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OBJECTIVE: The aim of this study was to evaluate fetal hemoglobin (HbF) and [alpha](1)-microglobulin (A1M) in maternal serum as first-trimester biomarkers for preeclampsia (PE). STUDY DESIGN: The design was a case-control study. We included 96 patients in the first trimester of pregnancy (60 with PE and 36 controls). Venous serum samples were analyzed for HbF and total hemoglobin (Hb) by enzyme-linked immunosorbent assay and for A1M by radioimmunoassay. Sensitivity and specificity was calculated by logistic regression and receiver operating characteristic curve analysis. RESULTS: The HbF/Hb ratio and A1M concentration were significantly elevated in serum from women with subsequent development of PE (P < .0001). The optimal sensitivity and specificity was obtained using the biomarkers in combination; 69% sensitivity for a 5% screen positive rate and 90% sensitivity for a 23% screen positive rate. CONCLUSION: The study suggests that HbF/Hb ratio in combination with A1M is predictive biomarkers for PE. [ABSTRACT FROM AUTHOR]

Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20-300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. A single heme exposure (20 µM) to muscle fibres decreased Ca 2+ -activated maximal (active) force (F o ) by about 50% and evoked an approximately 3-fold increase in Ca 2+ -independent (passive) force (F passive ). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations.

Intracellular free heme predisposes to oxidant-mediated tissue damage. We hypothesized that free heme causes alterations in myocardial contractility via disturbed structure and/or regulation of the contractile proteins. Isometric force production and its Ca(2+)-sensitivity (pCa50) were monitored in permeabilized human ventricular cardiomyocytes. Heme exposure altered cardiomyocyte morphology and evoked robust decreases in Ca(2+)-activated maximal active force (Fo) while increasing Ca(2+)-independent passive force (F passive). Heme treatments, either alone or in combination with H2O2, did not affect pCa50. The increase in F passive started at 3 µM heme exposure and could be partially reversed by the antioxidant dithiothreitol. Protein sulfhydryl (SH) groups of thick myofilament content decreased and sulfenic acid formation increased after treatment with heme. Partial restoration in the SH group content was observed in a protein running at 140 kDa after treatment with dithiothreitol, but not in other proteins, such as filamin C, myosin heavy chain, cardiac myosin binding protein C, and α-actinin. Importantly, binding of heme to hemopexin or alpha-1-microglobulin prevented its effects on cardiomyocyte contractility, suggesting an allosteric effect. In line with this, free heme directly bound to myosin light chain 1 in human cardiomyocytes. Our observations suggest that free heme modifies cardiac contractile proteins via posttranslational protein modifications and via binding to myosin light chain 1, leading to severe contractile dysfunction. This may contribute to systolic and diastolic cardiac dysfunctions in hemolytic diseases, heart failure, and myocardial ischemia-reperfusion injury., (Copyright © 2015. Published by Elsevier Inc.)

Preeclampsia (PE) is a serious pregnancy complication that manifests as hypertension and proteinuria after the 20(th) gestation week. Previously, fetal hemoglobin (HbF) has been identified as a plausible causative factor. Cell-free Hb and its degradation products are known to cause oxidative stress and tissue damage, typical of the PE placenta. A1M (α1-microglobulin) is an endogenous scavenger of radicals and heme. Here, the usefulness of A1M as a treatment for PE is investigated in the pregnant ewe PE model, in which starvation induces PE symptoms via hemolysis. Eleven ewes, in late pregnancy, were starved for 36 hours and then treated with A1M (n = 5) or placebo (n = 6) injections. After injections, the ewes were re-fed and observed for additional 72 hours. They were monitored for blood pressure, proteinuria, blood cell distribution and clinical and inflammation markers in plasma. Before termination, the utero-placental circulation was analyzed with Doppler velocimetry and the kidney glomerular function was analyzed by Ficoll sieving. At termination, blood, kidney and placenta samples were collected and analyzed for changes in gene expression and tissue structure. The starvation resulted in increased amounts of the hemolysis marker bilirubin in the blood, structural damages to the placenta and kidneys and an increased glomerular sieving coefficient indicating a defect filtration barrier. Treatment with A1M ameliorated these changes without signs of side-effects. In conclusion, A1M displayed positive therapeutic effects in the ewe starvation PE model, and was well tolerated. Therefore, we suggest A1M as a plausible treatment for PE in humans.

