Inter-Alpha Inhibitor Proteins Modify the Microvasculature after Exposure to Hypoxia-Ischemia and Hypoxia in Neonatal Rats.

Microvasculature develops during early brain development. Hypoxia-ischemia (HI) and hypoxia (H) predispose to brain injury in neonates. Inter-alpha inhibitor proteins (IAIPs) attenuate injury to the neonatal brain after exposure to HI. However, the effects of IAIPs on the brain microvasculature after exposure to HI have not been examined in neonates. Postnatal day-7 rats were exposed to sham treatment or right carotid artery ligation and 8% oxygen for 90 min. HI comprises hypoxia (H) and ischemia to the right hemisphere (HI-right) and hypoxia to the whole body, including the left hemisphere (H-left). Human IAIPs (hIAIPs, 30 mg/kg) or placebo were injected immediately, 24 and 48 h after HI/H. The brains were analyzed 72 h after HI/H to determine the effects of hIAIPs on the microvasculature by laminin immunohistochemistry and calculation of (1) the percentage area stained by laminin, (2) cumulative microvessel length, and (3) density of tunneling nanotubes (TNTs), which are sensitive indicators of the earliest phases of neo-vascularization/collateralization. hIAIPs mainly affected the percent of the laminin-stained area after HI/H, cumulative vessel length after H but not HI, and TNT density in females but not males. hIAIPs modify the effects of HI/H on the microvasculature after brain injury in neonatal rats and exhibit sex-related differential effects. Our findings suggest that treatment with hIAIPs after exposure to H and HI in neonatal rats affects the laminin content of the vessel basal lamina and angiogenic responses in a sex-related fashion.

High-mobility group box-1 and inter-alpha inhibitor proteins: In vitro binding and co-localization in cerebral cortex after hypoxic-ischemic injury.

The high-mobility group box-1 (HMGB1) protein is a transcription-regulating protein located in the nucleus. However, it serves as a damage-associated molecular pattern protein that activates immune cells and stimulates inflammatory cytokines to accentuate neuroinflammation after release from damaged cells. In contrast, Inter-alpha Inhibitor Proteins (IAIPs) are proteins with immunomodulatory effects including inhibition of pro-inflammatory cytokines. We have demonstrated that IAIPs exhibit neuroprotective properties in neonatal rats exposed to hypoxic-ischemic (HI) brain injury. In addition, previous studies have suggested that the light chain of IAIPs, bikunin, may exert its anti-inflammatory effects by inhibiting HMGB1 in a variety of different injury models in adult subjects. The objectives of the current study were to confirm whether HMGB1 is a target of IAIPs by investigating the potential binding characteristics of HMGB1 and IAIPs in vitro, and co-localization in vivo in cerebral cortices after exposure to HI injury. Solid-phase binding assays and surface plasmon resonance (SPR) were used to determine the physical binding characteristics between IAIPs and HMGB1. Cellular localizations of IAIPs-HMGB1 in neonatal rat cortex were visualized by double labeling with anti-IAIPs and anti-HMGB1 antibodies. Solid-phase binding and SPR demonstrated specific binding between IAIPs and HMGB1 in vitro. Cortical cytoplasmic and nuclear co-localization of IAIPs and HMGB1 were detected by immunofluorescent staining in control and rats immediately and 3 hours after HI. In conclusion, HMGB1 and IAIPs exhibit direct binding in vitro and co-localization in vivo in neonatal rats exposed to HI brain injury suggesting HMGB1 could be a target of IAIPs.

Changes in Cellular Localization of Inter-Alpha Inhibitor Proteins after Cerebral Ischemia in the Near-Term Ovine Fetus.

