In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.

Sepsis is a life-threatening response to infection associated with inflammation, oxidative stress and mitochondrial dysfunction. We investigated differential effects of three forms of vitamin E, which accumulate in different cellular compartments, on oxidative stress, mitochondrial function, mRNA and protein expression profiles associated with the human Toll-like receptor (TLR) -2 and -4 pathways. Human endothelial cells were exposed to lipopolysaccharide (LPS)/peptidoglycan G (PepG) to mimic sepsis, MitoVitE, α-tocopherol, or Trolox. Oxidative stress, mitochondrial function, mitochondrial membrane potential and metabolic activity were measured. NFκB-P65, total and phosphorylated inhibitor of NFκB alpha (NFκBIA), and STAT-3 in nuclear extracts, interleukin (IL)-6 and IL-8 production in culture supernatants and cellular mRNA expression of 32 genes involved in Toll-like receptor-2 and -4 pathways were measured. Exposure to LPS/PepG caused increased total radical production (p = 0.022), decreased glutathione ratio (p = 0.016), reduced membrane potential and metabolic activity (both p < 0.0001), increased nuclear NFκB-P65 expression (p = 0.016) and increased IL-6/8 secretion (both p < 0.0001). MitoVitE, α- tocopherol and Trolox were similar in reducing oxidative stress, NFκB activation and interleukin secretion. MitoVitE had widespread downregulatory effects on gene expression. Despite differences in site of actions, all forms of vitamin E were protective under conditions mimicking sepsis. These results challenge the concept that protection inside mitochondria provides better protection.

Many links between gut microbiota and disease development have been established in recent years, with particular bacterial strains emerging as potential therapeutics rather than causative agents. In this study we describe the immunostimulatory properties of Enterococcus gallinarum MRx0518, a candidate live biotherapeutic with proven anti-tumorigenic efficacy. Here we demonstrate that strain MRx0518 elicits a strong pro-inflammatory response in key components of the innate immune system but also in intestinal epithelial cells. Using a flagellin knock-out derivative and purified recombinant protein, MRx0518 flagellin was shown to be a TLR5 and NF-κB activator in reporter cells and an inducer of IL-8 production by HT29-MTX cells. E. gallinarum flagellin proteins display a high level of sequence diversity and the flagellin produced by MRx0518 was shown to be more potent than flagellin from E. gallinarum DSM100110. Collectively, these data infer that flagellin may play a role in the therapeutic properties of E. gallinarum MRx0518.

BackgroundReactive oxygen species and other free radicals, together with glucose and its metabolites are believed to play important roles in the aging process. The carbohydrate components of glycosylated proteins are important in mediating cell-cell interactions and a role has been suggested for them in the aging process. Erythrocytes are critical cells in the human body, heavily glycosylated and relatively easily available and so are good candidates to yield insights into how patterns of glycosylation change with age and disease. It has been claimed, based on a periodic acid Schiff assay, that human aging is associated with a decline of erythrocyte surface sialic acids. Plant lectins allow for more specific assays for glycans, including determining the linkage of sialic acids and analysis of single cells by flow cytometry. ResultsSialic acid specific plant lectins, Maackia amurensis lectin II (MAL), binding to α-2,3 linked sialic acids and Sambucus nigra (SNA), α-2,6 sialic acids, were used to show that erythrocyte surface sialic acids have no significant associations with donor age. A combination of storage and cellular aging produces a specific loss of α-2,6 sialic acids. By contrast, erythrocyte surface terminal fucoses increase significantly with age of donor. In order to determine which aspects of aging are important in determining this change, we investigated whether this novel human aging biomarker is associated with higher plasma glucose values, assessed by glycated hemoglobin (HbA1c) and reactive oxygen species (ROS) generation. Fucose levels were associated with HbA1c levels, but not ROS generation. ConclusionOur study identifies novel glycan-based biomarkers for human aging and disease. The simplicity of lectins are presented here as an attractive tool to study cellular medication in aging and disease.

We thank Drs Hegarty and Byrne for their interest in our paper and appreciate the opportunity to respond to their insightful comments [...]