Purpose of Review: Preeclampsia, one of the leading causes of pregnancy complications, affects 3-7% of pregnant women. This review summarizes the present knowledge of a new potential cause of the disease and suggests a method for its prediction/diagnosis and a possible treatment, both based on the recent findings on the involvement of fetal hemoglobin (HbF) and the heme and radical scavenging protein A1M (alpha-1-microglobulin)., Recent Findings: Gene and protein profiling studies have independently shown that increased amount of free HbF is accumulated in the preeclampsia placenta. As a result of a predominantly oxidative damage to the blood-placenta barrier, HbF leaks over to the maternal blood circulation. Elevated levels can be measured already in the first trimester, and later in pregnancy, the levels correlate with the blood pressure in women with preeclampsia. Ex-vivo data show that the human protein A1M, an endogeneous antioxidation protection protein, can prevent Hb-induced damage to the placenta, restore the blood-placental barrier and prevent maternal tissue damage., Summary: Free HbF may provide both a predictive and a diagnostic clinical biomarker from the first trimester. A1M has the potential as a future pharmacological treatment for preeclampsia.

Background: Cerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH., Methods: Using a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures., Results: Following IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH., Conclusions: Following preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

Purpose: To explore oxidative stress and the radical scavenger α(1)-microglobulin (A1M) in the vitreous body of human eyes with primary rhegmatogenous retinal detachment (RRD)., Methods: Levels of carbonyl groups, a marker of oxidative stress, and A1M were measured by ELISA and RIA in 14 vitreous samples derived from patients suffering from RRD, and compared with 14 samples from macula hole (MH) patients. Carbonyl group and A1M levels in RRD samples were statistically related to detachment characteristics. Analysis of total protein level, SDS-PAGE, and Western blotting of A1M was also performed. In a separate experiment, mRNA expression of A1M was measured by RT-PCR in rat retina explants., Results: Levels of carbonyl groups and A1M varied widely in RRD vitreous samples, but were significantly higher in samples derived from eyes with large detachment area and macula-off status, while the presence of vitreous hemorrhage did not show any significant correlation. Compared with MH samples, RRD samples displayed significantly higher levels of A1M, whereas changes in total protein levels and carbonyl groups were not significant. Novel forms of A1M, not previously seen in plasma, were found in the vitreous body by Western blotting. Furthermore, A1M expression was seen in rat retina explants and was upregulated after 24 h of culturing., Conclusion: Oxidative stress is a prominent feature of human eyes with primary RRD, and is directly related to detachment severity. Affected eyes can launch a protective response in the form of the radical scavenger A1M possibly derived from the retina. The results thus indicate potential therapeutic cell loss prevention in RRD by employing the endogeneous radical scavenger A1M.

Hemoglobin (Hb) is the major oxygen (O(2))-carrying system of the blood but has many potentially dangerous side effects due to oxidation and reduction reactions of the heme-bound iron and O(2). Extracellular Hb, resulting from hemolysis or exogenous infusion, is shown to be an important pathogenic factor in a growing number of diseases. This review briefly outlines the oxidative/reductive toxic reactions of Hb and its metabolites. It also describes physiological protection mechanisms that have evolved against extracellular Hb, with a focus on the most recently discovered: the heme- and radical-binding protein α(1)-microglobulin (A1M). This protein is found in all vertebrates, including man, and operates by rapidly clearing cytosols and extravascular fluids of heme groups and free radicals released from Hb. Five groups of pathological conditions with high concentrations of extracellular Hb are described: hemolytic anemias and transfusion reactions, the pregnancy complication pre-eclampsia, cerebral intraventricular hemorrhage of premature infants, chronic inflammatory leg ulcers, and infusion of Hb-based O(2) carriers as blood substitutes. Finally, possible treatments of these conditions are discussed, giving a special attention to the described protective effects of A1M.