Inter-alpha Inhibitor Proteins (IAIPs) are key immunomodulatory molecules. Endogenous IAIPs are present in human, rodent, and sheep brains, and are variably localized to the cytoplasm and nuclei at multiple developmental stages. We have previously reported that ischemia-reperfusion (I/R) reduces IAIP concentrations in the fetal sheep brain. In this study, we examined the effect of I/R on total, cytoplasmic, and nuclear expression of IAIPs in neurons (NeuN+), microglia (Iba1+), oligodendrocytes (Olig2+) and proliferating cells (Ki67+), and their co-localization with histones and the endoplasmic reticulum in fetal brain cells. At 128 days of gestation, fetal sheep were exposed to Sham (n = 6) or I/R induced by cerebral ischemia for 30 min with reperfusion for 7 days (n = 5). Although I/R did not change the total number of IAIP+ cells in the cerebral cortex or white matter, cells with IAIP+ cytoplasm decreased, whereas cells with IAIP+ nuclei increased in the cortex. I/R reduced total neuronal number but did not change the IAIP+ neuronal number. The proportion of cytoplasmic IAIP+ neurons was reduced, but there was no change in the number of nuclear IAIP+ neurons. I/R increased the number of microglia and decreased the total numbers of IAIP+ microglia and nuclear IAIP+ microglia, but not the number of cytoplasmic IAIP+ microglia. I/R was associated with reduced numbers of oligodendrocytes and increased proliferating cells, without changes in the subcellular IAIP localization. IAIPs co-localized with the endoplasmic reticulum and histones. In conclusion, I/R alters the subcellular localization of IAIPs in cortical neurons and microglia but not in oligodendrocytes or proliferating cells. Taken together with the known neuroprotective effects of exogenous IAIPs, we speculate that endogenous IAIPs may play a role during recovery from I/R.

Novel Neuroprotective Agents to Treat Neonatal Hypoxic-Ischemic Encephalopathy: Inter-Alpha Inhibitor Proteins.

Perinatal hypoxia-ischemia (HI) is a major cause of brain injury and mortality in neonates. Hypoxic-ischemic encephalopathy (HIE) predisposes infants to long-term cognitive deficits that influence their quality of life and place a large burden on society. The only approved treatment to protect the brain after HI is therapeutic hypothermia, which has limited effectiveness, a narrow therapeutic time window, and is not considered safe for treatment of premature infants. Alternative or adjunctive therapies are needed to improve outcomes of full-term and premature infants after exposure to HI. Inter-alpha inhibitor proteins (IAIPs) are immunomodulatory molecules that are proposed to limit the progression of neonatal inflammatory conditions, such as sepsis. Inflammation exacerbates neonatal HIE and suggests that IAIPs could attenuate HI-related brain injury and improve cognitive outcomes associated with HIE. Recent studies have shown that intraperitoneal treatment with IAIPs can decrease neuronal and non-neuronal cell death, attenuate glial responses and leukocyte invasion, and provide long-term behavioral benefits in neonatal rat models of HI-related brain injury. The present review summarizes these findings and outlines the remaining experimental analyses necessary to determine the clinical applicability of this promising neuroprotective treatment for neonatal HI-related brain injury.

Association between decreased cord blood inter-alpha inhibitor levels and neonatal encephalopathy at birth.

BACKGROUND: Inter-alpha inhibitor proteins (IAIPs) are structurally related proteins found in the systemic circulation with immunomodulatory anti-inflammatory properties. Reduced levels are found in inflammatory related conditions including sepsis and necrotizing enterocolitis, and in neonatal rodents after exposure to hypoxia ischemia. In the current study, cord blood IAIP levels were measured in neonates with and without exposure to hypoxic-ischemic encephalopathy (HIE). METHODS: This is a prospective cohort study including infants born ≥36 weeks over a one-year period. Term pregnancies were divided into two groups: a "reference control" (uncomplicated term deliveries), and "moderate to severe HIE" (qualifying for therapeutic hypothermia). IAIPs were quantified using a sensitive ELISA on the cord blood samples. RESULTS: The study included 57 newborns: Reference control group (n = 13) and moderate/severe HIE group (n = 44). Measurement of IAIP cord blood concentrations in moderate to severe HIE group [278.2 (138.0, 366.0) µg/ml] revealed significantly lower IAIP concentrations compared with the control group [418.6 (384.5, 445.0) µg/ml] (p = 0.002). CONCLUSIONS: These findings suggest a potential role for IAIPs as indicators of neonates at risk for HIE. IAIP levels could have diagnostic implications in the management of HIE. Future research is required to explore the relationship between HIE and IAIPs as biomarkers for disease severity. CATEGORY OF STUDY: Translational.

