ABSTRACT
Despite extensive investigations, an effective treatment for sepsis remains elusive and a better understanding of the inflammatory response to infection is required to identify potential new targets for therapy. In this study we have used RNAi technology to show, for the first time, that the inducible lysophosphatidylcholine acyltransferase 2 (LPCAT2) plays a key role in macrophage inflammatory gene expression in response to stimulation with bacterial ligands. Using siRNA- or shRNA-mediated knockdown, we demonstrate that, in contrast to the constitutive LPCAT1, LPCAT2 is required for macrophage cytokine gene expression and release in response to TLR4 and TLR2 ligand stimulation but not for TLR-independent stimuli. In addition, cells transfected to overexpress LPCAT2 exhibited increased expression of inflammatory genes in response to LPS and other bacterial ligands. Furthermore, we have used immunoprecipitation and Western blotting to show that in response to LPS, LPCAT2, but not LPCAT1, rapidly associates with TLR4 and translocates to membrane lipid raft domains. Our data thus suggest a novel mechanism for the regulation of inflammatory gene expression in response to bacterial stimuli and highlight LPCAT2 as a potential therapeutic target for development of anti-inflammatory and anti-sepsis therapies.
Subject(s)
1-Acylglycerophosphocholine O-Acyltransferase/metabolism , Macrophages, Peritoneal/immunology , Monocytes/immunology , Sepsis/immunology , 1-Acylglycerophosphocholine O-Acyltransferase/genetics , Animals , Cell Line, Tumor , Gene Expression Regulation/immunology , Gene Knockdown Techniques , Humans , Lipopolysaccharides/immunology , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/metabolism , Membrane Microdomains/metabolism , Mice , Monocytes/cytology , Monocytes/metabolism , Primary Cell Culture , RAW 264.7 Cells , RNA, Small Interfering/metabolism , Sepsis/microbiology , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 4/metabolismABSTRACT
Hydrogen peroxide is a key component of the innate immune response, regulating how a cell responds to a bacterial threat; however, being transient in nature makes it extremely difficult to detect. We show the development of an improved biosensor capable of the rapid detection of the hydrogen peroxide produced intracellularly in response to both smooth and rough lipopolysaccharides (LPS) structures. The arising signal and mass transport behaviour to the electrodes were characterised. This response was detected utilising a single walled carbon nanotube-based sensor that has been functionalised with an osmium complex for specificity and detecting the change in intracellular concentrations of hydrogen peroxide through chronoamperometry. This was conducted within murine macrophage (RAW264.7) cells and using ultra-pure LPS extracted from two different serotypes of bacteria (0111:B4 and Re495). This allowed the comparison of the immune response when infected with different structures of LPS. We demonstrate that the hydrogen peroxide signal can be electrochemically detected within 3 seconds post injection. Combining the nature of the mass transport of hydrogen peroxide and concentration characteristics, a bacterial 'fingerprint' was identified. The impact of this work will be demonstrated in allowing us to develop a rapid diagnostic for bacterial detection.
Subject(s)
Carbon/chemistry , Electrodes , Hydrogen Peroxide/metabolism , Lipopolysaccharides/metabolism , Nanotechnology , Animals , Biosensing Techniques , Mice , RAW 264.7 Cells , Reactive Oxygen Species/metabolismABSTRACT
Reactive oxygen species are highly reactive molecules that as well as being ubiquitously expressed throughout the body, are also known to be involved in many diseases and disorders including bacterial infection. Current technology has limited success in the accurate detection and identification of specific reactive oxygen species. To combat this, we have developed an electrochemical biosensor that is constructed from single walled carbon nanotubes that have been immobilised on an indium tin oxide surface functionalised with osmium-based compound. This sensor was integrated within mouse macrophage cells (RAW 264.7) with multiple serotypes of bacteria used to initiate an immune response. Intracellular hydrogen peroxide was then measured in response to the interaction of the lipopolysaccharides, present on the outer wall of Gram-negative bacteria, with the Toll-like Receptor 4. Additional controls of n-acetylcysteine and sodium pyruvate were implemented to prove the specificity of the sensor towards hydrogen peroxide. The sensors were found to have a lower limit of detection of 368â¯nM hydrogen peroxide. An increase in intracellular hydrogen peroxide was detected within 3 seconds of interaction of the bacteria with the macrophage cells. This low limit of detection combined with the rapid response of the sensor resulted in the unprecedented detection of hydrogen peroxide on a temporal level not previously seen in response to a bacterial threat. From the three serotypes of Gram-negative bacteria that were tested, there were distinct differences in hydrogen peroxide production. This proves that the innate immune system has the ability to respond dynamically and rapidly, after infection prior to the activation of the adaptive immune system.
