Effects of Recombinant α1-Microglobulin on Early Proteomic Response in Risk Organs after Exposure to 177Lu-Octreotate.

Recombinant α1-microglobulin (A1M) is proposed as a protector during 177Lu-octreotate treatment of neuroendocrine tumors, which is currently limited by bone marrow and renal toxicity. Co-administration of 177Lu-octreotate and A1M could result in a more effective treatment by protecting healthy tissue, but the radioprotective action of A1M is not fully understood. The aim of this study was to examine the proteomic response of kidneys and bone marrow early after 177Lu-octreotate and/or A1M administration. Mice were injected with 177Lu-octreotate and/or A1M, while control mice received saline or A1M vehicle solution. Bone marrow, kidney medulla, and kidney cortex were sampled after 24 h or 7 d. The differential protein expression was analyzed with tandem mass spectrometry. The dosimetric estimation was based on 177Lu activity in the kidney. PHLDA3 was the most prominent radiation-responsive protein in kidney tissue. In general, no statistically significant difference in the expression of radiation-related proteins was observed between the irradiated groups. Most canonical pathways were identified in bone marrow from the 177Lu-octreotate+A1M group. Altogether, a tissue-dependent proteomic response followed exposure to 177Lu-octreotate alone or together with A1M. Combining 177Lu-octreotate with A1M did not inhibit the radiation-induced protein expression early after exposure, and late effects should be further studied.

It's About Time: Time-Dependent Tissue Damage in the Adult Porcine Retina After Enucleation.

The ex vivo large animal retina is extensively used in research ranging from discovery of disease mechanisms to future treatment paradigms. Due to limited standardization when harvesting the tissue, the time after enucleation is often extended for several hours, a factor that so far has not yet been fully characterized. The purpose of this study was to investigate the relationship between time after enucleation and retinal tissue damage. Adult, porcine retinal explants were dissected and fixed 90 or 240 min after enucleation. In a separate experiment, explants were cultured for 48 h, following dissection either 90 or 240 min after enucleation. Retinas were analyzed morphologically using hematoxylin and eosin for overall tissue damage, TUNEL staining for detection of apoptosis, and RBPMS immunohistochemistry for evaluation of ganglion cell survival. In addition, medium from the cultured explants was sampled after 2, 24, and 48 h of culture and assessed for the cell damage marker lactate dehydrogenase (LDH). Retinas examined 240 min after enucleation displayed a significant increase in overall tissue damage, increased apoptosis, and decreased ganglion cell survival compared with 90-min counterparts. In the culture experiment, no significant difference in overall tissue damage was found between the 2 groups, however, apoptosis was significantly increased, and ganglion cell survival decreased in the cultured 240-min group. In addition, a significantly increased LDH medium activity was found in the 240-min group compared with the 90-min counterpart at all time points. The adult porcine retina is relatively resistant to tissue damage 90 min after enucleation but displays distinct signs of injury after 240 min. The importance of these time points is further highlighted when retinal explants are cultured. Our results strongly suggest that time after enucleation is a crucial factor that should be considered in experiments involving the ex vivo adult porcine retina.

Ferryl Hemoglobin and Heme Induce A1-Microglobulin in Hemorrhaged Atherosclerotic Lesions with Inhibitory Function against Hemoglobin and Lipid Oxidation.

Infiltration of red blood cells into atheromatous plaques and oxidation of hemoglobin (Hb) and lipoproteins are implicated in the pathogenesis of atherosclerosis. α1-microglobulin (A1M) is a radical-scavenging and heme-binding protein. In this work, we examined the origin and role of A1M in human atherosclerotic lesions. Using immunohistochemistry, we observed a significant A1M immunoreactivity in atheromas and hemorrhaged plaques of carotid arteries in smooth muscle cells (SMCs) and macrophages. The most prominent expression was detected in macrophages of organized hemorrhage. To reveal a possible inducer of A1M expression in ruptured lesions, we exposed aortic endothelial cells (ECs), SMCs and macrophages to heme, Oxy- and FerrylHb. Both heme and FerrylHb, but not OxyHb, upregulated A1M mRNA expression in all cell types. Importantly, only FerrylHb induced A1M protein secretion in aortic ECs, SMCs and macrophages. To assess the possible function of A1M in ruptured lesions, we analyzed Hb oxidation and heme-catalyzed lipid peroxidation in the presence of A1M. We showed that recombinant A1M markedly inhibited Hb oxidation and heme-driven oxidative modification of low-density lipoproteins as well plaque lipids derived from atheromas. These results demonstrate the presence of A1M in atherosclerotic plaques and suggest its induction by heme and FerrylHb in the resident cells.

The Role of α1-Microglobulin (A1M) in Erythropoiesis and Erythrocyte Homeostasis-Therapeutic Opportunities in Hemolytic Conditions.

α1-microglobulin (A1M) is a small protein present in vertebrates including humans. It has several physiologically relevant properties, including binding of heme and radicals as well as enzymatic reduction, that are used in the protection of cells and tissue. Research has revealed that A1M can ameliorate heme and ROS-induced injuries in cell cultures, organs, explants and animal models. Recently, it was shown that A1M could reduce hemolysis in vitro, observed with several different types of insults and sources of RBCs. In addition, in a recently published study, it was observed that mice lacking A1M (A1M-KO) developed a macrocytic anemia phenotype. Altogether, this suggests that A1M may have a role in RBC development, stability and turnover. This opens up the possibility of utilizing A1M for therapeutic purposes in pathological conditions involving erythropoietic and hemolytic abnormalities. Here, we provide an overview of A1M and its potential therapeutic effect in the context of the following erythropoietic and hemolytic conditions: Diamond-Blackfan anemia (DBA), 5q-minus myelodysplastic syndrome (5q-MDS), blood transfusions (including storage), intraventricular hemorrhage (IVH), preeclampsia (PE) and atherosclerosis.

α1-Microglobulin (A1M) Protects Human Proximal Tubule Epithelial Cells from Heme-Induced Damage In Vitro.

Oxidative stress is associated with many renal disorders, both acute and chronic, and has also been described to contribute to the disease progression. Therefore, oxidative stress is a potential therapeutic target. The human antioxidant α1-microglobulin (A1M) is a plasma and tissue protein with heme-binding, radical-scavenging and reductase activities. A1M can be internalized by cells, localized to the mitochondria and protect mitochondrial function. Due to its small size, A1M is filtered from the blood into the glomeruli, and taken up by the renal tubular epithelial cells. A1M has previously been described to reduce renal damage in animal models of preeclampsia, radiotherapy and rhabdomyolysis, and is proposed as a pharmacological agent for the treatment of kidney damage. In this paper, we examined the in vitro protective effects of recombinant human A1M (rA1M) in human proximal tubule epithelial cells. Moreover, rA1M was found to protect against heme-induced cell-death both in primary cells (RPTEC) and in a cell-line (HK-2). Expression of stress-related genes was upregulated in both cell cultures in response to heme exposure, as measured by qPCR and confirmed with in situ hybridization in HK-2 cells, whereas co-treatment with rA1M counteracted the upregulation. Mitochondrial respiration, analyzed with the Seahorse extracellular flux analyzer, was compromised following exposure to heme, but preserved by co-treatment with rA1M. Finally, heme addition to RPTE cells induced an upregulation of the endogenous cellular expression of A1M, via activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-pathway. Overall, data suggest that A1M/rA1M protects against stress-induced damage to tubule epithelial cells that, at least partly, can be attributed to maintaining mitochondrial function.

Heme-Induced Oxidation of Cysteine Groups of Myofilament Proteins Leads to Contractile Dysfunction of Permeabilized Human Skeletal Muscle Fibres.

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 Ca2+-activated maximal (active) force (Fo) by about 50% and evoked an approximately 3-fold increase in Ca2+-independent (passive) force (Fpassive). 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.

The heme and radical scavenger α1-microglobulin (A1M) confers early protection of the immature brain following preterm intraventricular hemorrhage.

BACKGROUND: Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with cerebro-cerebellar damage in very preterm infants, leading to neurodevelopmental impairment. Penetration, from the intraventricular space, of extravasated red blood cells and extracellular hemoglobin (Hb), to the periventricular parenchyma and the cerebellum has been shown to be causal in the development of brain injury following GM-IVH. Furthermore, the damage has been described to be associated with the cytotoxic nature of extracellular Hb-metabolites. To date, there is no therapy available to prevent infants from developing either hydrocephalus or serious neurological disability. Mechanisms previously described to cause brain damage following GM-IVH, i.e., oxidative stress and Hb-metabolite toxicity, suggest that the free radical and heme scavenger α1-microglobulin (A1M) may constitute a potential neuroprotective intervention. METHODS: Using a preterm rabbit pup model of IVH, where IVH was induced shortly after birth in pups delivered by cesarean section at E29 (3 days prior to term), we investigated the brain distribution of recombinant A1M (rA1M) following intracerebroventricular (i.c.v.) administration at 24 h post-IVH induction. Further, short-term functional protection of i.c.v.-administered human A1M (hA1M) following IVH in the preterm rabbit pup model was evaluated. RESULTS: Following i.c.v. administration, rA1M was distributed in periventricular white matter regions, throughout the fore- and midbrain and extending to the cerebellum. The regional distribution of rA1M was accompanied by a high co-existence of positive staining for extracellular Hb. Administration of i.c.v.-injected hA1M was associated with decreased structural tissue and mitochondrial damage and with reduced mRNA expression for proinflammatory and inflammatory signaling-related genes induced by IVH in periventricular brain tissue. CONCLUSIONS: The results of this study indicate that rA1M/hA1M is a potential candidate for neuroprotective treatment following preterm IVH.

Acute tissue reactions, inner segment pathology, and effects of the antioxidant α1-microglobulin in an in vitro model of retinal detachment.

The purpose of this study was to explore acute tissue reactions, ultrastructural photoreceptor morphology with emphasis on inner segments, and the effect of antioxidant treatment in an in vitro model of rhegmatogenous retinal detachment (RRD). A previously described method of RRD simulation was used with adult retinal porcine explants kept free-floating in culture medium with or without treatment with the radical scavenger α1-microglobulin (A1M). Explants were examined at 5 time points from 1 to 24 h using transmission electron microscopy as well as quantitative real-time PCR (RT-PCR) to quantify gene expression of the cell stress marker heat shock protein 70 (Hsp70) and oxidative stress marker heme oxygenase (HO-1). The culture medium level of the cell damage marker lactate dehydrogenase (LDH) and oxidative stress DNA damage marker 8-Oxo-2'-deoxyguanosine (8-OHdG) was also assessed at each time point. We found that the levels of Hsp70 and LDH rapidly increased in both groups, and at 3 and 6 h, Hsp70 was significantly higher in A1M treated retinas. At 24 h, Hsp70 and LDH, as well as 8-OHdG were significantly lower compared with controls, whereas the tissue level of HO-1 was significantly higher. Progressive ultrastructural photoreceptor changes were seen in untreated control explants from 1 h and onwards including outer segment shortening and loss, disruption of organelles within the inner segments and loss of perikarya in the outer nuclear layer. Inner segment pathology was more rapid and extensive in rods compared with in cones. In A1M treated counterparts, damage to rod inner segment mitochondria was significantly higher after 1 h of culture, but after this time, no statistical difference was found. At 24 h, cone inner segment mitochondrial disruption was significantly higher in control retinas and the number of surviving perikarya lower. From our results, we conclude that retinal explants elicit acute cell stress reactions when placed in culture without physical support simulating a detached retina floating in the vitreous space. Photoreceptors rapidly display degenerative changes including extensive damage to inner segment mitochondria indicating loss of energy transduction as an early key event. A1M increases initial mitochondrial stress in the rods, however, subsequent pathology is attenuated by the treatment, highlighting the dynamics of protective as well as disruptive oxidative stress reactions in the detached retina.

Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation.

