scholarly journals Development of an advanced diagnostic concept for intestinal inflammation: molecular visualisation of nitric oxide in macrophages by functional poly(lactic-co-glycolic acid) microspheres

2017 ◽  
Vol 8 ◽  
pp. 1637-1641
Author(s):  
Kathleen Lange ◽  
Christian Lautenschläger ◽  
Maria Wallert ◽  
Stefan Lorkowski ◽  
Andreas Stallmach ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Laser Scanning ◽  
Glycolic Acid ◽  
Intestinal Inflammation ◽  
Laser Scanning Microscopy ◽  
Hydrodynamic Diameter ◽  
New Approach ◽  
Early Evaluation ◽  
Griess Reaction ◽  
No Release

We here describe a new approach to visualise nitric oxide (NO) in living macrophages by fluorescent NO-sensitive microspheres based on poly(lactic-co-glycolic acid) (PLGA). PLGA microspheres loaded with NO550 dye were prepared through a modified solvent-evaporation method. Microparticles were characterized by a mean hydrodynamic diameter of 3000 nm, zeta potential of −26.000 ± 0.351 mV and a PDI of 0.828 ± 0.298. Under abiotic conditions, NO release was triggered through UV radiation (254 nm) of 10 mM sodium nitroprusside dehydrate (SNP). After incubation, AZO550 microspheres exhibited an about 8-fold increased emission at 550 nm compared to NO550 particles. For biotic NO release, RAW 264.7 murine macrophages were activated with lipopolysaccharide (LPS) of Salmonella typhimurium. After treatment with NO550 microparticles, only activated cells caused a green particle fluorescence and could be detected by laser scanning microscopy. NO release was confirmed indirectly with Griess reaction. Our functional NO550 particles enable a simple and early evaluation of inflammatory and immunological processes. Furthermore, our results on particle-based NO sensing and previous studies in targeting intestinal inflammation via (PLGA)-based microspheres demonstrate that an advanced concept for visualizing intestinal inflammation is tangible.

Synchronous detection of vascular tension and nitric oxide release in pulmonary artery: A combined application of confocal wire myograph with confocal laser scanning microscopy

Vascular ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 619-628
Author(s):  
Xiao-Ling Zhuang ◽  
Zhuang-Li Zhu ◽  
Jie-Ling Zhu ◽  
Su-Mei Lai ◽  
Long-Xin Gui ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pulmonary Artery ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Combined Application ◽  
M 10 ◽  
No Release

Objectives To detect the vascular tension and nitric oxide (NO) release synchronously in mice pulmonary artery, we perform two experiments and present a novel application of confocal wire myograph coupled with the confocal laser scanning microscopy. Methods In the first experiment, viable endothelium-intact mouse pulmonary artery (outer diameter 100–300 μM) rings underwent a one-hour preincubation with a NO-specific fluorescent dye, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate Calbiochem (2.5 μM), and then precontracted with phenylephrine (Phen, 10−6 M), and subsequently dilated in acetylcholine (ACh, 10−6 M – 10−4 M). The endothelium-dependent vasorelaxation and NO generation in pulmonary artery rings were simultaneously recorded. In the second experiment, after 30-min incubation with the former NO fluorescent dye, the qualified pulmonary artery rings were co-incubated for another 30 min with a nitric oxide synthase inhibitor, 10−4 M Nω-nitro-L-arginine-methyl-ester (L-NAME), and then pretreated with Phen (10−6 M) followed by ACh (10−5 M). The Ach-induced vasodilation and NO release were recorded simultaneously. Results ACh (10−6 M – 10−4 M) promoted pulmonary artery relaxation and intracellular NO release in a dose-dependent manner. Additionally, L-NAME (10−4 M) significantly attenuated the vasodilatation and the intracellular NO release. Conclusions This combined application visually confirms that the synchronous changes in Ach induced vasodilation and NO release, which provides a new method for cardiovascular research.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

scholarly journals Cell type-dependent release of nitric oxide and/or reactive nitrogenoxide species from intracellular SIN-1: effects on cellular NAD(P)H

2004 ◽  
Vol 385 (7) ◽  
Author(s):  
A.U. Swintek ◽  
S. Christoph ◽  
F. Petrat ◽  
H. de Groot ◽  
M. Kirsch
Keyword(s):  
Nitric Oxide ◽  
Cell Culture ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Culture Studies ◽  
Aortic Endothelial Cells ◽  
No Formation ◽  
Cell Culture Systems ◽  
Zone Electrophoresis ◽  
Capillary Zone

AbstractSIN-1 is frequently used in cell culture studies as an extracellularly operating generator of peroxynitrite. However, little is known about the nature of the reactive species produced intracellulary from SIN-1. SIN-1 can easily penetrate cells as exemplified for both L-929 mouse fibroblasts and bovine aortic endothelial cells (BAECs) by utilizing capillary zone electrophoresis. In L-929 cells, SIN-1 produced nitric oxide (NO) as monitored by the fluorescent NO scavenger FNOCT-1 and by means of a NO electrode, as well as reactive nitrogenoxide species (RNOS, e.g. peroxynitrite, nitrogen dioxide, dinitrogen trioxide), as detected with the fluorescent indicator DAF-2. Laser scanning microscopy revealed that in L-929 cells SIN-1-derived species initially oxidized the major fraction of the NAD(P)H within the cytosol and the nuclei, whereas the mitochondrial NAD(P)H level was somewhat increased. In marked contrast to this, in BAECs no evidence for NO formation was found although the intracellular amount of SIN-1 was fourfold higher than in L-929 cells. In BAECs, the level of NAD(P)H was slightly decreased within the first 10 min after administration of SIN-1 in both the cytosol/nuclei and mitochondria. These observations reflect the capability of SIN-1 to generate intracellularly either almost exclusively RNOS as in BAECs, or RNOS and freely diffusing NO as in L-929 cells. Nitric oxide as well as RNOS may decisively affect cellular metabolism as indicated by the alterations in the NAD(P)H level. Hence, care should be taken when applying SIN-1 as an exclusively peroxynitrite-generating compound in cell culture systems.