Subject(s)
Biosensing Techniques/methods , Gram-Negative Bacteria/immunology , Hydrogen Peroxide/analysis , Macrophages/chemistry , Macrophages/immunology , Animals , Electrochemical Techniques/methods , Gram-Negative Bacterial Infections/immunology , Hydrogen Peroxide/immunology , Immunity, Innate , Limit of Detection , Lipopolysaccharides/immunology , Macrophages/microbiology , Mice , Nanotubes, Carbon/chemistry , RAW 264.7 Cells , Reactive Oxygen Species/analysis , Reactive Oxygen Species/immunology , Toll-Like Receptor 4/immunologyABSTRACT
With the rapidly increasing demands for ultrasensitive biodetection, the design and applications of new nano-scale materials for development of sensors based on optical and electrochemical transducers have attracted substantial interest. In particular, given the comparable sizes of nanomaterials and biomolecules, there exist plenty of opportunities to develop functional nanoprobes with biomolecules for highly sensitive and selective biosensing, shedding new light on cellular behaviour. Towards this aim, herein we interface cells with patterned nano-arrays of carbon nanofibers forming a nanosensor-cell construct. We show that such a construct is capable of electrochemically communicating with the intracellular environment.
ABSTRACT
The recent discovery that mammalian nociceptors express Toll-like receptors (TLRs) has raised the possibility that these cells directly detect and respond to pathogens with implications for either direct nociceptor activation or sensitization. A range of neuronal TLRs have been identified, however a detailed description regarding the distribution of expression of these receptors within sub-populations of sensory neurons is lacking. There is also some debate as to the composition of the TLR4 receptor complex on sensory neurons. Here we use a range of techniques to quantify the expression of TLR4, TLR7 and some associated molecules within neurochemically-identified sub-populations of trigeminal (TG) and dorsal root (DRG) ganglion sensory neurons. We also detail the pattern of expression and co-expression of two isoforms of lysophosphatidylcholine acyltransferase (LPCAT), a phospholipid remodeling enzyme previously shown to be involved in the lipopolysaccharide-dependent TLR4 response in monocytes, within sensory ganglia. Immunohistochemistry shows that both TLR4 and TLR7 preferentially co-localize with transient receptor potential vallinoid 1 (TRPV1) and purinergic receptor P2X ligand-gated ion channel 3 (P2X3), markers of nociceptor populations, within both TG and DRG. A gene expression profile shows that TG sensory neurons express a range of TLR-associated molecules. LPCAT1 is expressed by a proportion of both nociceptors and non-nociceptive neurons. LPCAT2 immunostaining is absent from neuronal profiles within both TG and DRG and is confined to non-neuronal cell types under naïve conditions. Together, our results show that nociceptors express the molecular machinery required to directly respond to pathogenic challenge independently from the innate immune system.
