scholarly journals Epithelial Invasion by Escherichia coli Bearing Dr Fimbriae Is Controlled by Nitric Oxide-Regulated Expression of CD55

2004 ◽  
Vol 72 (5) ◽  
pp. 2907-2914 ◽  
Author(s):  
Li Fang ◽  
Bogdan J. Nowicki ◽  
Petri Urvil ◽  
Pawel Goluszko ◽  
Stella Nowicki ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Epithelial Cell Line ◽  
No Production ◽  
Dependent Manner ◽  
E Coli ◽  
Regulated Expression ◽  
Ishikawa Cells ◽  
Epithelial Invasion

ABSTRACT We previously reported that inhibition of nitric oxide (NO) increases the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr fimbria (Dr+). Epithelial binding and invasion by Dr+ E. coli has also been shown to be dependent upon the expression level of the cellular receptor decay-accelerating factor (DAF; CD55). Here, we hypothesize that NO-related severity of infection could be mediated by changes in DAF expression and in the rate of epithelial invasion. The cellular basis of NO effects on epithelial invasion with Dr+ E. coli was studied using Ishikawa endometrial carcinoma cells as an in vitro model of the human endometrial epithelium. Initially, we show that Ishikawa cells produce NO and express both NO synthase enzymes, NOS II and NOS III, and DAF protein. We next tested the abilities of both Dr+ E. coli and a Dr− E. coli mutant to invade Ishikawa cells, and invasion was seen only with Dr+ E. coli. Invasion by Dr+ E. coli was decreased by elevated NO production and increased by NO inhibition. Elevated NO production significantly decreased DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner. Here, we propose that in vitro invasion of an epithelial cell line is directly related to NO-regulated expression of DAF. The significance of NO-regulated receptor-ligand invasion is that it may represent a novel unrecognized phenomenon of epithelial defense against infection.

Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy

2001 ◽  
Vol 281 (6) ◽  
pp. C1819-C1824 ◽  
Author(s):  
Yao Song ◽  
Jay L. Zweier ◽  
Yong Xia
Keyword(s):  
Nitric Oxide ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Heat Shock Protein 90 ◽  
Tryptophan Fluorescence ◽  
Hsp90 Inhibitor ◽  
Resonance Spectroscopy ◽  
No Production ◽  
Dependent Manner ◽  
Intact Cells

Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca2+/calmodulin (Ca2+/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the K d of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90, P < 0.01). Ca2+ ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.

scholarly journals 617. Efficacy of Dimercaptosuccinic Acid (DMSA), a Zinc Chelator, in Combination with Imipenem Against Metallo-β-Lactamase Producing Escherichia coli in a Murine Peritonitis Model

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S287-S287
Author(s):  
Geoffrey Cheminet ◽  
Patrice Nordmann ◽  
Francoise Chau ◽  
Nicolas Kieffer ◽  
Katell Peoc’h ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Maximum Effect ◽  
Dependent Manner ◽  
Bacterial Strains ◽  
Concentration Dependent Manner ◽  
Bacterial Counts ◽  
E Coli ◽  
Zinc Chelator ◽  
Peritonitis Model