Neuroprotective efficacy of hypothermia and Inter-alpha Inhibitor Proteins after hypoxic ischemic brain injury in neonatal rats.

Therapeutic hypothermia is the standard of care for hypoxic-ischemic (HI) encephalopathy. Inter-alpha Inhibitor Proteins (IAIPs) attenuate brain injury after HI in neonatal rats. Human (h) IAIPs (60 â€‹mg/kg) or placebo (PL) were given 15 â€‹min, 24 and 48 â€‹h to postnatal (P) day-7 rats after carotid ligation and 8% oxygen for 90 â€‹min with (30 â€‹°C) and without (36 â€‹°C) exposure to hypothermia 1.5 â€‹h after HI for 3 â€‹h. Hemispheric volume atrophy (P14) and neurobehavioral tests including righting reflex (P8-P10), small open field (P13-P14), and negative geotaxis (P14) were determined. Hemispheric volume atrophy in males was reduced (P â€‹< â€‹0.05) by 41.9% in the normothermic-IAIP and 28.1% in the hypothermic-IAIP compared with the normothermic-PL group, and in females reduced (P â€‹< â€‹0.05) by 30.3% in the normothermic-IAIP, 45.7% in hypothermic-PL, and 55.2% in hypothermic-IAIP compared with the normothermic-PL group after HI. Hypothermia improved (P â€‹< â€‹0.05) the neuroprotective effects of hIAIPs in females. The neuroprotective efficacy of hIAIPs was comparable to hypothermia in female rats (P â€‹= â€‹0.183). Treatment with hIAIPs, hypothermia, and hIAIPs with hypothermia decreased (P â€‹< â€‹0.05) the latency to enter the peripheral zone in the small open field test in males. We conclude that hIAIPs provide neuroprotection from HI brain injury that is comparable to the protection by hypothermia, hypothermia increases the effects of hIAIPs in females, and hIAIPs and hypothermia exhibit some sex-related differential effects.

Inter-alpha Inhibitor Proteins Ameliorate Brain Injury and Improve Behavioral Outcomes in a Sex-Dependent Manner After Exposure to Neonatal Hypoxia Ischemia in Newborn and Young Adult Rats.

Hypoxic-ischemic (HI) brain injury is a major contributor to neurodevelopmental morbidities. Inter-alpha inhibitor proteins (IAIPs) have neuroprotective effects on HI-related brain injury in neonatal rats. However, the effects of treatment with IAIPs on sequential behavioral, MRI, and histopathological abnormalities in the young adult brain after treatment with IAIPs in neonates remain to be determined. The objective of this study was to examine the neuroprotective effects of IAIPs at different neurodevelopmental stages from newborn to young adults after exposure of neonates to HI injury. IAIPs were given as 11-sequential 30-mg/kg doses to postnatal (P) day 7-21 rats after right common carotid artery ligation and exposure to 90 min of 8% oxygen. The resulting brain edema and injury were examined by T2-weighted magnetic resonance imaging (MRI) and cresyl violet staining, respectively. The mean T2 values of the ipsilateral hemisphere from MRI slices 6 to 10 were reduced in IAIP-treated HI males + females on P8, P9, and P10 and females on P8, P9, P10, and P14. IAIP treatment reduced hemispheric volume atrophy by 44.5 ± 29.7% in adult male + female P42 rats and improved general locomotor abilities measured by the righting reflex over time at P7.5, P8, and P9 in males + females and males and muscle strength/endurance measured by wire hang on P16 in males + females and females. IAIPs provided beneficial effects during the learning phase of the Morris water maze with females exhibiting beneficial effects. IAIPs confer neuroprotection from HI-related brain injury in neonates and even in adult rats and beneficial MRI and behavioral benefits in a sex-dependent manner.