Subject(s)
Ganglia, Spinal/metabolism , Nociceptors/metabolism , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 7/metabolism , Trigeminal Ganglion/metabolism , 1-Acylglycerophosphocholine O-Acyltransferase/metabolism , Animals , Male , Rats , Rats, Sprague-DawleyABSTRACT
The ability to monitor intracellular events in real time is paramount to advancing fundamental biological and clinical science. We present the first demonstration of a direct interface of vertically aligned single-walled carbon nanotubes (VASWCNTs) with eukaryotic cells, RAW 264.7 mouse macrophage cell line. The cells were cultured on indium tin oxide with VASWCNTs. VASWCNTs entered the cells naturally without application of any external force and were shown to sense the intracellular presence of a redox active moiety, methylene blue. The technology developed provides an alluring platform to enable electrochemical study of an intracellular environment.
Subject(s)
Biosensing Techniques , Macrophages/metabolism , Nanotubes, Carbon , Tin Compounds/chemistry , Animals , Cell Line , Mice , Microscopy, Atomic Force , Photoelectron SpectroscopyABSTRACT
A water-based carbon screen-printing ink formulation, containing the redox mediator cobalt phthalocyanine (CoPC) and the enzyme glucose oxidase (GOx), was investigated for its suitability to fabricate glucose microbiosensors in a 96-well microplate format: (1) the biosensor ink was dip-coated onto a platinum (Pt) wire electrode, leading to satisfactory amperometric performance; (2) the ink was deposited onto the surface of a series of Pt microelectrodes (10-500 µm diameter) fabricated on a silicon substrate using MEMS (microelectromechanical systems) microfabrication techniques: capillary deposition proved to be successful; a Pt microdisc electrode of ≥100 µm was required for optimum biosensor performance; (3) MEMS processing was used to fabricate suitably sized metal (Pt) tracks and pads onto a silicon 96 well format base chip, and the glucose biosensor ink was screen-printed onto these pads to create glucose microbiosensors. When formed into microwells, using a 340 µl volume of buffer, the microbiosensors produced steady-state amperometric responses which showed linearity up to 5 mM glucose (CV=6% for n=5 biosensors). When coated, using an optimised protocol, with collagen in order to aid cell adhesion, the biosensors continued to show satisfactory performance in culture medium (linear range to 2 mM, dynamic range to 7 mM, CV=5.7% for n=4 biosensors). Finally, the operation of these collagen-coated microbiosensors, in 5-well 96-well format microwells, was tested using a 5-channel multipotentiostat. A relationship between amperometric response due to glucose, and cell number in the microwells, was observed. These results indicate that microphotolithography and screen-printing techniques can be combined successfully to produce microbiosensors capable of monitoring glucose metabolism in 96 well format cell cultures. The potential application areas for these microbiosensors are discussed.
Subject(s)
Biosensing Techniques/methods , Cell Culture Techniques , Glucose/isolation & purification , Microtechnology/methods , Cell Line , Electrochemistry/methods , Glucose/chemistry , Glucose Oxidase/chemistry , Indoles/chemistry , Microelectrodes , Organometallic Compounds/chemistry , Water/chemistryABSTRACT
A screen-printed carbon electrode (SPCE) incorporating the electrocatalyst cobalt phthalocyanine (CoPC), fabricated using a water-based ink formulation, has been investigated as the base transducer for a uric acid biosensor. A sandwich biosensor was fabricated by first depositing cellulose acetate (CA) onto this transducer (CoPC-SPCE), followed by uricase (UOX) and finally a polycarbonate (PC) membrane; this device is designated PC-UOX-CA-CoPC-SPCE. This biosensor was used in conjunction with chronoamperometry to optimize the conditions for the analysis of urine: temperature, 35°C; buffer, pH 9.2; ionic strength, 50 mM; uricase, 0.6 U; incubation time, 180 s. The proposed biosensor was applied to urine from a healthy subject. The precision determined on unspiked urine (n=6) was 5.82%. Urine was fortified with 0.225 mM UA, and the resulting precision and recovery were 4.21 and 97.3%, respectively. The linear working range of the biosensor was found to be 0.015 to 0.25 mM (the former represents the detection limit), and the sensitivity was calculated to be 2.10 µA/mM.
