme-linked immunosorbent assay (ELISA). We have found that LDN enhances maturation of BMDCs as evidenced by 1) up-regulating the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs; 2) down-regulating the rates of pinocytosis and phagocytosis accompanied by the results of decreased ACP, and FITC-dextran bio-assay; 3) mounting potential of BMDCs to drive T cell; and 4) inducing secretion of higher levels of IL-12 and TNF-α. It is therefore concluded that LDN can efficiently promote the maturation of BMDCs via precise modulation inside and outside BMDCs. Our study has provided meaningful mode of action on the role of LDN in immunoregulation, and rationale on future application of LDN for enhancing host immunity in cancer therapy and potent use in the design of DC-based vaccines for a number of diseases.
- Gd loading by hypotonic swelling: an efficient and safe route for cellular labeling. [Journal Article, Research Support, Non-U.S. Gov't]
- Contrast Media Mol Imaging 2013 Nov-Dec; 8(6):475-86.
Cells incubated in hypo-osmotic media swell and their membranes become leaky. The flow of water that enters the cells results in the net transport of molecules present in the incubation medium directly into the cell cytoplasm. This phenomenon has been exploited to label cells with MRI Gd-containing contrast agents. It has been found that, in the presence of 100 mM Gd-HPDO3A in an incubation medium characterized by an overall osmolarity of 160 mOsm l⁻¹, each cell is loaded with amounts of paramagnetic complex ranging from 2 × 10⁹ to 2 × 10¹⁰ depending on the cell type. To obtain more insight into the determinants of cellular labeling by the 'hypo-osmotic shock' methodology, a study on cell viability, proliferation rate and cell morphology was carried out on J774A.1 and K562 cells as representative of cells grown in adhesion and suspended ones, respectively. Moreover a comparison of the efficiency of the proposed method with established cell labeling procedures such as pinocytosis and electroporation was carried out. Finally, the effects of the residual electric charge, the size and some structural features of the metal complex were investigated. In summary, the 'hypotonic shock' methodology appears to be an efficient and promising tool to pursue cellular labeling with paramagnetic complexes. Its implementation is straightforward and one may foresee that it will be largely applied in in vitro cellular labeling of many cell types.
- Influence of nanoparticle shape, size, and surface functionalization on cellular uptake. [Journal Article, Research Support, Non-U.S. Gov't, Review]
- J Nanosci Nanotechnol 2013 Oct; 13(10):6485-98.
With the rapid development of biotechnology and nanomedicine, extensive research has focused on the investigations of delivering large-cargo molecules using nanoparticles through the cell membrane for disease diagnosis and treatment. Various inorganic and polymeric nanoparticles with optimized surface properties have been developed to carry these active cargo molecules such as organic molecules, oligonucleotides and proteins. Phagocytosis and pinocytosis have been suggested as the two major uptake mechanisms for nanoparticles to enter into cellular interior, but such mechanisms are still under debate. In order to enhance the efficiency of cellular uptake of nanoparticles and further understand the physiological process, it is important to investigate detailed interaction mechanisms between nanoparticles and cell membranes. Here, we will review the recent advances of the effect of nanoparticle properties (e.g., nanoparticle shape, size, charge, surface modification, etc.) on cellular uptake mechanisms. These will aid in the future design and development of nanoparticles with improved surface properties for drug and biomolecule delivery. Up to now, novel analytical techniques have been used to examine nanoparticle-cell membrane interactions, but their detailed uptake mechanisms and pathways still need more in-depth research. It is suggested that developing appropriate analytical techniques to study cellular uptake mechanisms of nanoparticles in real time is urgently desired.
- Mammalian cells preferentially internalize hydrogel nanodiscs over nanorods and use shape-specific uptake mechanisms. [Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.]
- Proc Natl Acad Sci U S A 2013 Oct 22; 110(43):17247-52.
Size, surface charge, and material compositions are known to influence cell uptake of nanoparticles. However, the effect of particle geometry, i.e., the interplay between nanoscale shape and size, is less understood. Here we show that when shape is decoupled from volume, charge, and material composition, under typical in vitro conditions, mammalian epithelial and immune cells preferentially internalize disc-shaped, negatively charged hydrophilic nanoparticles of high aspect ratios compared with nanorods and lower aspect-ratio nanodiscs. Endothelial cells also prefer nanodiscs, however those of intermediate aspect ratio. Interestingly, unlike nanospheres, larger-sized hydrogel nanodiscs and nanorods are internalized more efficiently than their smallest counterparts. Kinetics, efficiency, and mechanisms of uptake are all shape-dependent and cell type-specific. Although macropinocytosis is used by both epithelial and endothelial cells, epithelial cells uniquely internalize these nanoparticles using the caveolae-mediated pathway. Human umbilical vein endothelial cells, on the other hand, use clathrin-mediated uptake for all shapes and show significantly higher uptake efficiency compared with epithelial cells. Using results from both upright and inverted cultures, we propose that nanoparticle internalization is a complex manifestation of three shape- and size-dependent parameters: particle surface-to-cell membrane contact area, i.e., particle-cell adhesion, strain energy for membrane deformation, and sedimentation or local particle concentration at the cell membrane. These studies provide a fundamental understanding on how nanoparticle uptake in different mammalian cells is influenced by the nanoscale geometry and is critical for designing improved nanocarriers and predicting nanomaterial toxicity.