Abstract Background A strategy used by bacterial strains to resist β-lactam antibiotics is the expression of metallo-β-lactamases (MBL) requiring zinc for activity. The use of a zinc chelator may restore carbapenem activity against MBL-producing Enterobacteriaceae. DMSA is a heavy metal chelator approved in humans with a satisfactory safety record. Our objective was to evaluate the activity of DMSA in combination with carbapenems, in vitro and in a fatal murine peritonitis model, against MBL-producing Escherichia coli. Methods Isogenic derivatives of wild-type E. coli CFT073 producing the MBL NDM-1, VIM-2, IMP-1, and the serine carbapenemases OXA-48 and KPC-3 were constructed. Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem were determined against each strain alone or in combination with DMSA. Mice were infected with E. coli CFT073 or NDM-1 and treated intraperitoneally for 24 hours with imipenem 100 mg/kg every 4 hours, DMSA 200 mg/kg every 4 hours, or both. Mice survival rates and bacterial counts in peritoneal fluid (PF) and spleen were assessed at 24 hours. Results In vitro, DMSA in combination with each carbapenem permitted a significant decrease of the MICs against all MBL-producing strains, in a concentration-dependent manner. The maximum effect was found for the NDM-1 strain with a 6- to 8-fold MIC reduction, depending on the carbapenem used. NDM-1 strain became susceptible to carbapenems with concentrations of DMSA ≥6 mM. Increasing zinc concentrations above 1 mg/L (average human plasma concentration) did not alter this effect. No benefit of DMSA was observed against non-MBL strains. In vivo, when used alone, the DMSA regimen was not toxic in uninfected mice and ineffective against NDM-1-infected mice (100% mortality). Combination of imipenem and DMSA significantly reduced bacterial counts in PF and spleen as compared with imipenem alone (P < 0.001), and reduced mortality, although not significantly (11% vs. 37%, respectively, P = 0.12). No benefit of the combination was observed against CFT073. Conclusion DMSA is highly effective in vitro in reducing carbapenems MICs against MBL-producing E. coli and appears as a promising strategy in combination with carbapenems for the treatment of NDM-1-related infections. Disclosures All authors: No reported disclosures.

scholarly journals ANTI-INFLAMMATORY ACTIVITY OF TINOCRISPOSIDE BY INHIBITING NITRIC OXIDE PRODUCTION IN LIPOPOLYSACCHARIDES-STIMULATED RAW 264.7 CELLS

2018 ◽  
Vol 11 (4) ◽  
pp. 149 ◽  
Author(s):  
Adek Zamrud Adnan ◽  
Muhammad Taher ◽  
Tika Afriani ◽  
Annisa Fauzana ◽  
Dewi Imelda Roesma ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Diseases ◽  
Positive Control ◽  
Inflammatory Activity ◽  
Raw 264.7 Cells ◽  
No Production ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Anti Inflammatory

 Objective: The aim of this study was to investigate in vitro anti-inflammatory activity of tinocrisposide using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophage cells. Tinocrisposide is a furano diterpene glycoside that was isolated in our previous study from Tinospora crispa.Methods: Anti-inflammatory effect was quantified spectrometrically using Griess method by measuring nitric oxide (NO) production after the addition of Griess reagent.Results: The sample concentrations of 1, 5, 25, 50, and 100 μM and 100 μM of dexamethasone (positive control) have been tested against the LPS-stimulated RAW 264.7 cells, and the results showed NO level production of 39.23, 34.00, 28.9, 20.25, 16.3, and 13.68 μM, respectively, and the inhibition level of 22.67, 33.00, 43.03, 60.10, 68.00, and 73%, respectively.Conclusions: From the study, it could be concluded that tinocrisposide was able to inhibit the formation of NO in the LPS-stimulated RAW 264.7 cells in concentration activity-dependent manner, with half-maximal inhibition concentration 46.92 μM. It can be developed as anti-inflammatory candidate drug because NO is a reactive nitrogen species which is produced by NO synthase. The production of NO has been established as a mediator in inflammatory diseases.

Activation of Nitric Oxide Release and Oxidative Metabolism by Leukotrienes B4, C4, and D4 in Human Polymorphonuclear Leukocytes

Blood ◽  
1999 ◽  
Vol 93 (4) ◽  
pp. 1399-1405 ◽  
Author(s):  
Gerd Lärfars ◽  
Frédérique Lantoine ◽  
Marie-Aude Devynck ◽  
Jan Palmblad ◽  
Hans Gyllenhammar
Keyword(s):  
Nitric Oxide ◽  
Rank Order ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Kinetic Properties ◽  
No Production ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cysteinyl Leukotrienes ◽  
Mediators Of Inflammation