Subject(s)
Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Electrochemistry/instrumentation , Electrochemistry/methods , Uric Acid/urine , Urinalysis/instrumentation , Urinalysis/methods , Buffers , Calibration , HumansABSTRACT
Microband biosensors, screen-printed from a water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase (GOD) enzyme, were used to monitor glucose levels continuously in buffer and culture medium. Five biosensors were operated amperometrically (E(app) of +0.4V), in a 12-well tissue culture plate system at 37°C, using a multipotentiostat. After 24 h, a linear calibration plot was obtained from steady-state current responses for glucose concentrations up to 10 mM (dynamic range 30 mM). Within the linear region, a correlation coefficient (R(2)) of 0.981 was obtained between biosensor and spectrophotometric assays. Over 24 h, an estimated 0.15% (89 nmol) of the starting glucose concentration (24 mM) was consumed by the microbiosensor. The sensitivity of the biosensor response in full culture medium was stable between pHs 7.3 and 8.4. Amperometric responses for HepG2 monolayer cultures decreased with time in inverse proportionality to cell number (for 0 to 10(6) cell/ml), as glucose was being metabolised. HepG2 3D cultures (spheroids) were also shown to metabolise glucose, at a rate which was independent of spheroid age (between 6 and 15 days). Spheroids were used to assay the effect of a typical hepatotoxin, paracetamol. At 1 mM paracetamol, glucose uptake was inhibited by 95% after 6 h in culture; at 500 µM, around 15% inhibition was observed after 16 h. This microband biosensor culture system could form the basis for an in vitro toxicity testing system.
Subject(s)
Acetaminophen/toxicity , Biological Assay/instrumentation , Biosensing Techniques/instrumentation , Cell Survival/drug effects , Conductometry/instrumentation , Glucose/analysis , Computer Systems , Equipment Design , Equipment Failure Analysis , Hep G2 Cells , HumansABSTRACT
The upper respiratory tract commensal Neisseria lactamica (Nlac) induces protective humoral immunity against pathogenic Nmen serogroup B (Nmen), but whether it also affords anti-inflammatory mucosal protection, as reported for several gut commensals, has not been investigated. Here we demonstrate for the first time that Nlac weakly induces inflammatory responses compared with Nmen in the nasopharyngeal epithelial cell line, Detroit 562, and that Nlac achieves this by attenuation of secretory cytokine (TNF-α and IL-6) and to a lesser extent chemokine (IL-8 and RANTES) responses. Culture of Detroit cells with Nlac inhibited the induction of cytokine-chemokine mRNA by Nmen, reduced Nmen-induced NF-κß activity and increased constitutive PPAR-γ protein expression. Pretreatment of Detroit cells with a PPAR-γ antagonist abrogated the attenuation of inflammatory IL-6 by Nlac, as did heat-killing of the organisms and preventing their invasion with cytochalasin D. Inflammatory responses from Detroit cells were readily attenuated by Nlac following stimulation with pathogenic Nmen but more specifically following stimulation with the TLR-1/2 agonist Pam3Cys and pro-inflammatory cytokines (IL-1ß, TNF-α) but not LTA or LPS. These results indicate that Nlac plays an important role in suppressing pathogen-induced inflammation in the nasopharyngeal mucosa, mediated through TLR-1/2 stimulation, by activating PPAR-γ and inhibiting NF-κß activity.
Subject(s)
Cytokines/immunology , Epithelial Cells/microbiology , Nasopharynx/cytology , Neisseria lactamica/immunology , PPAR gamma/metabolism , Toll-Like Receptor 1/immunology , Toll-Like Receptor 2/immunology , Animals , Cell Line , Chemokines/immunology , Chemokines/metabolism , Cytokines/metabolism , Epithelial Cells/cytology , Epithelial Cells/immunology , Host-Pathogen Interactions , Humans , NF-kappa B/metabolismABSTRACT
Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37 degrees C, at an operating potential of +0.4V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4 nA/mM and a linear range of 0.45 to 9.0 mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55 kJ mol(-1) was calculated. In culture medium (pH 7.3), a sensitivity of 13 nA/mM was obtained and the response was linear up to 5 mM with a detection limit of 0.5 mM. The working concentration was up to 20 mM glucose with a precision of 11.3% for replicate biosensors (n=4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400 s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R(2)=0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20mM, with those determined spectrophotometrically (R(2)=0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9 nmol/(10(6) cells min) based on a 24-h period in culture.
