Exogenous inter-α inhibitor proteins prevent cell death and improve ischemic stroke outcomes in mice.

Inter-α inhibitor proteins (IAIPs) are a family of endogenous plasma and extracellular matrix molecules. IAIPs suppress proinflammatory cytokines, limit excess complement activation, and bind extracellular histones to form IAIP-histone complexes, leading to neutralization of histone-associated cytotoxicity in models of sepsis. Many of these detrimental processes also play critical roles in the pathophysiology of ischemic stroke. In this study, we first assessed the clinical relevance of IAIPs in stroke and then tested the therapeutic efficacy of exogenous IAIPs in several experimental stroke models. IAIP levels were reduced in both ischemic stroke patients and in mice subjected to experimental ischemic stroke when compared with controls. Post-stroke administration of IAIP significantly improved stroke outcomes across multiple stroke models, even when given 6 hours after stroke onset. Importantly, the beneficial effects of delayed IAIP treatment were observed in both young and aged mice. Using targeted gene expression analysis, we identified a receptor for complement activation, C5aR1, that was highly suppressed in both the blood and brain of IAIP-treated animals. Subsequent experiments using C5aR1-knockout mice demonstrated that the beneficial effects of IAIPs are mediated in part by C5aR1. These results indicate that IAIP is a potential therapeutic candidate for the treatment of ischemic stroke.

Effects of inter-alpha inhibitor proteins on brain injury after exposure of neonatal rats to severe hypoxia-ischemia.

Hypoxic-ischemic (HI) brain injury is one of the most common neurological problems occurring in premature and full-term infants after perinatal complications. Hypothermia is the only treatment approved for HI encephalopathy in newborns. However, this treatment is only partially protective, cannot be used to treat premature infants, and has limited efficacy to treat severe HI encephalopathy. Inflammation contributes to the evolution of HI brain injury in neonates. Inter-alpha Inhibitor Proteins (IAIPs) are immunomodulatory proteins that have neuroprotective properties after exposure to moderate HI in neonatal rats. The objective of the current study was to determine the neuroprotective efficacy of treatment with IAIPs starting immediately after or with a delay of one hour after exposure to severe HI of 120 min duration. One hundred and forty-six 7-day-old rat pups were randomized to sham control, HI and immediate treatment with IAIPs (60 mg/kg) or placebo (PL), and sham, HI and delayed treatment with IAIPs or PL. IAIPs or PL were given at zero, 24, and 48 h after HI or 1, 24 and 48 h after HI. Total brain infarct volume was determined 72 h after exposure to HI. Treatment with IAIPs immediately after HI decreased (P < 0.05) infarct volumes by 58.0% and 44.5% in male and female neonatal rats, respectively. Delayed treatment with IAIPs after HI decreased (P < 0.05) infarct volumes by 23.7% in male, but not in female rats. We conclude that IAIPs exert neuroprotective effects even after exposure to severe HI in neonatal rats and appear to exhibit some sex-related differential effects.

Immuno-modulator inter-alpha inhibitor proteins ameliorate complex auditory processing deficits in rats with neonatal hypoxic-ischemic brain injury.

Hypoxic-ischemic (HI) brain injury is recognized as a significant problem in the perinatal period, contributing to life-long language-learning and other cognitive impairments. Central auditory processing deficits are common in infants with hypoxic-ischemic encephalopathy and have been shown to predict language learning deficits in other at risk infant populations. Inter-alpha inhibitor proteins (IAIPs) are a family of structurally related plasma proteins that modulate the systemic inflammatory response to infection and have been shown to attenuate cell death and improve learning outcomes after neonatal brain injury in rats. Here, we show that systemic administration of IAIPs during the early HI injury cascade ameliorates complex auditory discrimination deficits as compared to untreated HI injured subjects, despite reductions in brain weight. These findings have significant clinical implications for improving central auditory processing deficits linked to language learning in neonates with HI related brain injury.

A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit.

Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only "cure" is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF) in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb) and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α1-microglobulin (A1M), removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co-administration of A1M. This study provides preclinical evidence supporting further examination of A1M as a potential new therapy for preeclampsia.

Inter-alpha inhibitor protein administration improves survival from neonatal sepsis in mice.

Inter-alpha inhibitor proteins (IaIp) are serine proteases inhibitors that modulate endogenous protease activity and have been shown to improve survival in adult models of sepsis. We evaluated the effect of IaIp on survival and systemic responses to sepsis in neonatal mice. Sepsis was induced in 2-d-old mice with lipopolysaccharide (LPS), Escherichia coli, and group B Streptococci. Sepsis was associated with 75% mortality. IaIp, given by i.p. administration at doses between 15 and 45 mg/kg from 1 to 6 h after the onset of sepsis, improved survival to approximately 90% (p = 0.0159) in both LPS-induced sepsis and with live bacterial infections. The greatest effect was on reversal of hemorrhagic pneumonitis. The effects were dose and time dependent. Systemic cytokine profile and tissue histology were examined. Survival was compared in IL-10 knock out animals. Systemic cytokine levels including TNF-[alpha] and IL-10 were increased after induction of sepsis and modulated significantly after IaIp administration. Because the effect of IaIp was still demonstrable in IL-10 deficient mice, we conclude the beneficial effects of IaIp is because of suppression of proinflammatory cytokines such as TNF-[alpha] rather than augmentation of IL-10. IaIp may offer significant benefits as a therapeutic

Delayed administration of human inter-alpha inhibitor proteins reduces mortality in sepsis.