- Activation of virus uptake through induction of macropinocytosis with a novel polymerizing peptide. [Journal Article, Research Support, N.I.H., Extramural, Research Support, N.I.H., Intramural]
- FASEB J 2014 Jan; 28(1):106-16.
A 27-aa peptide (P27) was previously shown to decrease the accumulation of human immunodeficiency virus type 1 (HIV-1) in the supernatant of chronically infected cells; however, the mechanism was not understood. Here, we show that P27 prevents virus accumulation by inducing macropinocytosis (MPC). Treatment of HIV-1- and human T-cell lymphotropic virus type 1 (HTLV-1)-infected cells with 2-10 μM P27 caused cell membrane ruffling and uptake of virus and polymerized forms of the peptide into large vacuoles. As demonstrated by electron microscopy, activation of MPC did not require virus or cells infected with virus, as P27 initiated its own uptake in the absence of virus. Inhibitors of MPC, Cytochalasin D and amiloride, decreased P27-mediated uptake of soluble dextran and inhibited P27-induced virus uptake by >60%, which provides further evidence that P27 induces MPC. In CD4(+) HeLa cells, HIV-1 infection was enhanced by P27 up to 4-fold, and P27 increased infection at concentrations as low as 20 nM. The 5-aa C-terminal domain of P27 was necessary for virus uptake and may be responsible for the polymerization of P27 into fibrils. These forms of P27 may play a key role in triggering MPC, making this peptide a useful tool for studying virus uptake and infection, as well as MPC of other macromolecules.
- Maturation inside and outside bone marrow dendritic cells (BMDCs) modulated by interferon-α (IFN-α). [Journal Article, Research Support, Non-U.S. Gov't]
- Int Immunopharmacol 2013 Nov; 17(3):843-9.
Interferons are made by cells in response to appropriate stimuli such as viruses, bacteria, parasites or tumor cells and are released into the surrounding medium. They then bind to receptors on target cells to allow for communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors. IFN-α is produced by leukocytes and is mainly involved in innate immune response against viral or bacterial infections and for tumor control. The aim of this work is to explore the detailed modulation of IFN-α on phenotypic and functional maturation inside and outside murine bone marrow derived dendritic cells (BMDCs). The maturity of BMDCs post treatment with IFN-α was evaluated with conventional light microscope and transmission electron microscopy (TEM) for morphology changes; flow cytometry (FCM) for changes of surface molecules on BMDCs; cytochemistry, acid phosphatase activity (ACP) test, and FITC-dextran bio-assay for biochemistry analysis and enzyme-linked immunosorbent assay (ELISA) for cytokine production by BMDCs. We have shown that IFN-α 1) up-regulates the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs; 2) down-regulates the rates of pinocytosis and phagocytosis by BMDCs as evidenced by the results of decreased ACP, and FITC-dextran bio-assay; 3) enhances the ability of BMDCs to drive T cell function; and 4) induces higher levels of IL-12 and TNF-α secreted by BMDCs. Therefore, we conclude that IFN-α can efficiently promote the maturation of BMDCs through detailed modulation inside and outside BMDCs. Our study has provided more detailed data on changes of BMDCs modulated by IFN-α, and rationale on future application of IFN-α for enhancing host immunity and potent adjuvant administration in the design of DC-based vaccines.
- P2Y4 receptor-mediated pinocytosis contributes to amyloid beta-induced self-uptake by microglia. [Journal Article, Research Support, Non-U.S. Gov't]
- Mol Cell Biol 2013 Nov; 33(21):4282-93.
Brain disturbances, like injuries or aberrant protein deposits, evoke nucleotide release or leakage from cells, leading to microglial chemotaxis and ingestion. Recent studies have identified P2Y12 purinergic receptors as triggers for microglial chemotaxis and P2Y6 receptors as mediators for phagocytosis. However, pinocytosis, known as the internalization of fluid-phase materials, has received much less attention. We found that ATP efficiently triggered pinocytosis in microglia. Pharmacological analysis and knockdown experiments demonstrated the involvement of P2Y4 receptors and the phosphatidylinositol 3-kinase/Akt cascade in the nucleotide-induced pinocytosis. Further evidence indicated that soluble amyloid beta peptide 1-42 induced self-uptake in microglia through pinocytosis, a process involving activation of P2Y4 receptors by autocrine ATP signaling. Our results demonstrate a previously unknown function of ATP as a "drink me" signal for microglia and P2Y4 receptors as a potential therapeutic target for the treatment of Alzheimer's disease.