Subject(s)
Biosensing Techniques/methods , Cell Count/instrumentation , Glucose/analysis , Biosensing Techniques/instrumentation , Cell Line, Tumor , Electrochemistry/instrumentation , Electrochemistry/methods , Humans , Reproducibility of ResultsABSTRACT
AIMS/HYPOTHESIS: Patients with type 1 diabetes mellitus are more susceptible than healthy individuals to exercise-induced oxidative stress and vascular endothelial dysfunction, which has important implications for the progression of disease. Thus, in the present study, we designed a randomised double-blind, placebo-controlled trial to test the original hypothesis that oral prophylaxis with vitamin C attenuates rest and exercise-induced free radical-mediated lipid peroxidation in type 1 diabetes mellitus. METHODS: All data were collected from hospitalised diabetic patients. The electron paramagnetic resonance spectroscopic detection of spin-trapped alpha-phenyl-tert-butylnitrone (PBN) adducts was combined with the use of supporting markers of lipid peroxidation and non-enzymatic antioxidants to assess exercise-induced oxidative stress in male patients with type 1 diabetes (HbA(1c) 7.9 +/- 1%, n = 12) and healthy controls (HbA(1c) 4.6 +/- 0.5%, n = 14). Following participant randomisation using numbers in a sealed envelope, venous blood samples were obtained at rest, after a maximal exercise challenge and before and 2 h after oral ingestion of 1 g ascorbate or placebo. Participants and lead investigators were blinded to the administration of either placebo or ascorbate treatments. Primary outcome was the difference in changes in free radicals following ascorbate ingestion. RESULTS: Six diabetic patients and seven healthy control participants were randomised to each of the placebo and ascorbate groups. Diabetic patients (n = 12) exhibited an elevated concentration of PBN adducts (p < 0.05 vs healthy, n = 14), which were confirmed as secondary, lipid-derived oxygen-centred alkoxyl (RO.) radicals (a(nitrogen) = 1.37 mT and abeta(hydrogen) = 0.18 mT). Lipid hydroperoxides were also selectively elevated and associated with a depression of retinol and lycopene (p < 0.05 vs healthy). Vitamin C supplementation increased plasma vitamin C concentration to a similar degree in both groups (p < 0.05 vs pre-supplementation) and attenuated the exercise-induced oxidative stress response (p < 0.05 vs healthy). There were no selective treatment differences between groups in the primary outcome variable. CONCLUSIONS/INTERPRETATION: These findings are the first to suggest that oral vitamin C supplementation provides an effective prophylaxis against exercise-induced free radical-mediated lipid peroxidation in human diabetic blood. CLINICAL TRIALS REGISTRATION NUMBER: ISRCTN96164937.