OBJECTIVE: We have recently shown that administration of human inter-alpha inhibitor proteins (IalphaIp) very early after the onset of sepsis maintains cardiovascular stability and reduced mortality. However, it remains unknown whether injection of IalphaIp at later time points of sepsis has any beneficial effects. We therefore hypothesized that IalphaIp and its active component bikunin are reduced in sepsis and that the delayed administration of IalphaIp also improves survival rate. DESIGN: : Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: : Male adult Sprague-Dawley rats. INTERVENTIONS: Rats were subjected either to polymicrobial sepsis by cecal ligation and puncture (CLP) or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). MEASUREMENTS AND MAIN RESULTS: : Bikunin gene expression in the liver was measured by reverse transcription polymerase chain reaction. Plasma concentrations of IalphaIp were determined by Western blot at 5 and 20 hrs after CLP. IalphaIp clearance was assessed by injecting radioactive IalphaIp at 12 hrs post-CLP, and the half-life was determined. In addition, IalphaIp (30 mg/kg of body weight) or vehicle was administered at 1, 5, or 10 hrs (single treatment) or at both 10 and 20 hrs (double treatment) post-CLP. The necrotic cecum was excised at 20 hrs post-CLP, and 10-day survival was recorded. The results indicate that bikunin gene expression decreased significantly at 20 hrs post-CLP. Moreover, IalphaIp concentrations decreased significantly at 5 and 20 hrs post-CLP, and its half-life increased from 5.6 +/- 0.3 hrs to 11.8 +/- 2.7 hrs (p <.05), suggesting down-regulation of IalphaIp in sepsis despite the decreased clearance. Administration of IalphaIp at 1 hr post-CLP improved the survival rate from 50% to 92% (p <.05), whereas there was no significant improvement when IalphaIp was administrated at 5 or 10 hrs post-CLP. However, double injection of IalphaIp at 10 and 20 hrs post-CLP (i.e., severe sepsis) increased the survival rate from 44% to 81% (p <.05). CONCLUSION: Since delayed but repeated administration of human IalphaIp improves survival after CLP, this compound appears to be a useful agent for the treatment of severe sepsis.

TNF/IL-1-inducible protein TSG-6 potentiates plasmin inhibition by inter-alpha-inhibitor and exerts a strong anti-inflammatory effect in vivo.

TNF-stimulated gene 6 (tsg6), encoding a 35-kDa secretory glycoprotein (TSG-6), is induced in fibroblasts, chondrocytes, synovial cells, and mononuclear cells by the proinflammatory cytokines TNF-alpha and IL-1, or by LPS. Large amounts of TSG-6 protein were found in synovial fluids of patients with rheumatoid arthritis. TSG-6 protein forms a stable complex with components of the serine protease inhibitor, inter-alpha-inhibitor (I alpha I). In this work, we show that TSG-6 potentiates the inhibitory effect of l alpha l on the protease activity of plasmin. The plasmin/plasminogen activator system is important in the protease network associated with inflammation. To test the hypothesis that through their cooperative inhibitory effect on plasmin TSG-6 and l alpha l can modulate the protease network and thus inhibit inflammation, we examined the effect of TSG-6 on experimentally induced inflammation. Human recombinant TSG-6 protein showed a potent anti-inflammatory activity in the murine air pouch model of carrageenan- or IL-1-induced acute inflammation. The inhibitory effect of locally administered TSG-6 on the IL-1-induced cellular infiltration was comparable with that of systemic dexamethasone treatment. Two mutant TSG-6 proteins with single amino acid substitutions close to the N terminus showed a complete or partial loss of anti-inflammatory activity. The anti-inflammatory effect of the TNF/IL-1-inducible TSG-6 protein, along with its ability to inhibit protease action through interaction with l alpha l, suggests that TSG-6 production during inflammation is part of a negative feedback loop operating through the protease network.

Immunosuppressive activity of IRA on the plaque-forming response to SRBC in vitro: reversal with educated cells.

The alpha-globulin-rich fraction of Cohn Fraction IV, designated IRA, suppresses the in vitro antibody response to sheep red blood cells (SRBC) without cytotoxicity. IRA was effective if added before or up to 24 hr after antigen exposure. The suppression could be reversed after 24-hr treatment by washing the cells two to three times; after 48 hr of IRA treatment, however, suppression could only be partially reversed. The addition of a population of thymus-derived cells educated to the antigen SRBC could effectively reverse the IRA-induced inhibition of antibody production, whereas BSA-educated T cells could not.