A novel red blood cell substitute based on crosslinked hemoglobin, superoxide dismutase, and catalase /

Modified hemoglobin red blood cell substitutes have a number of potential areas of application. In some of these applications, it will be important to lessen the pro-oxidant effects of hemoglobin and potential free radical-mediated toxicity. This research introduces a novel modified hemoglobin that is based on intermolecularly crosslinking hemoglobin, superoxide dismutase and catalase (PolyHb-SOD-CAT) with the bifunctional agent, glutaraldehyde. Superoxide dismutase and catalase catalyze the breakdown of superoxide radical and hydrogen peroxide respectively. Studies of structural and functional parameters reveal that PolyHb-SOD-CAT retains superoxide dismutase and catalase enzymatic activity, and consists of a mixture of molecular species ranging in molecular size and protein composition. Circulation time studies of PolyHb-SOD-CAT in rats show that hemoglobin, superoxide dismutase and catalase possess longer circulatory half-lives as compared to the free forms of these proteins. Studies also show that PolyHb-SOD-CAT prevents the formation of methemoglobin, ferrylhemoglobin, hydroxyl radical, free iron, and lipid peroxidation. Ischemia-reperfusion studies using isolated perfused hindlimbs and intestine of rat show that PolyHb-SOD-CAT reduced the formation of hydroxyl radical compared to PolyHb. Altogether, these results suggest that PolyHb-SOD-CAT is a potentially safer modified hemoglobin oxygen carrier by virtue of its ability to detoxify reactive oxygen species, and reduced propensity to promote and participate in oxidative processes

Extracellular Hb Enhances Cardiac Toxicity in Endotoxemic Guinea Pigs: Protective Role of Haptoglobin

Endotoxemia plays a major causative role in the myocardial injury and dysfunction associated with sepsis. Extracellular hemoglobin (Hb) has been shown to enhance the pathophysiology of endotoxemia. In the present study, we examined the myocardial pathophysiology in guinea pigs infused with lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, and purified Hb. We also examined whether the administration of the Hb scavenger haptoglobin (Hp) could protect against the effects observed. Here, we show that Hb infusion following LPS administration, but not either insult alone, increased myocardial iron deposition, heme oxygenase-1 expression, phagocyte activation and infiltration, as well as oxidative DNA damage and apoptosis assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) immunostaining, respectively. Co-administration of Hp significantly attenuated the myocardial events induced by the combination of LPS and Hb. These findings may have relevant therapeutic implications for the management of sepsis during concomitant disease or clinical interventions associated with the increased co-exposures to LPS and Hb, such as trauma, surgery or massive blood transfusions

The Large Clostridial Toxins from Clostridium sordellii and C. difficile Repress Glucocorticoid Receptor Activity▿

We have previously shown that Bacillus anthracis lethal toxin represses glucocorticoid receptor (GR) transactivation. We now report that repression of GR activity also occurs with the large clostridial toxins produced by Clostridium sordellii and C. difficile. This was demonstrated using a transient transfection assay system for GR transactivation. We also report that C. sordellii lethal toxin inhibited GR function in an ex vivo assay, where toxin reduced the dexamethasone suppression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Furthermore, the glucocorticoid antagonist RU-486 in combination with C. sordellii lethal toxin additively prevented glucocorticoid suppression of TNF-α. These findings corroborate the fact that GR is a target for the toxin and suggest a physiological role for toxin-associated GR repression in inflammation. Finally, we show that this repression is associated with toxins that inactivate p38 mitogen-activated protein kinase (MAPK)

Blood-Brain Barrier Disruption and Oxidative Stress in Guinea Pig after Systemic Exposure to Modified Cell-Free Hemoglobin

Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis