Project description:The technique of competitive double-labelling [H. Kaplan, K.J. Stevenson & B.S. Hartley, (1971) Biochem. J. 124, 289-299; L.P. Visentin & H. Kaplan (1975) Biochemistry 14, 463-468] was used to determine the reactivity of some amino groups towards acetic anhydride in deoxy-and liganded haemoglobin. Only those amino groups known to form salt bridges in deoxy-but not in liganded haemoglobin (i.e. the alpha-amino group of valine-1 alpha and the xi-amino group of lysine-40 alpha and lysine-127 alpha [M. F. Perutz (1970) Nature (London) 228, 726-739]) and different reactivities in the two structures.
Project description:One of the difficulties in creating a blood substitute on the basis of human haemoglobin (Hb) is the toxic nature of Hb when it is outside the safe environment of the red blood cells. The plasma protein haptoglobin (Hp) takes care of the Hb physiologically leaked into the plasma - it binds Hb and makes it much less toxic while retaining the Hb's high oxygen transporting capacity. We used Electron Paramagnetic Resonance (EPR) spectroscopy to show that the protein bound radical induced by H 2O 2 in Hb and Hp-Hb complex is formed on the same tyrosine residue(s), but, in the complex, the radical is found in a more hydrophobic environment and decays slower than in unbound Hb, thus mitigating its oxidative capacity. The data obtained in this study might set new directions in engineering blood substitutes for transfusion that would have the oxygen transporting efficiency typical of Hb, but which would be non-toxic.
Project description:Compound heterozygotes for sickle haemoglobin (HbS) and hereditary persistence of fetal haemoglobin (HPFH) have high fetal haemoglobin (HbF) levels but few, if any, sickle cell disease-related complications. We studied 30 cases of HbS-HPFH (types 1 and 2), confirmed by molecular analysis, and report the haematological features and change in HbF levels over time. These results were compared to those of patients with sickle cell anaemia or HbS-β(0) thalassaemia, including a subgroup of patients carrying the XmnI polymorphism, known to be associated with elevated HbF. Among the HbS-HPFH patients, HbF level was 50-90% during infancy and declined steeply within the first few years of life, stabilizing between ages 3 and 5years, at approximately 30%. Mean HbF of individuals age 5 or older was 31±3%, average haemoglobin concentration was 130±10g/l and average mean corpuscular volume (MCV) was 75±4 fl. Univariate and multivariate regression analyses significantly associated HbF with age, haemoglobin concentration, and MCV (P<0·001). There was a strong inverse association between HbF and age (r=-0·9, P<0·001). Despite having a much higher HbF level, patients with HbS-HPFH have a similar age-related pattern of HbF decline and associations as patients with sickle cell anaemia or HbS-β(0) thalassaemia.
Project description:BackgroundHaemolytic infection lyses red blood cells, releasing haemoglobin (Hb) into the plasma. Although recent studies showed that immune cells recognize redox-active cytotoxic extracellular Hb (metHb) bound to pathogen-associated molecular patterns (PAMPs), currently available information is limited to experiments performed in defined conditions using single cell lines. Therefore, a systemic approach targeting primary whole blood cells is required to better understand the cellular immune defence against metHb and PAMPs, when under a haemolytic infection.MethodsWe investigated how human white blood cells, including neutrophils, respond to metHb and lipoteichoic acid (LTA) by measuring reactive oxygen species (ROS), signalling mediators (ERK and p38), NF-?B, cytokines, elastase secretion and cell activation markers.FindingsmetHb activates NF-?B in TLR2-expressing HEK293 cells but not in normal or TLR9-expressing HEK293 cells. Treatment of isolated neutrophils with metHb increased production of ROS and expressions of IL-8, TNF?, and CD11b, which were further enhanced by metHb + LTA complex. While LTA stimulated the survival of neutrophils, it caused apoptotic cell death when complexed with metHb. The activation of neutrophils by metHb + LTA was subdued by the presence of other types of white blood cells.InterpretationmetHb and metHb + LTA complex are ligands of TLR2, inducing an unconventional TLR signalling pathway. Neutrophils are a highly sensitive cell type to metHb + LTA complex. During a haemolytic infection, white blood cells in the vicinity crosstalk to modulate neutrophil TLR-signalling induced by metHb and LTA.
Project description:We investigated the cytoprotective role of the dietary polyphenols on putative damage induced by Amadori adducts in Human Peritoneal Mesothelial Cells (HPMCs). Increased accumulation of early products of non-enzymatic protein glycation-Amadori adducts-in the peritoneal dialysis fluid due to their high glucose, induces severe damage in mesothelial cells during peritoneal dialysis. Dietary polyphenols reportedly have numerous health benefits in various diseases and have been used as an efficient antioxidant in the context of several oxidative stress-related pathologies. HPMCs isolated from different patients were exposed to Amadori adducts (highly glycated haemoglobin, at physiological concentrations), and subsequently treated with several polyphenols, mostly presented in our Mediterranean diet. We studied several Amadori-induced effects in pro-apoptotic and oxidative stress markers, as well as the expression of several pro-inflammatory genes (nuclear factor-kappaB, NF-kB; inducible Nitric Oxide synthetase, iNOS), different caspase-activities, level of P53 protein or production of different reactive oxygen species in the presence of different polyphenols. In fact, cytoprotective agents such as dietary polyphenols may represent an alternate approach to protect mesothelial cells from the cytotoxicity of Amadori adducts. The interference with the Amadori adducts-triggered mechanisms could represent a therapeutic tool to reduce complications associated with peritoneal dialysis in the peritoneum, helping to maintain peritoneal membrane function longer in patients undergoing peritoneal dialysis.