Lesley-Jane Eales-Reynolds

Reactive Oxygen Species Are Involved in Smoking-Induced Dysfunction of Nitric Oxide Biosynthesis and Upregulation of Endothelial Nitric Oxide Synthase An In Vitro Demonstration in Human Coronary Artery Endothelial Cells

Background—Our group has previously shown that human umbilical vein endothelial cells exposed to smokers’ serum decreased nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in the presence of increased eNOS expression. In the present study, we examined whether these observations extended to human coronary artery endothelial cells (HCAECs). In addition, the role of reactive oxygen species in the observed alterations was examined. Methods and Results—HCAECs were incubated with serum from 10 nonsmokers and 15 smokers for 12 hours with or without the addition of either polyethylene glycol-superoxide dismutase (PEG-SOD, 300 U/mL), PEG-SOD+PEG-catalase (1000 U/mL), chelerythrine (3 &mgr;mol/L), or tetrahydrobiopterin (20 &mgr;mol/L). At the end of incubation, NO, eNOS protein, and eNOS activity were measured from the same culture. HCAECs incubated with smokers’ serum alone showed significantly lower NO production (P <0.05) and eNOS activity (P <0.005) but higher eNOS expression (P <0.005) compared with nonsmokers. In smokers, addition of PEG-SOD, PEG-SOD+PEG-catalase, or tetrahydrobiopterin significantly (P <0.05) improved NO levels and eNOS activity. Interestingly, in the same smokers, a significant decrease in eNOS expression was only seen with the addition of PEG-SOD+PEG-catalase (P <0.05) and treatment with PEG-SOD alone insignificantly increased eNOS expression. Conclusions—The present study indicates that in vitro, HCAECs show similar changes in NO biosynthesis as human umbilical vein endothelial cells when exposed to smokers’ serum and also confirms that oxidative stress plays a central role in smoking-mediated dysfunction of NO biosynthesis in endothelial cells. Furthermore, these data support other studies suggesting a role for hydrogen peroxide in the upregulation of eNOS.

Generation and characterization of a PhoP homologue mutant of Neisseria meningitidis

Two‐component regulatory systems are important regulators of virulence genes in a number of bacteria. Genes encoding a two‐component regulator system, with homology to the phoP/phoQ system in salmonella, were identified in the meningococcal genome. Allele replacement was used to generate a meningococcal knock‐out mutant of the regulator component of this system, and its phenotype was examined. The mutant displayed many differences in protein profiles compared with wild type, consistent with it being a gene‐regulatory mutation. Many of the growth characteristics of the mutant were similar to those of phoP mutants of salmonella: it was unable to grow at low concentrations of magnesium and was sensitive to defensins and other environmental stresses. Magnesium‐regulated differences in protein expression were abrogated in the mutant, indicating that the meningococcal PhoP/PhoQ system may, as in salmonella, respond to changes in environmental magnesium levels. These results are consistent with the PhoP homologue playing a similar role in the meningococcus to PhoP in salmonella and suggest that it may similarly be involved in the regulation of virulence genes in response to environmental stimuli in the meningococcus. In support of this conclusion, we found the mutant grew was unable to grow in mouse serum and was attenuated in its ability to traverse through a layer of human epithelial cells. Identification of those genes regulated by the meningococcal PhoP may provide a route towards the identification of virulence genes in the meningococcus.

Monophosphoryl lipid A stimulated up-regulation of reactive oxygen intermediates in human monocytes in vitro

