scholarly journals 174 The effect of a microbial Muramidase on peptidoglycan content in the gut of swine using in-vitro and in-vivo measures

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 79-80
Author(s):  
Ursula M McCormack ◽  
Mikkel Klausen ◽  
Lisa A Laprade ◽  
Sonja Christian ◽  
Carsten Østergaard Frederiksen ◽  
...  
Keyword(s):  
Mixed Models ◽  
Fluorescein Isothiocyanate ◽  
Catalytic Performance ◽  
Intestinal Cells ◽  
Cell Wall Component ◽  
Structural Cell ◽  
Bacterial Division ◽  
Lactating Sows

Abstract Bacterial debris in the gastrointestinal tract (GIT) are continuously being produced by the microbiota present upon bacterial division and death. One of the most abundant components in bacterial debris are peptidoglycans (PGN), a structural cell wall component in gram- positive and negative bacteria. The objective of this work was to investigate if addition of a novel microbial muramidase (Muramidase 007; MUR) that hydrolyzes PGN, would reduce PGN adhesion to porcine intestinal cells in-vitro and hydrolyze PGN in the GIT of swine. Adhesion efficacy of intact and MUR hydrolyzed fluorescein-isothiocyanate (FITC)-labelled PGN were compared using fluorescence-microscopy (3 wells/condition). Catalytic performance of MUR on intact FITC-labelled PGN adhered to intestinal cells were also tested. In-vivo MUR-supplementation at 50,000 LSU/kg diet to gestating and lactating sows and/or their subsequent offspring for 42-d post-weaning was investigated. Mass-spectroscopy was used to quantify soluble and total muramic acid, which is only found in PGN, in the ileal and cecal digesta (8 piglets/treatment) to calculate percentage soluble-PGN. Cell-culture data were analyzed using GraphPad-Prism 8.0 and in-vivo data using mixed-models in JMP 14.0. MUR hydrolyzed PGN adhered 10x less to the IPEC-J2 cell line culture compared to intact-PGN (P< 0.05). In addition, data show that MUR hydrolyzed PGN attached to cell surfaces by 2x, as attached PGN were also reduced by 50% following MUR incubation (P< 0.05). Offering sows MUR-diets had no carryover effect on the percentage soluble PGN in their piglets’ digesta, and there were no interactions observed for sow x piglet x tract neither (P >0.05). Percentage soluble-PGN increased in piglets fed MUR compared to control-piglets (47.18 vs. 29.84% SEM:1.624; P< 0.001) and was higher in cecal digesta compared to ileal digesta (49.91 vs. 27.11%; SEM:1.634; P< 0.001), irrespective of MUR-supplementation. These results suggest that MUR may reduce PGN adhesion to intestinal cells and may hydrolyze PGN found in the GIT.

scholarly journals Inhibition of EZH2 by chidamide exerts antileukemia activity and increases chemosensitivity through Smo/Gli-1 pathway in acute myeloid leukemia

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Xuejie Jiang ◽  
Ling Jiang ◽  
Jiaying Cheng ◽  
Fang Chen ◽  
Jinle Ni ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Histone Deacetylases ◽  
Myeloid Leukemia ◽  
Hdac Inhibitors ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Gli 1 ◽  
Acute Myeloid

Abstract Background Epigenetic dysregulation plays important roles in leukemogenesis and the progression of acute myeloid leukemia (AML). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) reciprocally regulate the acetylation and deacetylation of nuclear histones. Aberrant activation of HDACs results in uncontrolled proliferation and blockade of differentiation, and HDAC inhibition has been investigated as epigenetic therapeutic strategy against AML. Methods Cell growth was assessed with CCK-8 assay, and apoptosis was evaluated by flow cytometry in AML cell lines and CD45 + and CD34 + CD38- cells from patient samples after staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). EZH2 was silenced with short hairpin RNA (shRNA) or overexpressed by lentiviral transfection. Changes in signaling pathways were detected by western blotting. The effect of chidamide or EZH2-specific shRNA (shEZH2) in combination with adriamycin was studied in vivo in leukemia-bearing nude mouse models. Results In this study, we investigated the antileukemia effects of HDAC inhibitor chidamide and its combinatorial activity with cytotoxic agent adriamycin in AML cells. We demonstrated that chidamide suppressed the levels of EZH2, H3K27me3 and DNMT3A, exerted potential antileukemia activity and increased the sensitivity to adriamycin through disruption of Smo/Gli-1 pathway and downstream signaling target p-AKT in AML cells and stem/progenitor cells. In addition to decreasing the levels of H3K27me3 and DNMT3A, inhibition of EZH2 either pharmacologically by chidamide or genetically by shEZH2 suppressed the activity of Smo/Gli-1 pathway and increased the antileukemia activity of adriamycin against AML in vitro and in vivo. Conclusions Inhibition of EZH2 by chidamide has antileukemia activity and increases the chemosensitivity to adriamycin through Smo/Gli-1 pathway in AML cells (Fig. 5). These findings support the rational combination of HDAC inhibitors and chemotherapy for the treatment of AML.

