- The microvascular alterations in frontal cortex during treatment with antipsychotics: a post-mortem study. [Journal Article]
- Rom J Morphol Embryol 2016; 57(2):501-6.
Schizophrenia is the most severe psychiatric illness, with a biological support in the brain. There is evidence that the adequate dopamine balance in the frontal cortex is associated with a better outcome of the disorder, while the alteration of dopamine mechanism at this level may affect the vascular system leading to secondary neuronal alterations. Our study was conducted post-mortem and its objective was to identify the alterations in the neuronal architecture, in the integrity of the microvascular unit in the frontal cortex of patients treated with potent and excessive D2-blocking antipsychotics.We studied post-mortem sections of the frontal cortex of three patients (two women and one man) diagnosed with schizophrenia or schizophrenia-spectrum disorders and treated with antipsychotics for the last 24 months. The slides were prepared according to the classical histopathological protocols.Various alterations were found at the neural and vascular levels in the frontal cortex. The most significant was the neural loss as the result of severe changes in the microvessels (diameter reduction, hyaline and collagen deposits, edema, pinocytosis and vacuolization).The evidences shown in our study highlight the fact that antipsychotics with potent antagonist action on D2 receptors may affect the neurovascular unit and small vessels in frontal cortex by altering the balance vasoconstriction-vasodilatation, thus reducing the blood flow and metabolism and generating structural microvascular changes proportional with the level of apoptosis at this level. The functional integrity of the dopaminergic system in frontal cortex depends on the vascular support and the capabilities of the neurovascular unit and any dysfunction increases the neuronal loss with clinically significant changes.The pathological data of our study raises the hypothesis for the pathogenic stages at the level of microvessels in the frontal cortex of the patients with schizophrenia or schizophrenia-spectrum disorders treated with D2-blocking antipsychotics: a stage with functional, reversible alterations that may be correlated with the impairments of working memory and presence of extrapyramidal symptoms and a lesional, irreversible stage with significant deterioration of cognition and global functioning. Further studies are needed to verify this hypothesis.
- Nox2-mediated PI3K and Cofilin Activation Confers Alternate Redox Control of Macrophage Pinocytosis. [JOURNAL ARTICLE]
- Antioxid Redox Signal 2016 Aug 3.
Internalization of extracellular fluid and its solute by macropinocytosis requires dynamic reorganization of actin cytoskeleton, membrane ruffling, and formation of large endocytic vacuolar compartments, called macropinosomes, inside the cell. Although instigators of macropinocytosis, such as growth factors and phorbol esters, stimulate NADPH oxidase (Nox) activation and signal transduction mediators upstream of Nox assembly, including Rac1 and protein kinase c (PKC), are involved in macropinocytosis, the role of Nox enzymes in macropinocytosis has never been investigated. This study was designed to examine the role of Nox2 and the potential downstream redox signaling involved in macropinocytosis.Phorbol myristate acetate activation of human and murine macrophages stimulated membrane ruffling, macropinosome formation, and subsequent uptake of macromolecules by macropinocytosis. Mechanistically, we found that pharmacological blockade of PKC, transcriptional knockdown of Nox2, and scavenging intracellular superoxide anion abolished phorbol ester-induced macropinocytosis. We observed that Nox2-derived reactive oxygen species via inhibition of phosphatase and tensin homolog and activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway, lead to activation of actin-binding protein cofilin, membrane ruffling, and macropinocytosis. Similarly, activation of macropinocytosis by M-CSF involves Nox2-mediated cofilin activation. Furthermore, peritoneal chimera experiments indicate that macropinocytotic uptake of lipids in hypercholesterolemic ApoE-/- mice was attenuated in Nox2y/- macrophages compared to wild type controls.In summary, these findings demonstrate a novel Nox2-mediated mechanism of solute uptake via macropinocytosis with broad implications for both general cellular physiology and pathological processes. The redox mechanism described herein may also identify new targets in atherosclerosis and other disease conditions involving macropinocytosis.