BACKGROUND: α1-Microglobulin (A1M) is a reductase and radical scavenger involved in physiological protection against oxidative damage. These functions were previously shown to be dependent upon cysteinyl-, C34, and lysyl side-chains, K(92, 118,130). A1M binds heme and the crystal structure suggests that C34 and H123 participate in a heme binding site. We have investigated the involvement of these five residues in the interactions with heme. METHODS: Four A1M-variants were expressed: with cysteine to serine substitution in position 34, lysine to threonine substitutions in positions (92, 118, 130), histidine to serine substitution in position 123 and a wt without mutations. Heme binding was investigated by tryptophan fluorescence quenching, UV-Vis spectrophotometry, circular dichroism, SPR, electrophoretic migration shift, gel filtration, catalase-like activity and molecular simulation. RESULTS: All A1M-variants bound to heme. Mutations in C34, H123 or K(92, 118, 130) resulted in significant absorbance changes, CD spectral changes, and catalase-like activity, suggesting involvement of these side-groups in coordination of the heme-iron. Molecular simulation support a model with two heme-binding sites in A1M involving the mutated residues. Binding of the first heme induces allosteric stabilization of the structure predisposing for a better fit of the second heme. CONCLUSIONS: The results suggest that one heme-binding site is located in the lipocalin pocket and a second binding site between loops 1 and 4. Reactions with the hemes involve the side-groups of C34, K(92, 118, 130) and H123. GENERAL SIGNIFICANCE: The model provides a structural basis for the functional activities of A1M: heme binding activity of A1M.

Influence of nanomechanical stress induced by ZnO nanoparticles of different shapes on the viability of cells.

There is growing interest in nanostructures interacting with living organisms. However, there are still no general rules for the design of biocompatible nanodevices. Here, we present a step towards understanding the interactions between nanostructures and living cells. We study the influence of nanomechanical stress induced by zinc oxide (ZnO) nanostructures of different shapes on the viability of both prokaryotic (Gram-negative bacteria: Escherichia coli and Enterobacter aerogenes, and Gram-positive bacteria: Staphylococcus epidermidis and Corynebacterium glutamicum) and eukaryotic cells (yeast Saccharomyces cerevisiae and liver cancer cell line HepG2). Nanoparticles (NPs) and nanorods (NRs) of matching crystallographic structure (P63mc) and active surface area (in the order of 5 × 10(-2)µm(2)) are almost non-toxic for cells under static conditions. However, under conditions that enable collisions between ZnO nanostructures and cells, NRs appear to be more damaging compared to NPs. This is due to the increased probability of mechanical damage caused by nanorods upon puncturing of the cell wall and membranes. Gram-positive bacteria, which have thicker cell walls, are more resistant to nanomechanical stress induced by NRs compared to Gram-negative strains and eukaryotic cells. The presented results may be exploited to improve the properties of nanotechnology based products such as implants, drug delivery systems, antibacterial emulsions and cosmetics.

First trimester prediction of preeclampsia.

Preeclampsia (PE) is a serious pregnancy-related condition that causes severe maternal and fetal morbidity and mortality. Within the recent years, there has been an increasing focus in predicting PE at the end of the first trimester of pregnancy. In this review, literature published between 2011 and 2015 was evaluated. In a total of six biomarker algorithms, for first and early second trimester, the prediction of preeclampsia is discussed. In addition, one randomized clinical trial was included. Several algorithms were based on placental biomarkers such as pregnancy-associated plasma protein A (PAPP-A), placental growth factor (PLGF), and soluble FMS-like tyrosine kinase 1 (s-FLT-1). The algorithms containing these biomarkers showed a high prediction rate (PR) for early onset PE, ranging from 44 to 92 % at 5 % false positive rate (FPR). New biomarkers suggest an alternative model based on free HbF and the heme scavenger alpha-1-microglobulin (A1M) with a prediction rate of 69 % at an FPR of 5 %. Interestingly, this model performs well without uterine artery Doppler pulsatility index (UtAD-PI), which is an advantage particularly if the screening method were to be implemented in developing countries. The randomized clinical trial showed a clear reduction in early onset PE as well as reducing preterm PE if identified high-risk pregnancies were treated with low-dose aspirin. In conclusion, PE prediction is now possible through several prediction algorithms and prophylaxis is beneficial in high-risk cases.

Human Anti-Oxidation Protein A1M--A Potential Kidney Protection Agent in Peptide Receptor Radionuclide Therapy.