Subject(s)
Ascorbic Acid/therapeutic use , Diabetes Mellitus, Type 1/blood , Exercise/physiology , Free Radicals/blood , Adolescent , Adult , Body Mass Index , Diabetes Mellitus, Type 1/drug therapy , Double-Blind Method , Electron Spin Resonance Spectroscopy , Energy Metabolism , Free Radicals/metabolism , Glycated Hemoglobin/metabolism , Humans , Hypoglycemic Agents/therapeutic use , Insulin/therapeutic use , Male , Oxidative Stress/drug effects , Patient Selection , Reference ValuesABSTRACT
We report proof of principle here of a gel encapsulation technique that departs from the minimum surface area to volume restriction of spherical microcapsules and allows gelation of preformed high-density (>or=2x10(4) cells/aggregate) 3-D HepG2 cell aggregates. The process involves forming a discoid 3-D cell aggregate in an ultrasound standing wave trap (USWT), which is subsequently recovered and encapsulated in alginate/CaCl2 hydrogel. The size of the ultrasound-formed aggregates was dependent upon the initial cell concentration, and was in the range of 0.4-2.6 mm in diameter (for cell concentrations ranging between 10(4) and 5x10(6)/ml). At low cell concentrations (
Subject(s)
Alginates , Carcinoma, Hepatocellular/pathology , Hepatocytes/pathology , Ultrasonics , Actins/metabolism , Albumins/metabolism , Biocompatible Materials , Calcium Chloride , Cell Aggregation/physiology , Cell Death , Cell Proliferation , Cell Survival/physiology , Hepatocytes/metabolism , Humans , Hydrogel, Polyethylene Glycol DimethacrylateABSTRACT
The major phospholipid in pulmonary surfactant dipalmitoyl phosphatidylcholine (DPPC) has been shown to modulate inflammatory responses. Using human monocytes, this study demonstrates that DPPC significantly increased PGE(2) (P<0.05) production by 2.5-fold when compared to untreated monocyte controls. Mechanistically, this effect was concomitant with an increase in COX-2 expression which was abrogated in the presence of a COX-2 inhibitor. The regulation of COX-2 expression was independent of NF-kappaB activity. Further, DPPC increased the phosphorylation of the cyclic AMP response element binding protein (CREB; an important nuclear transcription factor important in regulating COX-2 expression). In addition, we also show that changing the fatty acid groups of PC (e.g. using l-alpha-phosphatidylcholine beta-arachidonoyl-gamma-palmitoyl (PAPC)) has a profound effect on the regulation of COX-2 expression and CREB activation. This study provides new evidence for the anti-inflammatory activity of DPPC and that this activity is at least in part mediated via CREB activation of COX-2.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Cyclic AMP Response Element-Binding Protein/antagonists & inhibitors , Cyclooxygenase 2/metabolism , Monocytes/enzymology , Pulmonary Surfactants/metabolism , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/pharmacology , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclooxygenase 2/genetics , Dinoprostone/biosynthesis , Dinoprostone/metabolism , Humans , Monocytes/drug effects , Phosphorylation , Pulmonary Surfactants/chemistry , Pulmonary Surfactants/pharmacologyABSTRACT
AIMS: Type 2 diabetes mellitus (T2DM) is associated with enhanced platelet activation. We conducted a randomised double-blind study to compare the effects of combination metformin and rosiglitazone or metformin and gliclazide therapy on platelet function in persons with T2DM. METHODS: Fifty subjects on metformin monotherapy received either rosiglitazone 4 mg or gliclazide 80 mg. HbA1c, HOMA-R, markers of platelet activation, inflammation, endothelial activation and oxidative stress were measured at baseline and after 24 weeks of treatment. Separate in vitro platelet function studies were conducted on platelets pre-incubated with rosiglitazone and gliclazide. RESULTS: A significantly greater reduction in platelet aggregation was observed in the rosiglitazone treated group compared to gliclazide. HbA1c and markers of endothelial activation were reduced to a similar extent in both groups. A significant reduction in HOMA-R, markers of inflammation and oxidative stress was only observed with rosiglitazone. Reduction in platelet aggregation with rosiglitazone correlated with reduction in oxidative stress. In the in vitro study, rosiglitazone produced significantly greater reduction in platelet aggregation compared with gliclazide. CONCLUSION: Greater reduction in platelet activity observed with rosiglitazone may be related to reduced oxidative stress and a possible direct PPARgamma mediated effect on platelet function.