scholarly journals Isolation and Purification of Lipoarabinomannan from Urine of Adults with Active Tuberculosis

2021 ◽  
Author(s):  
Jason L. Cantera ◽  
Andrew A. Rashid ◽  
Lorraine L. Lillis ◽  
Roger B. Peck ◽  
Paul K. Drain ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Chemical Processes ◽  
Active Tuberculosis ◽  
Cell Wall Component ◽  
Isolation And Purification ◽  
Specific Antibodies ◽  
A Cell ◽  
Urine Specimens

AbstractLipoarabinomannan (LAM) is a cell wall component of Mycobacterium tuberculosis that is excreted in the urine of persons with active tuberculosis (TB). Limited diagnostic sensitivity of LAM immunoassays has been due to selecting antibodies against LAM derived from in vitro cultured M. tuberculosis, rather than LAM purified from in vivo clinical urine specimens. Urinary LAM (uLAM) is critical to enable the development of and/or screening of novel uLAM-specific antibodies but is typically dilute and in heterogeneous mixtures with other urine components. We used physical, enzymatic, and chemical processes for the scaled isolation and purification of uLAM. The purified material may then be used to develop more sensitive uLAM diagnostic tests for active TB disease.

Accutin, a New Disintegrin, Inhibits Angiogenesis In Vitro and In Vivo by Acting as Integrin vβ3 Antagonist and Inducing Apoptosis

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
Chia Hsin Yeh ◽  
Hui-Chin Peng ◽  
Tur-Fu Huang
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Fluorescein Isothiocyanate ◽  
Adenosine Diphosphate ◽  
Tube Formation ◽  
Platelet Glycoprotein ◽  
Dependent Manner ◽  
Antiangiogenic Effect

Abstract Endothelial integrins play an essential role in angiogenesis and cell survival. Accutin, a new member of disintegrin family derived from venom of Agkistrodon acutus, potently inhibited human platelet aggregation caused by various agonists (eg, thrombin, collagen, and, adenosine diphosphate [ADP]) through the blockade of fibrinogen binding to platelet glycoprotein IIb/IIIa (ie, integrin IIbβ3). In this report, we describe that accutin specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin vβ3, to human umbilical vein endothelial cells (HUVECs), but not those of other anti-integrin MoAbs such as 2β1, 3β1, and 5β1. Moreover, accutin, but not the control peptide GRGES, dose-dependently inhibited the 7E3 interaction with HUVECs. Both 7E3 and GRGDS, but not GRGES or Integrelin, significantly blocked fluorescein isothiocyanate-conjugated accutin binding to HUVEC. In functional studies, accutin exhibited inhibitory effects on HUVEC adhesion to immobilized fibrinogen, fibronectin and vitronectin, and the capillary-like tube formation on Matrigel in a dose- and RGD-dependent manner. In addition, it exhibited an effective antiangiogenic effect in vivo when assayed by using the 10-day-old embryo chick CAM model. Furthermore, it potently induced HUVEC apoptotic DNA fragmentation as examined by electrophoretic and flow cytometric assays. In conclusion, accutin inhibits angiogenesis in vivo and in vitro by blocking integrin vβ3 of endothelial cells and by inducing apoptosis. The antiangiogenic activity of disintegrins might be explored as the target of developing the potential antimetastatic agents. © 1998 by The American Society of Hematology.

Pentoxifylline inhibits FMLP-induced macromolecular leakage

1995 ◽  
Vol 269 (1) ◽  
pp. H239-H245
Author(s):  
K. Nakagawa ◽  
F. N. Miller ◽  
A. W. Knott ◽  
M. J. Edwards
Keyword(s):  
Inflammatory Responses ◽  
Fluorescein Isothiocyanate ◽  
Histamine Receptor ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Cyclic Monophosphate ◽  
Endogenous Histamine ◽  
Camp Analogue

