3-nitrotyrosine and Escherichia-coli-Infections

Parkinson's disease (PD) is the second most common neurodegenerative disorder of aging. The pathological hallmark of PD is neuronal inclusions termed Lewy bodies whose main component is alpha-synuclein protein. The finding of these Lewy bodies in the intestinal enteric nerves led to the hypothesis that the intestine might be an early site of PD disease in response to an environmental toxin or pathogen. One potential mechanism for environmental toxin(s) and proinflammatory luminal products to gain access to mucosal neuronal tissue and promote oxidative stress is compromised intestinal barrier integrity. However, the role of intestinal permeability in PD has never been tested. We hypothesized that PD subjects might exhibit increased intestinal permeability to proinflammatory bacterial products in the intestine. To test our hypothesis we evaluated intestinal permeability in subjects newly diagnosed with PD and compared their values to healthy subjects. In addition, we obtained intestinal biopsies from both groups and used immunohistochemistry to assess bacterial translocation, nitrotyrosine (oxidative stress), and alpha-synuclein. We also evaluated serum markers of endotoxin exposure including LPS binding protein (LBP). Our data show that our PD subjects exhibit significantly greater intestinal permeability (gut leakiness) than controls. In addition, this intestinal hyperpermeability significantly correlated with increased intestinal mucosa staining for E. coli bacteria, nitrotyrosine, and alpha-synuclein as well as serum LBP levels in PD subjects. These data represent not only the first demonstration of abnormal intestinal permeability in PD subjects but also the first correlation of increased intestinal permeability in PD with intestinal alpha-synuclein (the hallmark of PD), as well as staining for gram negative bacteria and tissue oxidative stress. Our study may thus shed new light on PD pathogenesis as well as provide a new method for earlier diagnosis of PD and suggests potential therapeutic targets in PD subjects.. Clinicaltrials.gov NCT01155492.

Topics: Acute-Phase Proteins; Aged; alpha-Synuclein; Biomarkers; Carrier Proteins; Endotoxins; Escherichia coli; Escherichia coli Infections; Female; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Intestines; Male; Membrane Glycoproteins; Middle Aged; Neurons; Oxidative Stress; Parkinson Disease; Permeability; Sucrose; Tyrosine

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO(-/-) mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO(-/-) mice unexpectedly had improved survival compared with wild-type (WT) mice within 5-12 h after intraperitoneal E. coli challenge. Lungs of MPO(-/-) mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO(-/-) mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO(-/-) mice. Inhibition of iNOS in MPO(-/-) mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO(-/-) mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.

Topics: Animals; Escherichia coli; Escherichia coli Infections; Gene Expression Regulation, Enzymologic; Lung; Lung Injury; Mice; Mice, Knockout; Neutrophils; Nitric Oxide; Nitric Oxide Synthase Type II; Oxidants; Peroxidase; Pulmonary Edema; Sepsis; Tyrosine

In the present study, we report the effect of vitamin A (Vit A, retinol palpitate) on kidney lipid peroxidation and 3-nitrotyrosine (3-NT) levels induced after Escherichia coli administration to guinea pigs. Vit A was administrated intraperitoneally (i.p.) to guinea pigs at a dose 15,000 IU/kg per day for 7 days prior to E. coli injection. On day 8, the animals were injected i.p. with E. coli dosed at 12 x10(9) colony forming units per kilogram. Kidneys were collected 6 h after administration of E. coli. Malondialdehyde (MDA) as a lipid peroxidation product, and 3-NT levels were measured by reverse phase high-performance liquid chromatography. There was a significant increase in MDA and 3-NT levels in lipopolysaccaharide-induced group (p<0.001). 3-NT was not detectable in kidney of normal control animals. However, Vit A administration prior to E. coli injection prevented 3-NT formation but did not prevent the rice in MDA level of kidney (p<0.001). Vit A alone did not alter the MDA level in the kidney of the control group.

