Natural antioxidants attenuate mycolactone toxicity to RAW 264.7 macrophages.

Mycobacterium ulcerans produces a macrolide exotoxin, mycolactone which suppresses immune cells activity, is toxic to most cells and the key virulence factor in the pathogenesis of Buruli ulcer disease. Mycolactone is reported to mediate the production of reactive oxygen species in keratinocytes; cells that play critical role in wound healing. Increased levels of reactive oxygen species have been shown to disrupt the well-ordered process of wound repair; hence, the function of wound-healing cells such as macrophages, keratinocytes, and fibroblast could be impaired in the presence of the reactive oxygen species mediator, mycolactone. To ensure regeneration of tissues in chronic ulcers, with proper and timely healing of the wounds, natural antioxidants that can combat the effects of induced reactive oxygen species in wound-healing cells ought to be investigated. Reactive oxygen species activity was determined in mycolactone-treated RAW 264.7 macrophages and the scavenging ability of the antioxidants (ascorbic acid, gallic acid, and green tea kombucha) against mycolactone-induced reactive oxygen species (superoxide anions) was assessed using fluorescein probe (DCF-DA) and nitroblue tetrazolium dye. Cytotoxicity of the antioxidants, mycolactone, and the protective effect of the antioxidants on the cells upon treatment with mycolactone were determined using the Alamar blue assay. The expression levels of endogenous antioxidant enzyme genes (superoxide dismutase, catalase, and glutathione peroxidase) in response to mycolactone-mediated reactive oxygen species were determined using RT-qPCR. Mycolactone induced the production of reactive oxygen species in RAW 264.7 macrophages, and the resulting superoxide anions were scavenged by some of the antioxidants. The selected endogenous antioxidant enzyme genes in the macrophages were upregulated in the presence of the antioxidants and mycolactone. The exogenously supplied ascorbic acid and green tea kombucha offered moderate protection to the macrophages against the toxicity of mycolactone. We conclude that the results provide insights into alternate and adjunct therapeutic approaches in Buruli ulcer treatment, which could significantly attenuate the toxicity of the pathogenic factor; mycolactone.

Antioxidant and free radical scavenging activity of iron chelators.

Inside the human body, reactive derivatives of oxygen, known as reactive oxygen species (ROS) such as the superoxide radical (O2•), hydroxyl radical (•OH) and hydrogen peroxide (H2O2), are constantly generated. The ROS easily cause oxidative damage to various biomolecules such as proteins, lipids and DNA leading to various disease conditions. Iron chelators function as antioxidants by scavenging ROS and also reduce the amount of available iron thereby decreasing the quantity of •OH generated by Fenton reactions. In this study, the antioxidant activity of the iron chelators: caffeic acid (CA), 2,3-dihydroxybenzoic acid (DHBA), desferroxamine B (FOB) and benzohydroxamic acid (BHA) were determined using five different in vitro antioxidant assays. The antioxidant assays used were: iron binding ability, reducing ability using the potassium ferricyanide reduction method, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, H2O2 scavenging activity and •OH scavenging activity. The standard used for the iron binding ability was Na2EDTA whereas vitamin C was used as a standard for the remaining assays. The iron chelators showed a concentration dependent increase in their radical scavenging activities as well as their reducing ability. At the concentration of 1 mM, FOB had the highest iron binding ability of 93.7% whereas DHBA had the lowest iron binding ability of 5.0% compared to the standard Na2EDTA which had 94.8%. The iron chelators, with the exception of BHA, showed good reducing ability than vitamin C. Caffeic acid showed significant DPPH, hydrogen peroxide and hydroxyl radical scavenging activities of 84.7%, 99.8% and 14.5%, respectively. All the iron chelators were observed to show significant activities in all five antioxidant assays.

Levels of soluble CD163 and severity of malaria in children in Ghana.

CD163 is an acute-phase-regulated monocyte/macrophage membrane receptor expressed late in inflammation. It is involved in the haptoglobin-mediated removal of free hemoglobin from plasma, has been identified as a naturally soluble plasma glycoprotein with potential anti-inflammatory properties, and is possibly linked to an individual's haptoglobin phenotype. High levels of soluble CD163 (sCD163) in a malaria episode may therefore downregulate inflammation and curb disease severity. In order to verify this, the relationships between sCD163 levels, malaria severity, and selected inflammatory mediators (tumor necrosis factor alpha [TNF-alpha], interleukin-6 [IL-6], and IL-10) were assessed by enzyme-linked immunosorbent assay using plasma samples obtained from pediatric malaria patients with uncomplicated malaria (UM [n = 38]), cerebral malaria (CM [n = 52]), and severe malarial anemia (SA [n = 55]) during two consecutive malaria transmission seasons (2002 and 2003). Median sCD163 levels were higher in UM (11.9 microg/ml) patients than in SA (7.7 microg/ml; P = 0.010) and CM (8.0 microg/ml; P = 0.031) patients. Levels of sCD163 were also higher in all patient groups than in a group of 81 age-matched healthy controls. The higher sCD163/TNF-alpha ratio in UM patients, coupled with the fact that sCD163 levels correlated with TNF-alpha levels in UM patients but not in CM and SA patients, suggests inflammatory dysregulation in the complicated cases. The study showed that sCD163 levels are elevated during acute malaria. High sCD163 levels in UM patients may be due to the induction of higher-level anti-inflammatory responses, enabling them to avoid disease complications. It is also possible that UM patients simply lost their CD163 receptors from macrophages in inflammatory sites while complicated-malaria patients still had their receptors attached to activated macrophages, reflecting ongoing and higher-level inflammation associated with complicated malaria.