- Poly (l-γ-glutamylglutamine) Polymer Enhances Doxorubicin Accumulation in Multidrug Resistant Breast Cancer Cells. [Journal Article]
- Molecules 2016; 21(6)
Drug resistance is one of the bottlenecks of cancer chemotherapy in the clinic. Polymeric nanomedicine is one of the most promising strategies for overcoming poor chemotherapy responses due to the multidrug resistance (MDR).In this study, a new polymer-based drug delivery system, poly (l-γ-glutamylglutamine)-doxorubicin (PGG-Dox) conjugate, was studied in both drug-induced resistant human breast cancer MDA-MB-231/MDR cells and their parent human breast cancer MDA-MB-231 cells. The effect of PGG on facilitating the growth inhibition of Dox against multidrug resistant cells were investigated by evaluating the cytotoxicity of PGG-Dox conjugate, PGG/Dox unconjugated complex and free Dox on both cells. The underlying mechanisms in resistant cells were further studied via the intracellular traffic studies.Both conjugated and unconjugated PGG significantly increased Dox uptake, prolonged Dox retention and reduced Dox efflux in the MDA-MB-231/MDR cells. The PGG-Dox conjugate is taken up by tumor cells mainly by pinocytosis pathway, in which PGG-Dox conjugate-containing vesicles are formed and enter the cells.This study indicated that both polymer-drug conjugate and unconjugated complex are promising strategies of overcoming resistance of anti-tumor drugs.
- Distinct Cellular Pathways for Induction of CD4+ T Cell-Dependent Antibody Responses to Antigen Expressed by Intact Bacteria Versus Isolated Soluble Antigen. [Journal Article]
- J Immunol 2016 May 15; 196(10):4204-13.
Uptake of intact bacteria and soluble Ags by APCs is mediated by phagocytosis and endocytosis or pinocytosis, respectively. Thus, we predicted that injection of clodronate-containing liposomes (CLs), which selectively deplete cells efficient in phagocytosis, would inhibit murine CD4(+) T cell-dependent IgG responses to Ags expressed by intact bacteria but not isolated soluble Ags. Surprisingly, injection of CLs markedly inhibited protein-specific IgG responses to intact, heat-killed Streptococcus pneumoniae, as well as a soluble OVA-polysaccharide conjugate or OVA alone. IgG anti-polysaccharide responses to bacteria and conjugate were also reduced, but more modestly. In both instances, CL-mediated inhibition was associated with a significant reduction in induced germinal centers and CD4(+) germinal center T follicular helper cells. However, CL injection, which largely abrogated the proliferative response of adoptively transferred OVA peptide-specific-transgenic CD4(+) T cells in response to immunization with S. pneumoniae expressing OVA peptide, did not inhibit T cell proliferation in response to OVA-polysaccharide conjugate or OVA. In this regard, monocyte-derived cells, depleted by CLs, internalized S. pneumoniae in vivo, whereas CD11c(low) dendritic cells, unaffected by CL injection, internalized soluble OVA. Ex vivo isolation and coculture of these respective APCs from S. pneumoniae- or OVA-immunized mice with OVA-specific T cells, in the absence of exogenous Ag, demonstrated their selective ability to induce T cell activation. These data suggest that, although distinct APCs initiate CD4(+) T cell activation in response to Ag expressed by intact bacteria versus Ag in soluble form, CL-sensitive cells appear to be necessary for the subsequent IgG responses to both forms of Ag.
- Lipoprotein X Causes Renal Disease in LCAT Deficiency. [Journal Article]
- PLoS One 2016; 11(2):e0150083.
Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.
- Experimental study of super paramagnetic iron oxide labeled synovial mesenchymal stem cells. [Journal Article]
- Int J Clin Exp Med 2015; 8(11):20309-15.
To investigate the feasibility and changes of biological characteristics before and after synovial mesenchymal stem cells (SMSCs) labelled by super paramagnetic iron oxide (SPIO). The rabbit SMSCs were isolated, cultured, purified and identified in vitro. After adding the different concentrations of SPIO-labelled liquid, the cells were incubated 24 h in 37°C carbon dioxide incubator. The labeled-cell samples were observed by Prussian blue staining, transmission electron microscope (TEM) and the cell biology before and after the labeling was compared. The blue stained particles could be seen in the cytoplasm; the SPIO label was positive in 95% SMSC cells. With the concentration of the label liquid increasing, the blue-stained cytoplasm became darker. A large number of high electron density particles could be seen in the cytoplasm and in the pinocytosis vesicles by TEM, which suggested SPIO label positive. When the SPIO concentration was (12.5~50) μg/mL, the differences in cell proliferation and cell viability between the SMSCs after labelling and the SMSCs before labelling were not significant; when the concentration was over 100 μg/mL, the cell proliferation and cell viability were inhibited. A certain concentration range of SPIO can safely label the rabbit SMSC according to this study, which is important for solving the problem of tracing SMSCs in the joints.
- D-Alanylation of Teichoic Acids and Loss of Poly-N-Acetyl Glucosamine in Staphylococcus aureus during Exponential Growth Phase Enhance IL-12 Production in Murine Dendritic Cells. [Journal Article, Research Support, Non-U.S. Gov't]
- PLoS One 2016; 11(2):e0149092.