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.

Inter-alpha inhibitor proteins attenuate lipopolysaccharide-induced blood-brain barrier disruption and downregulate circulating interleukin 6 in mice.

Circulating levels of inter-alpha inhibitor proteins change dramatically in acute inflammatory disorders, which suggest an important contribution to the immunomodulatory system. Human blood-derived inter-alpha inhibitor proteins are neuroprotective and improve survival of neonatal mice exposed to lipopolysaccharide. Lipopolysaccharide augments inflammatory conditions and disrupts the blood-brain barrier. There is a paucity of therapeutic strategies to treat blood-brain barrier dysfunction, and the neuroprotective effects of human blood-derived inter-alpha inhibitor proteins are not fully understood. To examine the therapeutic potential of inter-alpha inhibitor proteins, we administered human blood-derived inter-alpha inhibitor proteins to male and female CD-1 mice after lipopolysaccharide exposure and quantified blood-brain barrier permeability of intravenously injected 14C-sucrose and 99mTc-albumin. We hypothesized that human blood-derived inter-alpha inhibitor protein treatment would attenuate lipopolysaccharide-induced blood-brain barrier disruption and associated inflammation. Lipopolysaccharide increased blood-brain barrier permeability to both 14C-sucrose and 99mTc-albumin, but human blood-derived inter-alpha inhibitor protein treatment only attenuated increases in 14C-sucrose blood-brain barrier permeability in male mice. Lipopolysaccharide stimulated a more robust elevation of male serum inter-alpha inhibitor protein concentration compared to the elevation measured in female serum. Lipopolysaccharide administration also increased multiple inflammatory factors in serum and brain tissue, including interleukin 6. Human blood-derived inter-alpha inhibitor protein treatment downregulated serum interleukin 6 levels, which were inversely correlated with serum inter-alpha inhibitor protein concentration. We conclude that inter-alpha inhibitor proteins may be neuroprotective through mechanisms of blood-brain barrier disruption associated with systemic inflammation.

Effects of Juvenile or Adolescent Working Memory Experience and Inter-Alpha Inhibitor Protein Treatment after Neonatal Hypoxia-Ischemia.

Hypoxic-Ischemic (HI) brain injury in the neonate contributes to life-long cognitive impairment. Early diagnosis and therapeutic interventions are critical but limited. We previously reported in a rat model of HI two interventional approaches that improve cognitive and sensory function: administration of Inter-alpha Inhibitor Proteins (IAIPs) and early experience in an eight-arm radial water maze (RWM) task. Here, we expanded these studies to examine the combined effects of IAIPs and multiple weeks of RWM assessment beginning with juvenile or adolescent rats to evaluate optimal age windows for behavioral interventions. Subjects were divided into treatment groups; HI with vehicle, sham surgery with vehicle, and HI with IAIPs, and received either juvenile (P31 initiation) or adolescent (P52 initiation) RWM testing, followed by adult retesting. Error rates on the RWM decreased across weeks for all conditions. Whereas, HI injury impaired global performance as compared to shams. IAIP-treated HI subjects tested as juveniles made fewer errors as compared to their untreated HI counterparts. The juvenile group made significantly fewer errors on moderate demand trials and showed improved retention as compared to the adolescent group during the first week of adult retesting. Together, results support and extend our previous findings that combining behavioral and anti-inflammatory interventions in the presence of HI improves subsequent learning performance. Results further indicate sensitive periods for behavioral interventions to improve cognitive outcomes. Specifically, early life cognitive experience can improve long-term learning performance even in the presence of HI injury. Results from this study provide insight into typical brain development and the impact of developmentally targeted therapeutics and task-specific experience on subsequent cognitive processing.

Pharmacokinetics of Inter-Alpha Inhibitor Proteins and Effects on Hemostasis After Hypoxic-Ischemic Brain Injury in Neonatal Rats.