Topics: Animals; Chromatography, High Pressure Liquid; Escherichia coli Infections; Guinea Pigs; Kidney; Lipid Peroxidation; Malondialdehyde; Tyrosine; Vitamin A

In animal models of endotoxin, the excess production of NO and the reactive nitrogen species (RNS), are potent oxidant and nitrating agents, lead to lipid peroxidation, apoptosis, tissue dysfunction and injury and inactivate enzymes in many cell types. Although liver functions are well known to deteriorate following bacterial infection, the underlying specific mechanism(s) remain a matter of considerable debate. Therefore, the aim of the present study was to determine the in vivo effect of bacterial lipopolysaccharides (LPS) on Na+,K+-ATPase activity of guinea pig liver, and to investigate the possible contribution of RNS by measuring of iNOS activity and 3-nitrotyrosine (nTyr) levels. Liver Na+,K+-ATPase activity were maximally inhibited 6 h after LPS injection (p < 0.001 ). nTyr was not detectable in liver of normal control animals, but was detected markedly in LPS exposed animals. LPS treatment significantly increased iNOS activity of liver (p < 0.001). The regression analysis revealed a very close correlation between Na+,K+-ATPase activity and nTyr levels of LPS treated animals (r = -0.863, p < 0.001). Na+, K+-ATPase activity were also negatively correlated with iNOS activity (r = -0.823, p < 0.003) in inflamed tissues. Our results have strongly suggested that bacterial LPS disturbs activity of membrane Na+,K+-ATPase that may be an important component leading to the pathological consequences such as hepatocyte cell loss and dysfunction in which the production of RNS are increased as in the case of LPS challenge.

Topics: Animals; Cell Death; Enzyme Activation; Escherichia coli; Escherichia coli Infections; Guinea Pigs; Lipopolysaccharides; Liver; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Reactive Nitrogen Species; Reactive Oxygen Species; Sodium-Potassium-Exchanging ATPase; Tyrosine

Inducible nitric oxide synthase (iNOS)-deficient mice were used to examine the role of iNOS in Escherichia coli-induced urinary tract infection (UTI). The toxicity of nitric oxide (NO)/peroxynitrite to bacteria and host was also investigated. The nitrite levels in urine of iNOS+/+ but not iNOS/ mice increased after infection. No differences in bacterial clearance or persistence were noted between the genotypes. In vitro, the uropathogenic E. coli 1177 was sensitive to 3-morpholinosydnonimine, whereas the avirulent E. coli HB101 was sensitive to both NO and 3-morpholinosydnonimine. E. coli HB101 was statistically (P < 0.05) more sensitive to peroxynitrite than E. coli 1177. Nitrotyrosine immunoreactivity was observed in infected bladders of both genotypes and in infected kidneys of iNOS+/+ mice. Myeloperoxidase, neuronal (n)NOS, and endothelial (e)NOS immunoreactivity was observed in inflammatory cells of both genotypes. Our results indicate that iNOS/ and iNOS+/+ mice are equally susceptible to E. coli-induced UTI and that the toxicity of NO to E. coli depends on bacterial virulence. Furthermore, myeloperoxidase and nNOS/eNOS may contribute to nitrotyrosine formation in the absence of iNOS.

Topics: Animals; Antibodies; Colony Count, Microbial; Escherichia coli; Escherichia coli Infections; Female; Genotype; Immunohistochemistry; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nephritis; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Peroxidase; Peroxynitrous Acid; Tyrosine; Urinary Tract Infections

The aim of this study was to determine the effect of Escherichia coli (E. coli)-derived lipopolysaccharide on rat plasma low density lipoprotein (LDL), malondialdehyde and 3-nitrotyrosine levels (an indicator of protein nitration). Six hours after intraperitoneal administration of E.coli, plasma LDL was measured electrophoretically and malondialdehyde level was measured by spectrophotometric method. Plasma malondialdehyde was significantly (p<0.001) elevated in E. coli-injected rats (4.97 +/- 1.33; n=10) in comparison to control animals (1.83 +/- 0.5; n=10). In addition, plasma 3-nitrotyrosine level, determined by reverse-phase HPLC, was also increased in the infected group (2.84 +/- 1.17 to 0.22 +/- 0.13; n=10). This increase was statistically significant (p<0.001). An increased level of oxidation of lipids and 3-nitrotyrosine was observed as a result of free radical-mediated damage in plasma. In conclusion, asymptomatic infections may increase the risk of atherosclerosis by inducing free radical formation and a consequent increase in the oxidation of LDL.

