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Free hemoglobin (Hb) during autoxidation increases 8-iso-prostaglandin-F2-alpha (8-isoprostane) formation in vitro. Because 8-isoprostane and plasma Hb are elevated in chronic renal failure (CRF), we evaluated the role of Hb in this isoprostane synthesis in vivo. By monitoring correlations between Hb, haptoglobin (Hp), CD163-Hb-scavenger receptor, and 8-isoprostane that is known to induce CD163 shedding, we examined whether 8-isoprostane blocks Hb catabolism in CRF. Additionally, by studying the effect of 8-isoprostane on human coronary artery endothelium (HCAEC) in vitro and its impact on intercellular adhesion molecule-1 (ICAM-1) in vivo, we tested its role in promotion of cardiovascular events in CRF. Twenty-two never-dialyzed CRF patients and 18 control patients were screened for renal function, plasma and urine 8-isoprostane, and plasma Hb, Hp, thiobarbituric-acid-reactants (TBARS), C-reactive-protein (CRP), and soluble (s) ICAM-1 and sCD163. HCAEC exposed to 8-isoprostane were tested for ICAM-1 and apoptosis. In CRF, urine 8-isoprostane was significantly elevated and correlated with free-Hb and TBARS. The increased free-Hb, Hp, and sCD163 in CRF suggested 8-isoprostane-mediated suppression of Hb catabolism through CD163 receptor shedding. 8-Isoprostane enhanced ICAM-1 expression and apoptosis in HCAEC. CRF patients showed elevated sICAM-1. In conclusion, free-Hb, via 8-isoprostane, paradoxically blocks its own catabolism. Free-Hb and/or 8-isoprostane may intensify cardiovascular events in CRF.

Chemically modified hemoglobin (Hb) solutions are under current investigation as potential red cell substitutes. Researchers at Texas Tech University have developed a novel free Hb based blood substitute product. This blood substitute is composed of purified bovine Hb cross-linked intramolecularly with o-adenosine-5'-triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione (GSH). In this study, we compared the effects of our novel blood substitute and unmodified (U) Hb, by using allogenic plasma as the control, on human blood components: red blood cells (RBCs), platelets, monocytes (Mo), and low-density lipoproteins (LDLs). The pro-oxidant potential of both Hb solutions on RBCs was examined by the measurement of osmotic and mechanical fragility, conjugated dienes (CD), lipid hydroperoxides (LOOH), thiobarbituric acid reactants (TBAR-S), isoprostanes (8-iso PGF2alpha) and intracellular GSH. The oxidative modification of LDLs was assessed by CD, LOOH, and TBAR-S, and the degree of apolipoprotein (apo) B cross-linking. The effects of Hb on platelets have been studied by monitoring their responses to the aggregation agonists: collagen, ADP, epinephrine, and arachidonic acid. Monocytes were cultured with Hb solutions or plasma and tested for TNF-alpha and IL-1beta release, then examined by electron microscopy. Results indicate that native UHb initiates oxidative stress of many blood components and aggravates inflammatory responses of Mo. It also caused an increase in RBC osmotic and mechanical fragility (p < 0.001). While the level of GSH was slightly changed, the lipid peroxidation of RBC increased (p < 0.001). UHb was found to be a stimulator of 8-iso PGF2alpha synthesis, a potent modulator of LDLs, and an effective potentiator of agonist induced platelet aggregation. Contrarily, our novel blood substitute did not seem to induce oxidative stress nor to increase Mo inflammatory reactions. The osmotic and mechanical fragility of RBCs was similar to that of the control. Such modified Hb failed to alter LDLs, increase the production of 8-iso PGF2alpha, but markedly inhibited platelet aggregation. The effect of this novel blood substitute can be linked with the cytoprotective and anti-inflammatory properties of adenosine, which is used as a cross-linker and surface modifier, and a modification procedure that lowers the hemoglobin pro-oxidant potential.