Staphylococcus aureus is a major human pathogen that has evolved very efficient immune evading strategies leading to persistent colonization. During different stages of growth, S. aureus express various surface molecules, which may affect the immune stimulating properties, but very little is known about their role in immune stimulation and evasion. Depending on the growth phase, S. aureus may affect antigen presenting cells differently. Here, the impact of growth phases and the surface molecules lipoteichoic acid, peptidoglycan and poly-N-acetyl glucosamine on the induction of IL-12 imperative for an efficient clearance of S. aureus was studied in dendritic cells (DCs). Exponential phase (EP) S. aureus was superior to stationary phase (SP) bacteria in induction of IL-12, which required actin-mediated endocytosis and endosomal acidification. Moreover, addition of staphylococcal cell wall derived peptidoglycan to EP S. aureus stimulated cells increased bacterial uptake but abrogated IL-12 induction, while addition of lipoteichoic acid increased IL-12 production but had no effect on the bacterial uptake. Depletion of the capability to produce poly-N-acetyl glucosamine increased the IL-12 inducing activity of EP bacteria. Furthermore, the mutant dltA unable to produce D-alanylated teichoic acids failed to induce IL-12 but like peptidoglycan and the toll-like receptor (TLR) ligands LPS and Pam3CSK4 the mutant stimulated increased macropinocytosis. In conclusion, the IL-12 response by DCs against S. aureus is highly growth phase dependent, relies on cell wall D-alanylation, endocytosis and subsequent endosomal degradation, and is abrogated by receptor induced macropinocytosis.
- C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages. [Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.]
- Nanoscale 2016 Feb 21; 8(7):4134-44.
There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.
- Into rather unexplored terrain-transcellular transport across the blood-brain barrier. [Journal Article, Review]
- Glia 2016 Jul; 64(7):1097-123.
Efficient neuronal signaling in the central nervous system strictly depends on a well-balanced microenvironment around glial cells, synapses, and axons. Unique features of the blood-brain barrier (BBB) endothelium largely determine the composition of this micro-milieu and are dependent on the tight interplay with surrounding astrocytes and pericytes. BBB endothelial cells are endowed with a highly restrictive junctional complex that occludes the intercellular cleft, thereby preventing paracellular diffusion. The paracellular pathway is subject to extensive research as integrity loss of the junctional complex is associated with many neuropathologies, inflammation, and edema. Another important feature of the BBB endothelium is the low prevalence of nonspecific, transcytotic events, including (macro)pinocytosis, clathrin-dependent and caveolin-dependent endocytosis and the subsequent trafficking of vesicles to the opposite membrane. Although less studied, evidence is accruing that this pathway importantly contributes to increased BBB permeability, often when the junctional complex remains intact. Here, we review current knowledge on the contribution of the transcellular pathway to the BBB leak observed in different pathologic conditions. In addition, we hypothesize that nonselective, large pore connexin and pannexin channels may contribute to transcellular transport, either by providing a direct diffusion pathway across the endothelial monolayer, or indirectly, by exerting control over intracellular levels of the signaling ion Ca(2+) that is involved in many steps of the vesicular pathway. We conclude that transcytotic events at the BBB, despite being less acknowledged, cannot be simply dismissed as done in the past, but actively contribute to BBB leakage in many different pathologies. GLIA 2016;64:1097-1123.
- [THE DYNAMICS OF IMMUNOLOGICAL RESULTS OF PATIENTS WITH T-CELL SKIN LYMPHOMAS AND PSORIASIS BY THE THERAPY OF ACTIVATION MECHANISMS SANOGENESIS METHODS]. [English Abstract, Journal Article]
- Lik Sprava 2015 Apr-Jun; (3-4):31-8.
The therapy T-cell skin lymphoma and psoriasis by the application of activation mechanisms sanogenesis methods, such as: original--a treatment plasmapheresis, a standard heparin infusion; used for the first time--wobenzym; solutions of acid acetic food and sodium bicarbonate; known--the basic sanitations of concomitant diseases, photopheresis caused remissions in 79.6% patients with different stages T-cell skin lymphoma (observed over an 8-year span), and in 67% of patients with psoriasis (observed over an 6-year span). Depuration reactions (phagocytosis, pinocytosis, toxin neutralization) has been activated by detoxication of treatment plasmapheresis and heparin infusions. The topical therapy with wobenzym, solutions of acid acetic food and sodium bicarbonate renewed natural immune barrier of skin. Basic therapy of concomitant diseases enhanced of patient state of health and mobilized compensatory resources. Photopheresis initiated autoimmunization processes by malignant CD4+ lymphocytes. When remission was achieved, the parameters of cellular and humoral immunity returned to normal levels, or the parameters made worse in the absence of remission.