Peptide receptor radionuclide therapy (PRRT) has been in clinical use for 15 years to treat metastatic neuroendocrine tumors. PRRT is limited by reabsorption and retention of the administered radiolabeled somatostatin analogues in the proximal tubule. Consequently, it is essential to develop and employ methods to protect the kidneys during PRRT. Today, infusion of positively charged amino acids is the standard method of kidney protection. Other methods, such as administration of amifostine, are still under evaluation and show promising results. α1-microglobulin (A1M) is a reductase and radical scavenging protein ubiquitously present in plasma and extravascular tissue. Human A1M has antioxidation properties and has been shown to prevent radiation-induced in vitro cell damage and protect non-irradiated surrounding cells. It has recently been shown in mice that exogenously infused A1M and the somatostatin analogue octreotide are co-localized in proximal tubules of the kidney after intravenous infusion. In this review we describe the current situation of kidney protection during PRRT, discuss the necessity and implications of more precise dosimetry and present A1M as a new, potential candidate for renal protection during PRRT and related targeted radionuclide therapies.

Extracellular fetal hemoglobin induces increases in glomerular permeability: inhibition with α1-microglobulin and tempol.

Extracellular fetal hemoglobin (HbF) and adult hemoglobin (HbA) are proinflammatory and generate ROS. Increased plasma levels of extracellular HbF have recently been reported to occur in early preeclampsia. α1-Microglobulin (A1M) is a physiological heme-binding protein and radical scavenger that has been shown to counteract vascular permeability increases induced by HbA in the perfused placenta. The present study was performed to investigate whether HbF and HbA will increase glomerular permeability in vivo and to test whether A1M and tempol, a ROS scavenger, can prevent their effects. Anesthetized Wistar rats were continuously infused intravenously with either HbA, HbF, or cyano-inactivated HbF together with FITC-Ficoll-70/400, inulin, and (51)Cr-labeled EDTA for 2 h. Plasma samples and urine samples (left ureter) were taken repeatedly and analyzed by high-performance size exclusion chromatography to assess glomerular sieving coefficients for Ficoll of radius 10-80 Å. In separate experiments, A1M or tempol was given before and during Hb infusions. Extracellular HbF caused rapid, transient increases in glomerular permeability to large Ficoll molecules (50-80Å), contrary to the effects of HbA and cyano-inactivated HbF. For HbF, glomerular sieving coefficients for Ficoll of radius 60Å increased from 3.85 ± 0.85 × 10(-5) to 2.60 ± 0.96 × 10(-4) at 15 min, changes that were abrogated by tempol and reduced by A1M. In conclusion, our data demonstrate that extracellular HbF, infused systemically, can acutely increase glomerular permeability through inducing oxidative stress.

Extracellular hemoglobin - mediator of inflammation and cell death in the choroid plexus following preterm intraventricular hemorrhage.

BACKGROUND: Intraventricular hemorrhage (IVH) with post-hemorrhagic ventricular dilatation (PHVD) is a major cause of neurodevelopmental impairment and mortality in preterm infants. The mechanisms leading to PHVD and brain damage remain largely unknown. The choroid plexus and the ependyma, which constitute an essential part of the blood-brain barrier (BBB), are the first structures to encounter the damaging effects of extravasated blood. The breakdown of the BBB is a critical upstream event leading to brain damage following IVH. In this study we investigated the impact of hemorrhage and hemoglobin (Hb) metabolites on the choroid plexus epithelium. METHODS: Using a preterm rabbit pup model of IVH, the structural and functional integrity, cellular, inflammatory and oxidative response of the choroid plexus, at 24 and 72 hours following IVH + PHVD, were investigated. In order to further characterize cellular and molecular mechanisms, primary human choroid plexus epithelial cells were exposed to cerebrospinal fluid (CSF) from preterm infants with IVH as well as to Hb-metabolites. Finally, the blocking effects of the Hb-scavenger haptoglobin (Hp) were investigated both in vivo and in vitro. RESULTS: Following IVH + PHVD, an up-regulation of mRNA for the receptor-related genes TLR-4, IL1R1, FAS, the transcription factor NF-Κß and for the pro-inflammatory and chemotactic effector molecules, IL-1ß, TNFα, MCP-1, IL-8, and IL-6 was observed in the choroid plexus at 24 and 72 hours. This was associated with structural disintegration, caspase activation and cell death in the choroid plexus epithelium. In vitro characterization of choroid plexus epithelial cells, following exposure to hemorrhagic CSF and to the Hb-metabolites metHb and heme, displayed apoptotic and necrotic cell death and an up-regulation of receptor-related and inflammatory effector molecules similar to that observed in vivo following IVH + PHVD. Intraventricular injection of the Hb-scavenger Hp in vivo and co-incubation with Hp in vitro reversed or reduced the cellular activation, inflammatory response, structural damage and cell death. CONCLUSION: Hb-metabolites are important causal initiators of cell death following IVH and removal or scavenging of Hb-metabolites may present an efficient means to reduce the damage to the immature brain following IVH.