BACKGROUND: Hypoxic-ischemic (HI) brain injury is a leading cause of long-term neurodevelopmental morbidities in neonates. Human plasma-derived Inter-Alpha Inhibitor Proteins (hIAIPs) are neuroprotective after HI brain injury in neonatal rats. The light chain (bikunin) of hIAIPs inhibits proteases involved in the coagulation of blood. Newborns exposed to HI can be at risk for significant bleeding in the brain and other organs. OBJECTIVE: The objectives of the present study were to assess the pharmacokinetics (PK) and the duration of bleeding after intraperitoneal (IP) administration of hIAIPs in HI-exposed male and female neonatal rats. METHODS: HI was induced with the Rice-Vannucci method in postnatal (P) day-7 rats. After the right common carotid artery ligation, rats were exposed to 90 min of 8% oxygen. hIAIPs (30 mg/kg, IP) were given immediately after Sham or HI exposure in the PK study and serum was collected 1, 6, 12, 24, or 36 h after the injections. Serum hIAIP concentrations were measured with a competitive ELISA. ADAPT5 software was used to fit the pooled PK data considering first-order absorption and disposition. hIAIPs (60 mg/kg, IP) were given in the bleeding time studies at 0, 24 and 48 h after HI with tail bleeding times measured 72 h after HI. RESULTS: IP administration yielded significant systemic exposure to hIAIPs with PK being affected markedly including primarily faster absorption and reduced elimination as a result of HI and modestly of sex-related differences. hIAIP administration did not affect bleeding times after HI. CONCLUSION: These results will help to inform hIAIP dosing regimen schedules in studies of neuroprotection in neonates exposed to HI.

Inter-alpha Inhibitor Proteins Modulate Neuroinflammatory Biomarkers After Hypoxia-Ischemia in Neonatal Rats.

Neuroinflammation contributes to hypoxic-ischemic (HI) brain injury. Inter-alpha inhibitor proteins (IAIPs) have important immunomodulatory properties. Human (h) plasma-derived IAIPs reduce brain injury and improve neurobehavioral outcomes after HI. However, the effects of hIAIPs on neuroinflammatory biomarkers after HI have not been examined. We determined whether hIAIPs attenuated HI-related neuroinflammation. Postnatal day-7 rats exposed to sham-placebo, or right carotid ligation and 8% oxygen for 90 minutes with placebo, and hIAIP treatment were studied. hIAIPs (30 mg/kg) or PL was injected intraperitoneally immediately, 24, and 48 hours after HI. Rat complete blood counts and sex were determined. Brain tissue and peripheral blood were prepared for analysis 72 hours after HI. The effects of hIAIPs on HI-induced neuroinflammation were quantified by image analysis of positively stained astrocytic (glial fibrillary acid protein [GFAP]), microglial (ionized calcium binding adaptor molecule-1 [Iba-1]), neutrophilic (myeloperoxidase [MPO]), matrix metalloproteinase-9 (MMP9), and MMP9-MPO cellular markers in brain regions. hIAIPs reduced quantities of cortical GFAP, hippocampal Iba-1-positive microglia, corpus callosum MPO, and cortical MMP9-MPO cells and the percent of neutrophils in peripheral blood after HI in male, but not female rats. hIAIPs modulate neuroinflammatory biomarkers in the neonatal brain after HI and may exhibit sex-related differential effects.

Alterations in inter-alpha inhibitor protein expression after hypoxic-ischemic brain injury in neonatal rats.