Abstract Because arachidonate metabolites are potent mediators of inflammation, we have studied the effects of leukotriene B4(LTB4) and the cysteinyl leukotrienes C4 and D4 (LTC4 and LTD4) on the release of nitric oxide (NO), in vitro, by human polymorphonuclear granulocytes (PMN). Two independent and highly sensitive real-time methods were used for these studies, ie, the NO-dependent oxidation of oxyhemoglobin (HbO2) to methemoglobin and a NO-sensitive microelectrode. When activated with LTB4, LTC4, or LTD4, but not with other lipoxygenase products such as 5S-HETE, 5-oxo-ETE or 5S,12S-diHETE, PMN produced NO in a stimulus- and concentration-dependent manner. The rank order of potency was LTB4 = LTC4 &gt; LTD4, corresponding to 232 ± 50 pmol of NO/106 PMN for 100 nmol/L LTB4 after 30 minutes. The kinetic properties of the responses were similar for all three leukotrienes with a maximum response at 13 ± 3 minutes. Cysteinyl leukotriene and LTB4 antagonists inhibited the agonist-induced NO production by 70%, and treatment with Bordetella pertussis toxin, or chelation of cytosolic Ca2+, [Ca2+]i, also efficiently inhibited this response. In contrast, treatment of PMN with cytochalasin B (5 μg/mL) enhanced the LTB4-induced NO formation by 86%. Thus, this is the first demonstration that the cysteinyl leukotrienes LTC4 and LTD4, as well as LTB4, activate NO release from human PMN by surface receptor, G-protein and [Ca2+]i-dependent mechanisms. This effect differs from activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, for which only LTB4is an activator.

Single oral inoculation with Escherichia coli (ATCC 25922) stimulates generalised production of nitric oxide in mice

2009 ◽  
Vol 57 (1) ◽  
pp. 127-138 ◽  
Author(s):  
Ana Nemec ◽  
Zlatko Pavlica ◽  
David Crossley ◽  
Irena Zdovc ◽  
Damijan Eržen ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Spin Trap ◽  
Oral Route ◽  
No Production ◽  
Paramagnetic Resonance ◽  
E Coli ◽  
Oral Inoculation ◽  
Nos Inhibitors ◽  
Oxide Synthase

Nitric oxide (NO) production was investigated in the lungs, thoracic aorta, heart, liver, spleen, kidneys and brain of mice inoculated orally withEscherichia coliATCC 25922. Detection of NO was performed by electron paramagnetic resonance (EPR) using diethyldithiocarbamate (DETC) spin trap. Nitric oxide synthase (NOS) inhibitors [nonselective: L-NAME and inducible NOS (iNOS) selective: 1400W] were used to determine the source of NO. Spin-trap only and untreated mice were included as controls. Within 2.5 hours (h) of a single oral inoculation withE. colihalf of the animals had increased NO levels in all investigated organs. Thereafter the signals dropped before increasing again to reach maximal median values by 25 h in all organs of all inoculated mice. The most intense response occurred in livers, followed by aorta and lungs. Early (2.5 h) inhibition of the signal was achieved using both NOS inhibitors. L-NAME was also effective at 25 h, while 1400W-treated mice had increased NO levels beyond 7 h. The generalised increase in NO production in the short and longer term indicates a host response toE. coliadministered by the oral route of infection.

scholarly journals Dimercaptosuccinic acid in combination with carbapenems against isogenic strains of Escherichia coli producing or not producing a metallo-β-lactamase in vitro and in murine peritonitis

2020 ◽  
Vol 75 (12) ◽  
pp. 3593-3600 ◽  
Author(s):  
G Cheminet ◽  
V de Lastours ◽  
L Poirel ◽  
F Chau ◽  
K Peoc’h ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Peritoneal Fluid ◽  
Dimercaptosuccinic Acid ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bacterial Counts ◽  
E Coli ◽  
Time Kill ◽  
Isogenic Strains