Hematological and renal toxicity in mice after three cycles of high activity [177Lu]Lu-PSMA-617 with or without human α1-microglobulin.

Radioligand therapy with [177Lu]Lu-PSMA-617 can be used to prolong life and reduce tumor burden in terminally ill castration resistant prostate cancer patients. Still, accumulation in healthy tissue limits the activity that can be administered. Therefore, fractionated therapy is used to lower toxicity. However, there might be a need to reduce toxicity even further with e.g. radioprotectors. The aim of this study was to (i). establish a preclinical mouse model with fractionated high activity therapy of three consecutive doses of 200 MBq [177Lu]Lu-PSMA-617 in which we aimed to (ii). achieve measurable hematotoxicity and nephrotoxicity and to (iii). analyze the potential protective effect of co-injecting recombinant α1-microglobulin (rA1M), a human antioxidant previously shown to have radioprotective effects. In both groups, three cycles resulted in increased albuminuria for each cycle, with large individual variation. Another marker of kidney injury, serum blood urea nitrogen (BUN), was only significantly increased compared to control animals after the third cycle. The number of white and red blood cells decreased significantly and did not reach the levels of control animals during the experiment. rA1M did reduce absorbed dose to kidney but did not show significant protection here, but future studies are warranted due to the recent clinical studies showing a significant renoprotective effect in patients.

Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation.

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-1ß, 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.

Fetal hemoglobin in preeclampsia: a new causative factor, a tool for prediction/diagnosis and a potential target for therapy.

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.

Vitreous levels of oxidative stress biomarkers and the radical-scavenger α1-microglobulin/A1M in human rhegmatogenous retinal detachment.

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.

Positional identification of Ncf1 as a gene that regulates arthritis severity in rats.

The identification of genes underlying quantitative-trait loci (QTL) for complex diseases, such as rheumatoid arthritis, is a challenging and difficult task for the human genome project. Through positional cloning of the Pia4 QTL in rats, we found that a naturally occurring polymorphism of Ncf1 (encoding neutrophil cytosolic factor 1, a component of the NADPH oxidase complex) regulates arthritis severity. The disease-related allele of Ncf1 has reduced oxidative burst response and promotes activation of arthritogenic T cells. Pharmacological treatment with substances that activate the NADPH oxidase complex is shown to ameliorate arthritis. Hence, Ncf1 is associated with a new autoimmune mechanism leading to severe destructive arthritis, notably similar to rheumatoid arthritis in humans.

Co-administration with A1M does not influence apoptotic response of 177Lu-octreotate in GOT1 neuroendocrine tumors.

Recombinant α1-microglobulin (A1M) is a proposed radioprotector during 177Lu-octreotate therapy of neuroendocrine tumors (NETs). To ensure a maintained therapeutic effect, we previously demonstrated that A1M does not affect the 177Lu-octreotate induced decrease in GOT1 tumor volume. However, the underlying biological events of these findings are still unknown. The aim of this work was to examine the regulation of apoptosis-related genes in GOT1 tumors short-time after i.v. administration of 177Lu-octreotate with and without A1M or A1M alone. Human GOT1 tumor-bearing mice received 30 MBq 177Lu-octreotate or 5 mg/kg A1M or co-treatment with both. Animals were sacrificed after 1 or 7 days. Gene expression analysis of apoptosis-related genes in GOT1 tissue was performed with RT-PCR. In general, similar expression patterns of pro- and anti-apoptotic genes were found after 177Lu-octreotate exposure with or without co-administration of A1M. The highest regulated genes in both irradiated groups compared to untreated controls were FAS and TNFSFRS10B. Administration of A1M alone only resulted in significantly regulated genes after 7 days. Co-administration of A1M did not negatively affect the transcriptional apoptotic response of 177Lu-octreotate in GOT1 tumors.