Using Confocal Laser Scanning Microscopy to Study an Ambers, a New Approach

2019 ◽  
Vol 806 ◽  
pp. 197-202
Author(s):  
Alexander Mikhailovich Zakharenko ◽  
Igor Yurevich Chekryzhov ◽  
Kirill Sergeevich Golokhvast
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Far East ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Northern Spain ◽  
New Approach ◽  
Laser Bleaching

Currently, in the scientific literature, there is no a sole opinion on mechanisms governing phenomenon of blue amber. In specimens sampled in Dominican Republic it is caused by presence of perylene; whereas, specimens sampled in northern Spain this has not been confirmed. We have obtained specimens from a third, earlier not studied, source of blue amber in the Far East of Russia that was described in 2015. This sample was investigated with a laser confocal microscopy and a laser bleaching studies. We found fluorescence pattern that is typical for perylene and bleaching showed rapid diffusion that is consistent with the perylene hypothesis. Moreover we demonstrate that confocal laser scanning microscopy could be successfully used for rapid search of the organisms in the dark part of the amber samples.

Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide

2008 ◽  
Vol 294 (1) ◽  
pp. G109-G119 ◽  
Author(s):  
Rahul J. Anand ◽  
Shipan Dai ◽  
Christopher Rippel ◽  
Cynthia Leaphart ◽  
Faisal Qureshi ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Gap Junctions ◽  
Gap Junction ◽  
Intestinal Inflammation ◽  
Lucifer Yellow ◽  
Close Association ◽  
Activated Macrophages ◽  
Intracellular Location ◽  
Gap Junction Communication ◽  
No Release

Enterocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication. We now hypothesize that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that “activation” of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine ω-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS−/− mice, implicating NO in the process. Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso- N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.

scholarly journals NO Candida auris: Nitric Oxide in Nanotherapeutics to Combat Emerging Fungal Pathogen Candida auris

2020 ◽  
Vol 6 (2) ◽  
pp. 85 ◽  
Author(s):  
Levi G. Cleare ◽  
Kevin L. Li ◽  
Waleed M. Abuzeid ◽  
Parimala Nacharaju ◽  
Joel M. Friedman ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Fungal Pathogen ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Fungal Species ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Candida Auris ◽  
Colony Forming Unit ◽  
Antifungal Effects

Candida auris (C. auris) is an emerging pathogenic fungal species that is especially worrisome due to its high mortality rates and widespread antifungal resistance. Previous studies have demonstrated the efficacy of nitric oxide (NO) nanoparticles on Candida species, and, to our knowledge, this is the first study to investigate the antifungal effects of a NO-generating nanoparticle on C. auris. Six C. auris strains were incubated with a nanoparticle (NAC-SNO-np), which releases N-acetylcysteine S-nitrosothiol (NAC-SNO) and N-acetylcysteine (NAC), and generates NO, through colony forming unit (CFU) assays, and confocal laser scanning microscopy. NAC-SNO-np effectively eradicates planktonic and biofilm C. auris. Across all six strains, 10 mg/mL NAC-SNO-np significantly reduced the number of CFUs (p < 0.05) and demonstrated a >70% decrease in biofilm viability (p < 0.05). NAC-SNO-np effectively eradicates planktonic C. auris and significantly reduces C. auris biofilm formation. Hence, this novel NO-releasing nanoparticle shows promise as a future therapeutic.

scholarly journals Transport of ultrasmall gold nanoparticles (2 nm) across the blood–brain barrier in a six-cell brain spheroid model

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Viktoriya Sokolova ◽  
Gehad Mekky ◽  
Selina Beatrice van der Meer ◽  
Michael C. Seeds ◽  
Anthony J. Atala ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Brain Barrier ◽  
Confocal Laser ◽  
Hydrodynamic Diameter ◽  
Core Diameter ◽  
Blood Brain

Abstract The blood–brain barrier (BBB) is an efficient barrier for molecules and drugs. Multicellular 3D spheroids display reproducible BBB features and functions. The spheroids used here were composed of six brain cell types: Astrocytes, pericytes, endothelial cells, microglia cells, oligodendrocytes, and neurons. They form an in vitro BBB that regulates the transport of compounds into the spheroid. The penetration of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm; hydrodynamic diameter 3–4 nm) across the BBB was studied as a function of time by confocal laser scanning microscopy, with the dissolved fluorescent dye (FAM-alkyne) as a control. The nanoparticles readily entered the interior of the spheroid, whereas the dissolved dye alone did not penetrate the BBB. We present a model that is based on a time-dependent opening of the BBB for nanoparticles, followed by a rapid diffusion into the center of the spheroid. After the spheroids underwent hypoxia (0.1% O2; 24 h), the BBB was more permeable, permitting the uptake of more nanoparticles and also of dissolved dye molecules. Together with our previous observations that such nanoparticles can easily enter cells and even the cell nucleus, these data provide evidence that ultrasmall nanoparticle can cross the blood brain barrier.

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