Subject(s)
Blood Platelets/drug effects , CD40 Ligand/drug effects , Diabetes Mellitus, Type 2/drug therapy , Gliclazide/pharmacology , Platelet Aggregation/drug effects , Thiazolidinediones/pharmacology , Adult , Aged , Blood Glucose/drug effects , Diabetes Mellitus, Type 2/blood , Female , Humans , Hypoglycemic Agents , Male , Middle Aged , Oxidative Stress/drug effects , PPAR gamma/drug effects , PPAR gamma/metabolism , RosiglitazoneABSTRACT
BACKGROUND: Endothelial dysfunction (ED) has been described in Type 2 diabetes (T2DM). We have described previously a diminution of flow-mediated arterial dilatation and, by implication, further ED in T2DM in response to postprandial lipaemia (PPL) at 4 h. This is possibly mediated by oxidative stress/alteration of the nitric oxide (NO) pathway. T2DM subjects tend to exhibit both exaggerated and prolonged PPL. We therefore studied the relationship of PPL to the duration of ED in T2DM subjects and oxidative stress with or without the antioxidant, vitamin C. METHODS: Twenty subjects with T2DM with moderate glycaemic control (mean HbA1c 8.4%) were studied. After an overnight fast, all subjects consumed a standard fat meal. Endothelial function (EF), lipid profiles, and venous free radicals were measured in the fasting, peak lipaemic phase (4 h) and postprandially to 8 h. The study was repeated in a double-blinded manner with placebo, vitamin C (1 g) therapy for 2 days prior to re-testing and with the fat meal. Oxidative stress was assessed by lipid-derived free radicals in plasma, ex vivo by electron paramagnetic resonance spectroscopy (EPR) and by markers of lipid peroxidation (TBARS). Endothelial function was assessed by flow-mediated vasodilatation (FMD) of the brachial artery. RESULTS: There was a significant decrease in endothelial function in response to PPL from baseline (B) 1.3 +/- 1.3% to 4 h 0.22 +/- 1.1% (P < 0.05) and 8 h 0.7 +/- 0.9% (P < 0.05) (mean +/- sem). The endothelial dysfunction seen was attenuated at each time point with vitamin C. Baseline EF with vitamin C changed from (fasting) 3.8 +/- 0.9-2.8 +/- 0.8 (at 4 h) and 2.9 +/- 1.3 (at 8 h) in response to PPL. Vitamin C attenuated postprandial (PP) oxidative stress significantly only at the 4-h time point [301.1 +/- 118 (B) to 224.7 +/- 72 P < 0.05] and not at 8 h 301.1 +/- 118 (B) to 260 +/- 183 (P = NS). There were no changes with placebo treatment in any variable. PPL was associated with a PP rise in TG levels (in mmol/l) from (B) 1.8 +/- 1 to 2.7 +/- 1 at 4 h and 1.95 +/- 1.2 at 8 h (P = 0.0002 and 0.33, respectively). CONCLUSION: PPL is associated with prolonged endothelial dysfunction for at least 8 h after a fatty meal. Vitamin C treatment improves endothelial dysfunction at all time points and attenuates PPL-induced oxidative stress. This highlights the importance of low-fat meals in T2DM and suggests a role for vitamin C therapy to improve endothelial function during meal ingestion.
Subject(s)
Antioxidants/administration & dosage , Ascorbic Acid/administration & dosage , Diabetes Mellitus, Type 2/physiopathology , Endothelium/physiopathology , Lipids/blood , Administration, Oral , Adult , Aged , Area Under Curve , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/drug therapy , Dietary Fats/administration & dosage , Endothelium/drug effects , Female , Humans , Lipid Peroxidation/drug effects , Lipid Peroxidation/physiology , Lipoproteins, VLDL/blood , Male , Middle Aged , Oxidative Stress/drug effects , Oxidative Stress/physiology , Postprandial Period , Triglycerides/blood , Vasodilation/drug effects , Vasodilation/physiologyABSTRACT
Congestive heart failure (CHF) is associated with oxidative stress. Platelet responsiveness to nitric oxide (NO) donors, are impaired in patients with angina pectoris, possibly by increasing oxidative stress. We investigated the occurrence of platelet resistance to NO in patients, with ischaemic or non-ischaemic cardiomyopathy compared with normal subjects. Anti-aggregatory effects of sodium nitroprusside (SNP), oxidative stress and whole blood superoxide anion content were determined, with correlates of responsiveness to SNP. Inhibition of platelet aggregation by SNP was 65.4+/-3.55% in controls and 59.3+/-4.1% in CHF (P=ns) despite increased oxidative stress and post-aggregation O2- in CHF patients. However, subsets of CHF patients have NO-resistant platelets: this is associated with increasing age and/or increased oxidative stress (both p<0.05).