The production of reactive oxygen and nitrogen intermediates is a common response to infectious challenge in vivo. These agents have been implicated in the modulation of cytokine responses and are produced in large amounts in response to endotoxins produced by a number of infectious agents. The antigen‐presenting cell activation caused by these lipopolysacchardies (LPS) has been exploited in the use of these agents as adjuvants. In recent years, less‐toxic derivatives have been sought. One such agent, monophosphoryl lipid A (MPL), has been used increasingly in vivo as an adjuvant and as a modulator of the inflammatory process. It is known that this agent modulates the inflammatory response and cytokine production. In addition, we have shown its effect on the production of reactive nitrogen intermediates. In this paper, we show that MPL stimulates the release of high levels of superoxide (O2−) and hydrogen peroxide (H2O2), the latter being greater than that seen with LPS and appearing to be related to the inability of MPL to stimulate catalase activity. When cells were pretreated with LPS or MPL and subsequently challenged with LPS, the production of O2− and H2O2 was inhibited significantly by LPS and MPL. The concentration of MPL required to induce significant hyporesponsiveness to subsequent LPS challenge was 10 times lower than that of LPS. Hyporesponsiveness was greatest when induced by 10 μg/ml MPL, the same concentration that induced the maximum release of H2O2 in primary stimulation. In addition, we have shown that following MPL pretreatment, LPS stimulation does not cause the loss of cytoplasmic IκBα, which occurs when human monocytes are cultured with LPS. From our results, we propose a model for the reduced toxicity of MPL.

Endogenous free radical generating sources are involved in smoking-mediated dysfunction of nitric oxide biosynthesis in human coronary artery endothelial cells: An in vitro demonstration

Background: Vulnerable plaques contain abundant macrophages. We developed a novel photodynamic agent, chlorin, conjugated with maleylated albumin, (ce6-mal-alb) that concentrates in macrophage-rich plaques and has a high fluorescent yield. As such, we tested hypothesis that experimental atherosclerotic lesions (ATHERO) can be detected using ce6-mal-alb and an intravascular fluorescence spectroscopy catheter. Methods: ATHERO were induced in New Zealand rabbits by infradiaphragmatic aortic balloon-injury followed by high cholesterol diet. At IO weeks, ce6-mal-alb was administered to 7 atherosclerotic and 7 control animals. 24 hours later, aortic uptake of the ce6mal-alb uptake was measured in situ, using an intravascular fluorescence spectroscopy catheter. Thereafter, the aortas were excised and dissolved in NaOH/SDS for spectrofluorimetric determination of ce6 content. Results: Intravascular measurements of ce6-mal-alb fluorescence were higher in ATHERO vs. non-athero segments, (8.8i4.8 vs. 2.2k2.2 X103AU, P<O.OOl, figure). Further, ce6-mal-alb concentration was higher within the ATHERO aortas (5.2t3.2 vs. 1.9+1.2, ce6 fluorescencelgm tissue x IOs, p=<O.Ol). Conclusion: CeG-mal-alb can be employed for intravascular characterization of atherosclerotic plaques. Because this novel PDT compound is selectively toxic to macrophages when light-activated, this agent may be useful for both the detection and therapeutic modification of macrophage-rich plaques.

Making a level playing field at Master’s level — an application of self-directed learning

Abstract This study was designed to investigate the ability of a self-directed learning (SDL) approach to overcome the difficulties encountered in delivering a basic module in immunology to students studying for a Masters degree in Medical Microbiology. Difficulties arise from the wide range of academic and experiential learning skills exhibited by the students on this programme, the part-time, modular structure of the programme and the fact that each module is only provided once every two years necessitating that first and second year students are taught simultaneously. It was hoped that an SDL approach would provide an appropriate outcome for all students (i.e. one which reflected their general academic ability) and would enhance retention/application of the knowledge obtained. The programme was designed using Grow’s model of structured SDL making the assumption that the majority of students would be classed as interested learners (Grow, 1991). Outcomes of this approach were assessed by comparing the results obtained by this cohort of students with a previous one taught didactically. Analysis was performed not only for this module but also for a subsequent practical module in which the students are expected to apply the knowledge obtained in the theoretical module. The results show that despite being an academically weaker cohort, the students taught immunology using the SDL approach (MEW) showed similar results to the cohort taught didactically (OLD). In addition, the results of the OLD cohort were skewed artificially by the abnormally high results obtained in their coursework assignment. Taken together, these results suggest that the SDL approach improved student performance. When the results for the practical module were considered, it was found that students taught didactically tended to do worse in their practical module than their taught module. Fewer students who learnt through the SDL approach showed a decrease in performance in their practical module, the remainder showing a significant increase. Thus, it is concluded that the SDL approach to immunology has improved student performance and retention/application. However, the model needs some refining to allow for individual variation in learning styles.