The acute inflammatory responses to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and the effects of pentoxifylline (PTXF) on the responses in vivo were studied. We used intravital microscopy with rat cremaster muscle preparation to determine inflammatory responses of microcirculation. Macromolecular leakage from postcapillary venules was evaluated by quantifying the extravasation of fluorescein isothiocyanate conjugated to bovine serum albumin. FMLP induced a rapid increase in macromolecular leakage, an increase in leukocyte-endothelium adhesion, and a decrease in blood flow in the microcirculation. PTXF inhibited FMLP-induced responses in a dose-dependent manner but failed to block the histamine-dependent leakage induced by compound 48/80. In addition, diphenhydramine, a histamine-receptor blocker, did not affect the macromolecular leakage induced by FMLP. The cell-permeable adenosine 3',5'-cyclic monophosphate (cAMP) analogue N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate mimicked PTXF's effects on the microcirculation and also inhibited FMLP-induced macromolecular leakage. PTXF is known to inhibit phosphodiesterase and increase intracellular cAMP, which modulates functions of endothelial cells, smooth muscle cells, and neutrophils in vitro. Our findings suggest that FMLP induces acute inflammatory responses through activation of neutrophils, independent of endogenous histamine release, and that PTXF inhibits these responses through elevated intracellular cAMP.

scholarly journals Sickling-induced binding of immunoglobulin to sickle erythrocytes

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 636-639 ◽  
Author(s):  
GA Green ◽  
VK Kalra
Keyword(s):  
Flow Cytometry ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Low Density ◽  
Autologous Plasma ◽  
Sickle Cells ◽  
Sickle Erythrocytes

Abstract Previously we demonstrated that sickle erythrocytes sedimenting at high densities after gradient centrifugation contain higher levels of surface immunoglobulin bound in vivo in comparison to low-density erythrocytes from the same patient. The present study examines the possibility that binding of autologous IgG to sickle erythrocytes may be associated with the sickling phenomenon. In the present study we subjected low-density erythrocytes to prolonged sickling under nitrogen in the presence of platelet-poor autologous plasma with added glucose for 24 hours (37 degrees C). After reoxygenation IgG bound in vitro was quantified by a nonequilibrium 125iodinated protein A-binding assay and by flow cytometry. Results show that sickle erythrocytes incubated under nitrogen bound significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per cell (mean +/- SD) compared with sickle cells incubated under oxygenation (227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26 molecules IgG per cell for untreated sickle cells. In contrast, normal erythrocytes incubated in autologous plasma exhibited no detectable IgG binding in vitro under either oxygenation or deoxygenation. Flow cytometry shows that deoxygenation of sickle cells generated a two-to-sixfold increase in the subpopulation of brightly fluorescent IgG-positive cells in comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD) increase in median fluorescence intensity for fluorescein isothiocyanate-labeled deoxygenated sickled cells compared with labeled oxygenated sickle cells. Our studies demonstrate that prolonged sickling will induce in vitro binding of autologous IgG to sickle erythrocytes. These findings indicate that sickle erythrocytes may be unique when compared with erythrocytes from other nonimmunologic hemolytic anemias or senescent red cells in that the primary events producing surface antigens recognized by autoantibody may include the sickling process. These findings also suggest that sickling in vivo may generate membrane alterations in sickle erythrocytes that lead to cumulative binding of autoantibody in vivo.

scholarly journals Oral Delivery of Nucleic Acids with Passive and Active Targeting to the Intestinal Tissue Using Polymer-Based Nanocarriers

Pharmaceutics ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1075
Author(s):  
Sagun Poudel ◽  
Prabhat R. Napit ◽  
Karen P. Briski ◽  
George Mattheolabakis
Keyword(s):  
Nucleic Acids ◽  
Oral Delivery ◽  
Parent Compound ◽  
Genetic Material ◽  
Simulated Gastric Fluid ◽  
Intestinal Cells ◽  
Long Term Treatment ◽  
Fluid Environment

Despite the apparent advantages for long-term treatment and local therapies against intestinal diseases, the oral delivery of nucleic acids has been challenging due to unfavorable physiological conditions for their stability. In this study, a novel nanodelivery system of PEG-PCL nanoparticles with encapsulated nucleic acids–mannosylated PEI (Man-PEI) complexes was developed for intestinal delivery. We complexed model nucleic acids with Man-PEI at the optimal N/P ratio of 20:1 for in vitro and in vivo analyses. Cells were transfected in vitro and analyzed for gene expression, receptor-mediated uptake, and PEG-PCL nanoparticles’ toxicity. We also evaluated the nucleic acid’s stability in the nanocarrier during formulation, and under simulated gastrointestinal environments or the presence of nucleases. Finally, we assessed the biodistribution for the PEG-PCL nanoparticles with encapsulated complexes and their ability to transfect intestinal cells in vivo. Nucleic acids complexed with Man-PEI were protected from degradation against nucleases. In comparison to the parent compound PEI, Man-PEI transfected the cells with an overall higher potency. Competition assay indicated receptor-mediated endocytosis promoted by mannose receptors. The PEG-PCL nanoparticles with Man-PEI/plasmid complexes indicated minimal cytotoxicity. The nanocarrier successfully protected the complexes in a simulated gastric fluid environment and released them in a simulated intestinal fluid environment, promoted by the presence of lipases. The oral administration of the PEG-PCL nanoparticles with encapsulated Man-PEI/plasmid complexes transfected intestinal cells with the plasmid in vivo, while presenting a time-dependent progression through the intestines. Conclusively, our carrier system can deliver genetic material to the GI tract and actively target mannose receptor overexpressing cells.