Subject(s)
Heart Failure/physiopathology , Nitric Oxide Donors/pharmacology , Nitroprusside/pharmacology , Oxidative Stress/physiology , Platelet Aggregation/drug effects , Adult , Age Factors , Aged , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Case-Control Studies , Cell Culture Techniques , Female , Heart Failure/drug therapy , Humans , Male , Middle Aged , Severity of Illness IndexABSTRACT
Although many aspects of firefly bioluminescence are understood, the mechanism by which adult fireflies produce light as discrete rapid flashes is not. Here we examine the most postulated theory, that flashing is controlled by gating oxygen access to the light-emitting cells (photocytes). According to this theory, the dark state represents repression of bioluminescence by limiting oxygen, which is required for bioluminescence; relief from this repression by transiently allowing oxygen access to the photocytes allows the flash. We show that normobaric hyperoxia releases the repression of light emission in the dark state of both spontaneously flashing and non-flashing fireflies, causing continual glowing, and we measure the kinetics of this process. Secondly, we determine the length of the barriers to oxygen diffusion to the photocytes in the aqueous and gas phases. Thirdly, we provide constraints upon the distance between any gas-phase gating structure(s) and the photocytes. We conclude from these data that the flash of the adult firefly is controlled by gating of oxygen to the photocytes, and demonstrate that this control mechanism is likely to act by modulating the levels of fluid in the tracheoles supplying photocytes, providing a variable barrier to oxygen diffusion.
Subject(s)
Coleoptera/physiology , Luminescent Measurements , Oxygen/physiology , Periodicity , Adenosine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Coleoptera/anatomy & histology , Diffusion , Electric Stimulation , Firefly Luciferin/metabolism , Kinetics , Luciferases/metabolism , Oxygen/administration & dosageABSTRACT
Phosphatidylcholine (PC) is the major phospholipid of pulmonary surfactant and it is hypothesized that PC and its subspecies modulate the functions of alveolar macrophages. The most abundant of these subspecies is dipalmitoylphosphatidylcholine (DPPC). This study was undertaken to determine the effect of PC on monocyte function using a human monocytic cell line, MonoMac-6 (MM6). This study showed that preincubation of MM6 cells with DPPC at 125 microg/ml for 2 h inhibited the oxidative response to either zymosan or phorbol-12-myristate-13-acetate (PMA) by 30% (P < 0.001). This inhibition with DPPC was independent of LPS priming. When DPPC was replaced with 1-palmitoyl-2-arachidonoyl phosphatidylcholine (PAPC) there was no inhibition and in contrast a significant increase in oxidant production was observed. We also demonstrated that total PC (tPC; a heterogeneous species of PC from egg) and DPPC but not PAPC significantly inhibited the release of TNF-alpha from MM6 cells (P < 0.05). DPPC did not inhibit phosphorylation of the mitogen activated protein kinases (MAPKs) p44/p42 or p38 in stimulated cells. Measurements of membrane fluidity with spin label EPR spectroscopy indicate that DPPC incorporation significantly alters the membrane fluidity of MM6 cells. These results suggest that DPPC, the major component of pulmonary surfactant, may play a role in modulating leucocyte inflammatory responses in the lung. This may in part be related to membrane effects but does not include alterations in p44/p42 or p38 MAPK signalling.