The authors have previously documented that appropriate chemical and pharmacologic modification of the hemoglobin molecule are required to attenuate certain pathophysiologic reactions of the reticuloendothelium. The current study further investigates the molecular responses of human coronary artery endothelial cells to a high concentration (0.4 mmol) of 1) unmodified bovine hemoglobin; and 2) an improved blood substitute that comprises hemoglobin cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione. In this study, the scavenging effect of hemoglobins toward nitric oxide (NO) was evaluated by the measurement of nitrite (NO2-) and nitrate (NO3-) formation. The pro-oxidant effect of hemoglobin on endothelial cells was examined by the measurement of intracellular reduced glutathione, and by monitoring the formation of lipid hydroperoxides and 8-iso prostaglandin F2alpha, a novel potent vasoconstrictor, which is produced by a noncyclooxygenase mechanism involving free radical catalyzed peroxidation of arachidonic acid. The inflammatory reactions of endothelial cells were evaluated by the expression of the adhesion molecule, intracellular adhesion molecule-1, and the activation of nuclear transcription factor, nuclear factor kappaB. In additional, endothelial cell responses were investigated by analysis of intracellular ionized calcium concentrations. Results indicate that unmodified hemoglobin in a concentration of 0.4 mmol/L can aggravate endothelial cell oxidative and inflammatory responses. This hemoglobin produced a significant (p < 0.01) depletion of reduced glutathione, acceleration of lipid peroxidation, and a greater influx of Ca2+. The formation of 8-iso prostaglandin F2alpha increased compared with the control cells (p < 0.01). Unmodified hemoglobin was found to be a potent scavenger of NO, great activator of nuclear factor kappaB, and a stimulator of intracellular adhesion molecule-1 expression. Contrarily, the improved blood substitute did not appear to induce oxidative stress nor to increase the intracellular Ca2+. The concentration of 8-iso prostaglandin F2alpha was similar to that in the control cells, whereas the formation of NO2-/NO3- was much lower (p < 0.05) than in the unmodified hemoglobin group. The effect of an improved blood substitute can be linked with the anti-inflammatory and cytoprotective properties of adenosine, which is used as a cross-linker and surface modifier, and the type of the chemical modification procedure that lowers hemoglobin pro-oxidant potential.

Objective: The study of the placental HIV infection in cases of seropositive pregnant women after exclusion of maternal contamination of chorionic villi samples by variable number of tandem repeats (VNTR) analysis., Methods: We studied 30 HIV-positive women: 17 terminated their pregnancy (11 in the first trimester and six in the second) and 13 delivered at term (one was a twin gestation). We selected chorionic villi and ruled out maternal contamination by VNTR analysis. DNA from chorionic villi and cord and maternal blood were tested for HIV by PCR. All infants underwent a paediatric follow-up., Results: All maternal blood samples tested positive for HIV-1 by polymerase chain reaction. No maternal contamination was revealed and HIV was found in six out of 11 first trimester placentas, in all second trimester samples, and in 10 out of 14 at term. Cord blood tested positive in all second trimester cases and in seven out of 14 liveborns. In no case was HIV found in cord blood without infection of the corresponding placenta; conversely, three placentas tested positive but cord blood was negative. Two infants were HIV-positive, 11 were uninfected (one case was lost to follow-up)., Conclusion: Our study indicates that HIV-1 can infect the placenta from first trimester onwards. HIV was found in two-thirds of our cord blood samples but it is possible that some viral DNA in cord blood may have come from infected placental cells. Additional studies are needed to assess the source of HIV in cord blood and the possible contribution of placental or maternal cells infected with HIV to vertical transmission of the virus.

The actual hemoglobin (Hb) contribution to endothelin-1 (ET-1) production in human umbilical vein endothelial cells (EC) was investigated. Cells were incubated with 0.1 mmol or 0.3 mmol of bovine: 1) unmodified (U) ferrous-Hb; 2) U-ferric-Hb; 3) U-ferryl-Hb; 4) polymerized low molecular weight (m.w.) Hb with chemically modified surface (< 400 kDa); and 5) glutaraldehyde polymerized, high m.w. Hb (< 1020 kDa). The incubation medium was tested at 6 and 24 hr for lactate dehydrogenase (index of cellular injury), and for ET-1 release by the cells. Before radioimmunoassay, the ET-1 was extracted from cell culture medium by a two-step purification procedure: 1) ultrafiltration, and 2) column extraction with C18 cartridges. The data suggested that the oxidation status of Hb and its concentration play an important role in causing EC injury. The highest toxicity was observed when EC were incubated with 0.1 mmol of ferryl-Hb, and there was no toxicity with 0.3 mmol of ferric-Hb. These results indicate that the ferric-Hb and low m.w. polymerized Hb at a concentration of 0.1 mmol did not alter ET-1 synthesis and produced a level similar to that of the control. However, it was found that ferryl-Hb and ferrous-Hb in a concentration of 0.1 mmol significantly reduced ET-1 release. All Hbs at a concentration of 0.3 mmol markedly inhibited the production of ET-1. The greatest decrease in ET-1 levels was produced by ferryl-Hb, and the lowest by ferric-Hb and low m.w. polymerized Hb. The Hb's inhibitory effect was more pronounced at 24 hr of incubation. It was also found that although Hb molecules showed a high degree of cross-reactivity with polyclonal anti ET-1 antibodies, the presence of different Hb solutions in the EC culture medium did not change the immunologic properties of ET-1 peptide. In conclusion, Hb inhibitory activity toward ET-1 production might be related to Hb mediated endothelial oxidative injury.