Leukotriene B4-induced neutrophil-mediated endothelial leakage in vitro and in vivo

1991 ◽  
Vol 71 (4) ◽  
pp. 1322-1330 ◽  
Author(s):  
S. Rosengren ◽  
A. M. Olofsson ◽  
U. H. von Andrian ◽  
E. Lundgren-Akerlund ◽  
K. E. Arfors
Keyword(s):  
Umbilical Vein ◽  
Neutrophil Migration ◽  
Fluorescein Isothiocyanate ◽  
Leukotriene B4 ◽  
Vascular Leakage ◽  
Neutrophil Granulocytes ◽  
Vitro Assay ◽  
Umbilical Vein Endothelial Cells

The vascular leakage of macromolecules seen in several models after application of leukotriene B4 (LTB4) is mediated by neutrophil granulocytes. We describe here an in vitro assay for this event. Human umbilical vein endothelial cells were grown on polycarbonate filters separating luminal and abluminal compartments of fluid. Both clearance rate of fluorescein isothiocyanate albumin and neutrophil migration through the endothelial monolayer were increased when LTB4 (10–100 nM) was added to the abluminal compartment. However, if LTB4 was instead added to the luminal compartments together with the neutrophils, no migration or change in clearance could be detected. These findings were confirmed in vivo in the cheek pouches of anesthetized hamsters, where extravascular application of LTB4 induced intravascular adhesion of neutrophils, accompanied by neutrophil-dependent vascular leakage. On the other hand, intravascular deposition of LTB4 with micropipettes induced adhesion of leukocytes but no leakage. In conclusion, the presence of neutrophils adhering to endothelium does not necessarily imply the development of neutrophil-mediated vascular leakage. Instead, the leakage appears connected to the process of neutrophil chemotaxis.

scholarly journals Developing Body-Components-Based Theranostic Nanoparticles for Targeting Ovarian Cancer

Pharmaceutics ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 216 ◽  
Author(s):  
Ravit Edelman ◽  
Yehuda G. Assaraf ◽  
Anton Slavkin ◽  
Tamar Dolev ◽  
Tal Shahar ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Fluorescein Isothiocyanate ◽  
Ovarian Cancer Cells ◽  
Long Wavelength ◽  
Self Assembling ◽  
Theranostic Nanoparticles ◽  
Body Components

Ovarian cancer mortality is the highest among gynecologic malignancies. Hence, the major challenges are early diagnosis and efficient targeted therapy. Herein, we devised model theranostic nanoparticles (NPs) for combined diagnostics and delivery of chemotherapeutics, targeted to ovarian cancer cells. These NPs were made of natural biocompatible and biodegradable body components: hyaluronic acid (HA) and serum albumin (SA). The hydrophilic HA served as the targeting ligand for cancer cells overexpressing CD44, the HA receptor. SA, the natural carrier of various ligands through the blood, served as the hydrophobic block of the self-assembling block copolymeric Maillard-conjugates. We show the successful construction of fluorescently-labeled SA-HA conjugate-based theranostic NPs, their loading with paclitaxel (PTX) (association constant (8.6 ± 0.8) × 103 M−1, maximal loading capacity of 4:1 PTX:BSA, and 96% encapsulation efficiency), selective internalization and cytotoxicity to CD44-overexpressing ovarian cancer cells (IC50: 26.4 ± 2.3 nM, compared to 115.0 ± 17.4 of free PTX, and to 58.6 ± 19.7 nM for CD44-lacking cognate ovarian cancer cells). Fluorescein isothiocyanate (FITC) was used for in vitro imaging, whereas long wavelength fluorophores or other suitable tracers would be used for future in vivo diagnostic imaging. Collectively, our findings demonstrate that fluorescent HA-SA NPs harboring a cytotoxic drug cargo can specifically target, label CD44-expressing ovarian cancer cells and efficiently eradicate them.

scholarly journals FtsZ filament capping by MciZ, a developmental regulator of bacterial division

2015 ◽  
Vol 112 (17) ◽  
pp. E2130-E2138 ◽  
Author(s):  
Alexandre W. Bisson-Filho ◽  
Karen F. Discola ◽  
Patrícia Castellen ◽  
Valdir Blasios ◽  
Alexandre Martins ◽  
...  
Keyword(s):  
Cell Division ◽  
Regulatory Proteins ◽  
In Vitro Assays ◽  
Temperature Sensitive ◽  
X Ray ◽  
Bacterial Division ◽  
Bacterial Homolog ◽  
Cell Division Inhibitor

Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.

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