Abstract Background Carbapenemase-producing Enterobacterales represent a major therapeutic challenge. MBLs, requiring zinc at their catalytic site, could be inhibited by meso-dimercaptosuccinic acid (DMSA), a heavy metal chelator already widely used for treating lead intoxication. Objectives To evaluate the activity of carbapenems alone or combined with DMSA against MBL-producing Escherichia coli in a severe murine peritonitis model. Methods Isogenic strains of wild-type E. coli CFT073 producing the MBLs NDM-1, VIM-2 and IMP-1, and the control serine carbapenemases OXA-48 and KPC-3 were constructed. MIC determinations and time–kill assays were performed for imipenem, meropenem and ertapenem alone or in combination with DMSA. Infected mice were treated intraperitoneally for 24 h with imipenem, DMSA or their combination. Bacterial counts in peritoneal fluid and spleen were assessed at 24 h. Results DMSA in combination with each carbapenem caused a significant decrease in the MICs for all MBL-producing strains, in a concentration-dependent manner, but did not provide benefit against non-MBL strains. In mice infected with the NDM-1-producing strain, the combination of imipenem and DMSA significantly reduced bacterial counts in peritoneal fluid (P = 0.0006) and spleen (P &lt; 0.0001), as compared with imipenem alone, with no benefit against the KPC-3-producing and CFT073 strains. DMSA concentrations in plasma of mice were comparable to those obtained in humans with a standard oral dose. Conclusions DMSA restores the activity of carbapenems against MBL-producing strains, and its combination with carbapenems appears to be a promising strategy for the treatment of NDM-producing E. coli infections.

Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity

1992 ◽  
Vol 70 (1) ◽  
pp. 43-48 ◽  
Author(s):  
S. S. Ghosh ◽  
Richard C. Franson
Keyword(s):  
Escherichia Coli ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Phospholipase A ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Streptomyces Chromofuscus ◽  
Hydrolysis Of

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0–8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 μM) or propanolol (IC50 = 180 μM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.Key words: Escherichia coli, substrate, phospholipase D, Streptomyces chromofuscus, sodium fluoride, propranolol.

scholarly journals In Vitro Reduction of Interleukin-8 Response to Enterococcus faecalis by Escherichia coli Strains Isolated from the Same Polymicrobial Urines

2021 ◽  
Vol 9 (7) ◽  
pp. 1501
Author(s):  
Gabriella Piatti ◽  
Laura De Ferrari ◽  
Anna Maria Schito ◽  
Anna Maria Riccio ◽  
Susanna Penco ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Enterococcus Faecalis ◽  
Interleukin 8 ◽  
Fluoroquinolone Resistance ◽  
Epithelial Cell Line ◽  
E Coli ◽  
Tract Infections ◽  
K 12

Urinary tract infections are often polymicrobial and are mainly due to uropathogenic Escherichia coli (UPEC). We previously demonstrated a link among clinical fluoroquinolone susceptible E. coli reducing in vitro urothelial interleukin-8 (CXCL8) induced by E. coli K-12, polymicrobial cystitis, and pyuria absence. Here, we evaluated whether fifteen clinical fluoroquinolone susceptible UPEC were able to reduce CXCL8 induced by Enterococcus faecalis that had been isolated from the same mixed urines, other than CXCL8 induced by E. coli K-12. We also evaluated the connection between fluoroquinolone susceptibility and pathogenicity by evaluating the immune modulation of isogenic gyrA, a mutant UPEC resistant to ciprofloxacin. Using the 5637 bladder epithelial cell line, we observed that lower CXCL8 induced the most UPEC isolates than K-12 and the corresponding E. faecalis. During coinfections of UPEC/K-12 and UPEC/E. faecalis, we observed lower CXCL8 than during infections caused by K-12 and E. faecalis alone. UPEC strains showed host–pathogen and pathogen–pathogen interaction, which in part explained their persistence in the human urinary tract and coinfections, respectively. Mutant UPEC showed lower modulating activity with respect to the wildtypes, confirming the connection between acquired fluoroquinolone resistance and the decrease of innate microbial properties.