Objective: The aim of this study was to evaluate fetal hemoglobin (HbF) and α(1)-microglobulin (A1M) in maternal serum as first-trimester biomarkers for preeclampsia (PE)., Study Design: The design was a case-control study. We included 96 patients in the first trimester of pregnancy (60 with PE and 36 controls). Venous serum samples were analyzed for HbF and total hemoglobin (Hb) by enzyme-linked immunosorbent assay and for A1M by radioimmunoassay. Sensitivity and specificity was calculated by logistic regression and receiver operating characteristic curve analysis., Results: The HbF/Hb ratio and A1M concentration were significantly elevated in serum from women with subsequent development of PE (P < .0001). The optimal sensitivity and specificity was obtained using the biomarkers in combination; 69% sensitivity for a 5% screen positive rate and 90% sensitivity for a 23% screen positive rate., Conclusion: The study suggests that HbF/Hb ratio in combination with A1M is predictive biomarkers for PE., (Copyright © 2011 Mosby, Inc. All rights reserved.)

During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α(1)-microglobulin (A1M) was recently shown to have reductase properties, reducing heme-proteins and other substrates, and to scavenge heme and radicals. We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (K(d) = 0.96×10(-6) M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding.

Alpha-particle irradiation of cells damages not only the irradiated cells but also nontargeted bystander cells. It has been proposed that the bystander effect is caused by oxidants and free radicals generated by the radiation. Recent studies have shown that α(1)-microglobulin protects against cell damage caused by oxidants and free radicals. Using a novel experimental system that allows irradiation of 0.02% of a human hepatoma monolayer, leaving 99.98% as bystander cells, we investigated the influence of oxidative stress and the cell-protective effects of α(1)-microglobulin during α-particle irradiation. The results showed an increase in cell death in both irradiated cells and bystander cells. A significant increase in apoptosis, oxidation markers and expression of the stress response genes heme oxygenase 1, superoxide dismutase, catalase, glutathione peroxidase 1, p21 and p53 were observed. Addition of α(1)-microglobulin reduced the amount of dead cells and inhibited apoptosis, formation of oxidation markers, and up-regulation of stress response genes. The results emphasize the role of oxidative stress in promoting bystander effects. Furthermore, the results suggest that α(1)-microglobulin protects nonirradiated cells by eliminating oxidants and free radicals generated by radiation and imply that α(1)-microglobulin can be used in radiation therapy of tumors to minimize damage to surrounding tissues.

Preeclampsia is a major cause of morbidity and mortality during pregnancy. To date, the pathogenesis of the disease is not fully understood. Recent studies show that preeclampsia is associated with overexpression of the hemoglobin genes alpha2 and gamma and accumulation of the protein in the vascular lumen of the placenta. Hypothesizing that cell-free hemoglobin leaks from the placenta into the maternal circulation and contributes to the endothelial damage and symptoms by inducing oxidative stress, we analyzed fetal and adult hemoglobin (HbF, HbA), haptoglobin, oxidation markers, and the heme scavenger and antioxidant alpha(1)-microglobulin in plasma, urine, and placenta in preeclamptic women (n=28) and women with normal pregnancy (n=27). The mean plasma concentrations of HbF, HbA, protein carbonyl groups, membrane peroxidation capacity, and alpha(1)-microglobulin were significantly increased in preeclamptic women. The levels of total plasma Hb correlated strongly with the systolic blood pressure. The plasma haptoglobin concentrations of women with preeclampsia were significantly depressed. Increased amounts of alpha(1)-microglobulin mRNA and protein were found in placenta from preeclamptic women, and the levels of plasma and placenta alpha(1)-microglobulin correlated with the plasma Hb concentrations. The heme-degrading form t-alpha(1)-microglobulin was significantly increased in urine in preeclampsia. These results support the idea that hemoglobin-induced oxidative stress is a pathogenic factor in preeclampsia., (Copyright 2009. Published by Elsevier Inc.)