Topics: Animals; Escherichia coli; Escherichia coli Infections; Free Radicals; Lipid Peroxidation; Lipopolysaccharides; Lipoproteins, LDL; Malondialdehyde; Oxidation-Reduction; Rats; Tyrosine

Endotoxin-induced peroxynitrite formation has been demonstrated in plasma. The aim of this study is to evaluate whether this has an effect on erythrocytes. For this purpose erythrocyte 3-nitrotyrosine (3-NT) level, Na+-K+ ATPase and glutathione peroxidase activities were measured both in vivo and in vitro. In vivo peroxynitrite formation was induced in rats by intraperitoneal Escherichia coli (E.coli) injection. Erythrocytes were directly incubated with peroxynitrite in the in vitro experiment. 3-NT levels were measured by reverse-phase HPLC, glutathione peroxidase, and Na+-K+ ATPase activities were measured by spectrophotometric techniques. There was a marked increase in the 3-NT levels in both experiments. However, glutathione peroxidase activity was significantly increased in in vivo experiments, while decreasing in in vitro conditions. Although Na+-K+ ATPase activities were significantly reduced by peroxynitrite in vitro, Na+-K+ ATPase activities were similar in control and E.coli-injected rat erythrocytes. Although nitrating effect of peroxynitrite does not seem to be preventable by endogenous antioxidants, this effect of peroxynitrite may not endanger erythrocytes if the oxidative damage of peroxynitrite is prevented.

Topics: Alleles; Animals; Chromatography, High Pressure Liquid; Enzyme Induction; Erythrocytes; Escherichia coli; Escherichia coli Infections; Glutathione Peroxidase; Oxidative Stress; Peroxynitrous Acid; Rats; Sodium-Potassium-Exchanging ATPase; Spectrophotometry; Tyrosine

To assess the contribution of poly (adenosine 5'-diphosphate ribose) synthetase (PARS) to the development of bacterial lipopolysaccharide (LPS)-induced acute lung injury and vascular failure in pigs.. Four groups of anesthetized, paralyzed, and mechanically ventilated domestic white pigs. Group 1 served as control, whereas Escherichia coli LPS (20 microg/kg/h) was continuously infused in group 2. Group 3 received 20 mg/kg injection of 3-aminobenzamide (a selective inhibitor of PARS activity) 15 minutes before LPS infusion. Only 3-aminobenzamide and not LPS was injected in group 4. All animals were examined for 180 minutes. Systemic and pulmonary hemodynamics and lung mechanics were measured during the experimental period. Lung wet/dry ratio, bronchoalveolar lavage (BAL) protein levels and cell counts and lung nitrotyrosine (footprint of peroxynitrite) immunostaining were also measured in a few animals.. LPS infusion evoked a progressive decline in systemic arterial pressure, a small increase in cardiac output, and biphasic elevation of pulmonary arterial pressure. Lung compliance declined progressively, whereas lung and total respiratory resistance rose significantly after LPS infusion. Prominent nitrotyrosine immunostaining was detected around small airways and pulmonary endothelium of LPS-infused animals. No significant changes in lung wet/dry ratio and BAL protein levels and cell counts were produced by LPS infusion. Pretreatment with 3-aminobenzamide did not alter the systemic and pulmonary hemodynamic responses to LPS infusion but eliminated the rise in pulmonary and total respiratory resistance.. We concluded that PARS activation plays an important role in the changes of lung mechanics associated with LPS-induced acute lung injury but had no role in vascular failure.

Topics: Animals; Benzamides; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Enzyme Inhibitors; Escherichia coli Infections; Female; Hemodynamics; Immunohistochemistry; Lipopolysaccharides; Male; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proteins; Pulmonary Circulation; Respiratory Distress Syndrome; Respiratory Mechanics; Shock, Septic; Swine; Tyrosine