Hypoxic-ischemic (HI) brain injury is frequently associated with premature and/or full-term birth-related complications that reflect widespread damage to cerebral cortical structures. Inflammation has been implicated in the long-term evolution and severity of HI brain injury. Inter-Alpha Inhibitor Proteins (IAIPs) are immune modulator proteins that are reduced in systemic neonatal inflammatory states. We have shown that endogenous IAIPs are present in neurons, astrocytes and microglia and that exogenous treatment with human plasma purified IAIPs decreases neuronal injury and improves behavioral outcomes in neonatal rats with HI brain injury. In addition, we have shown that endogenous IAIPs are reduced in the brain of the ovine fetus shortly after ischemic injury. However, the effect of HI on changes in circulating and endogenous brain IAIPs has not been examined in neonatal rats. In the current study, we examined changes in endogenous IAIPs in the systemic circulation and brain of neonatal rats after exposure to HI brain injury. Postnatal day 7 rats were exposed to right carotid artery ligation and 8% oxygen for 2h. Sera were obtained immediately, 3, 12, 24, and 48h and brains 3 and 24h after HI. IAIPs levels were determined by a competitive enzyme-linked immunosorbent assay (ELISA) in sera and by Western immunoblots in cerebral cortices. Serum IAIPs were decreased 3h after HI and remained lower than in non-ischemic rats up to 7days after HI. IAIP expression increased in the ipsilateral cerebral cortices 24h after HI brain injury and in the hypoxic contralateral cortices. However, 3h after hypoxia alone the 250kDa IAIP moiety was reduced in the contralateral cortices. We speculate that changes in endogenous IAIPs levels in blood and brain represent constituents of endogenous anti-inflammatory neuroprotective mechanism(s) after HI in neonatal rats.

Ischemia reduces inter-alpha inhibitor proteins in the brain of the ovine fetus.

Hypoxic-ischemic (HI) brain injury is a major cause of neurological abnormalities in the perinatal period. Inflammation contributes to the evolution of HI brain injury. Inter-alpha inhibitor proteins (IAIPs) are a family of proteins that are part of the innate immune system. We have reported that endogenous IAIPs exhibit developmental changes in ovine brain and that exogenous IAIP treatment reduces neuronal death in HI neonatal rats. However, the effects of HI on endogenous IAIPs in brain have not been previously examined. In this study, we examined the effects of ischemia-reperfusion on endogenous IAIPs levels in fetal sheep brain. Cerebral cortex, cerebellum, cervical spinal cord, choroid plexus, and CSF were snap frozen from sham control fetuses at 127 days gestation and after 30-min of carotid occlusion and 4-, 24-, and 48-h of reperfusion. IAIP levels were determined by Western immunoblot. IAIP expressions of the 250 kDa Inter-alpha inhibitor (IaI) and 125 kDa Pre-alpha inhibitor (PaI) in cerebral cortex and PaI in cerebellum were reduced (p < 0.05) 4-h after ischemia compared with controls and returned toward control levels 24- and 48-h after ischemia. CSF PaI and IaI were reduced 48 h after ischemia. We conclude that IAIPs in cerebral cortex and cerebellum are reduced by brain ischemia, and return toward control levels between 24 and 48 h after ischemia. However, changes in CSF IAIPs were delayed, exhibiting decreases 48 h after ischemia. We speculate that the decreases in endogenous IAIPs reflect increased utilization, potentially suggesting that they have endogenous neuroprotective properties. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 726-737, 2017.

ProThera Biologics, Inc.: a novel immunomodulator and biomarker for life-threatening diseases.

ProThera Biologics is a development stage bio-therapeutics company in East Providence, Rhode Island. The company was founded in 2002 to focus on the critical role and commercial potential of Inter-alpha Inhibitor Proteins (IAIP) for treating acute life-threatening inflammatory diseases. The discovery research originated in the basic research laboratories of the co-founders, Yow-Pin Lim, MD, PhD, and Douglas C. Hixson, PhD, at Rhode Island Hospital, a Lifespan partner. The company is backed by the Slater Technology Fund and has received research grants from the Rhode Island State Science and Technology Council (RI STAC) as well as continuous funding from the National Institutes of Health (NIH), with several Phase I and II Small Business Innovation Research (SBIR) grants over the past 10 years. ProThera has developed a novel process to purify Inter-alpha Inhibitor Proteins from source material, and has conducted groundbreaking research into the usage of IAIP to fight systemic inflammation.