Finegoldia magna is an anaerobic bacterial species that is part of the normal human flora on all nonsterile body surfaces, but it is also a significant opportunistic pathogen causing a wide range of infections. Some isolates of F. magna that are more frequently associated with clinical infection express protein L, a surface protein containing multiple homologous domains (B1-B5) that bind Igs through interactions with Ig L chains. The present study shows that the N-terminal A domain of protein L binds S100A8/A9, antibacterial proteins present in large amounts in the cytoplasm of neutrophils, but also extracellularly in tissues during inflammation. As a result, protein L-expressing F. magna are protected against killing by S100A8/A9. Igs and S100A8/A9 were found to interact independently with protein L, demonstrating that this bacterial surface protein is capable of manipulating both adaptive and innate immune defense mechanisms.

Background and Purpose: Preeclampsia is a major obstetric problem of unknown etiology. The fact that removal of the placenta is the only cure for preeclampsia, has led to the well-established hypothesis, that the placenta is central in the etiology. Gene profiling and proteomics studies have suggested oxidative stress caused by reperfusion and free oxygen radicals as a potential pathophysiological mechanism in preeclampsia. In this study, the dual placental perfusion model was used in order to evaluate the damaging effects of oxidative stress induced by xanthine/xanthine oxides and free hemoglobin., Material and Methods: The dual placenta perfusion model is a well-established in vitro model for functional placental studies. Placentas were perfused with medium containing either xanthine/xanthine oxidase or erythrocytes as a source of free hemoglobin. Concentration of free hemoglobin in the medium was measured by means of ELISA. Whole genome microarray technique and bioinformatics were used to evaluate the gene expression profile in the two groups., Results: Substantial levels of free adult hemoglobin were detected in the perfusions. A total of 58 genes showed altered gene expression, the most altered were hemoglobin alpha, beta and gamma, tissue factor pathway inhibitor 2 and superoxide dismutase 2. Bioinformatics revealed that biological processes related to oxidative stress, anti-apoptosis and iron ion binding were significantly altered., Conclusions: The results suggest that perfusion with xanthine/xanthine oxidase and free hemoglobin induce changes in gene expression similar to what has been described for the preeclamptic placenta.

Lazarillo, a glycoprotein involved in axon growth and guidance in the grasshopper embryo, is the only member of the lipocalin family that is attached to the cell surface by a GPI anchor. Recently, the study of Lazarillo homologous genes in Drosophila and mouse has revealed new functions in the regulation of lifespan, stress resistance and neurodegeneration. Here we report an analysis of biochemical properties of Lazarillo to gain insight into the molecular basis of its physiological function. Recombinant forms of the grasshopper protein were expressed in two different systems to test: (1) potential binding of several hydrophobic ligands; (2) protein-protein homophilic interactions; and (3) whether interaction with the function-blocking mAb 10E6 interferes with ligand binding. We tested 10 candidate ligands (retinoic acid, heme, bilirubin, biliverdin, ecdysterone, juvenile hormone, farnesol, arachidonic acid, linoleic acid and palmitic acid), and monitored binding using electrophoretic mobility shift, absorbance spectrum, and fluorimetry assays. Our work indicates binding to heme and retinoic acid, resulting in increased electrophoretic mobility, as well as to fatty acids, resulting in multimerization. Retinoic acid and fatty acids binding were confirmed by fluorescence titration, and heme binding was confirmed with absorbance spectrum assays. We demonstrate that Lazarillo oligomerizes in solution and can form clusters in the plasma membrane when expressed and GPI-anchored to the cell surface, however it is unable to mediate cell-cell adhesion. Finally, by ligand-mAb competition experiments we show that ligand-binding alone cannot be the key factor for Lazarillo to perform its function during axonal growth in the grasshopper embryo., (Copyright (c) 2008 John Wiley & Sons, Ltd.)