Nitric oxide induces apoptosis in renal tubular epithelial cells through activation of caspase-8

2006 ◽  
Vol 290 (5) ◽  
pp. F1044-F1054 ◽  
Author(s):  
Caigan Du ◽  
Qiunong Guan ◽  
Hong Diao ◽  
Ziqin Yin ◽  
Anthony M. Jevnikar
Keyword(s):  
Nitric Oxide ◽  
Epithelial Cells ◽  
Proinflammatory Cytokines ◽  
Mononuclear Cells ◽  
Caspase 8 ◽  
No Production ◽  
Dependent Manner ◽  
Tubular Epithelial Cells ◽  
Inhibitory Protein

The susceptibility or resistance of tubular epithelial cells (TEC) to apoptosis is pivotal to the long-term maintenance of kidney function following episodes of inflammation, such as graft rejection. TEC apoptosis can occur with ischemia as well as with proinflammatory cytokines and nitric oxide (NO), produced by infiltrating mononuclear cells. TEC can also produce abundant amounts of NO during inflammation but the role and regulation of NO-induced injury of TEC are not well understood. Apoptosis in TEC in vitro was determined by FACS analysis with annexin-V and propidium iodide staining. NO in culture supernatants was measured by Greiss reagent, and protein expression of inducible NO synthetase (NOS2/iNOS) and caspase-8 was examined by Western blot analysis. Here, we showed that murine TEC produced abundant amounts of NO in response to proinflammatory cytokines (IFN-γ/TNF-α) through upregulation of NOS2, and inhibition of endogenous NO production by l-NMMA reduced TEC apoptosis in cytokine-stimulated cultures. Addition of exogenous NO (sodium nitroprusside) induced TEC apoptosis as well as caspase-8 activation in a dose-dependent manner. The key role of caspase-8 in NO-induced TEC apoptosis was demonstrated by that NO-induced TEC apoptosis can be blocked by caspase-8 inhibition using z-IETD-fmk, caspase-8 silencing with shRNA or by overexpressing the endogenous caspase-8 inhibitor c-FLIP (cellular Flice-inhibitory protein). In conclusion, endogenous NO from NOS2 activity as well as exogenous NO can contribute to renal injury through apoptosis of TEC. Activation of caspase-8 plays a central role in NO-induced apoptosis and caspase-8 inhibition may be an important therapeutic target during renal inflammation.

scholarly journals Ablation of IL-17A leads to severe colitis in IL-10-deficient mice: implications of myeloid-derived suppressor cells and NO production

2019 ◽  
Vol 32 (3) ◽  
pp. 187-201
Author(s):  
Masashi Tachibana ◽  
Nobumasa Watanabe ◽  
Yuzo Koda ◽  
Yukako Oya ◽  
Osamu Kaminuma ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Inflammatory Diseases ◽  
Genetic Defect ◽  
Suppressor Cells ◽  
T Cell Response ◽  
No Production ◽  
Dependent Manner ◽  
Myeloid Derived Suppressor Cells ◽  
Severe Colitis

Abstract IL-10 is an immune regulatory cytokine and its genetic defect leads to gastrointestinal inflammation in humans and mice. Moreover, the IL-23/Th17 axis is known to be involved in these inflammatory disorders. IL-17A, a representative cytokine produced by Th17 cells, has an important role for the pathological process of inflammatory diseases. However, the precise function of IL-17A in inflammatory bowel disease (IBD) remains controversial. In this study, we evaluated the effect of IL-17A on colitis in IL-10-deficient (Il10−/−) mice. Mice lacking both IL-10 and IL-17A (Il10−/−Il17a−/−) suffered from fatal wasting and manifested more severe colitis compared with Il10−/−Il17a+/− mice. Moreover, we found that CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) accumulated in the bone marrow, spleen and peripheral blood of Il10−/−Il17a−/− mice. These MDSCs highly expressed inducible nitric oxide synthase (iNOS) (Nos2) and suppressed the T-cell response in vitro in a NOS-dependent manner. In correlation with these effects, the concentration of nitric oxide was elevated in the serum of Il10−/−Il17a−/− mice. Surprisingly, the severe colitis observed in Il10−/−Il17a−/− mice was ameliorated in Il10−/−Il17a−/−Nos2−/− mice. Our findings suggest that IL-17A plays suppressive roles against spontaneous colitis in Il10−/− mice in an iNOS-dependent manner and inhibits MDSC differentiation and/or proliferation.

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