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Chen X.,Brown University | Rivard L.,Brown University | Naqvi S.,Brown University | Nakada S.,Brown University | And 5 more authors.
Neuroscience | Year: 2016

Inter-alpha Inhibitor Proteins (IAIPs) are a family of related serine protease inhibitors. IAIPs are important components of the systemic innate immune system. We have identified endogenous IAIPs in the central nervous system (CNS) of sheep during development and shown that treatment with IAIPs reduces neuronal cell death and improves behavioral outcomes in neonatal rats after hypoxic-ischemic brain injury. The presence of IAIPs in CNS along with their exogenous neuroprotective properties suggests that endogenous IAIPs could be part of the innate immune system in CNS. The purpose of this study was to characterize expression and localization of IAIPs in CNS. We examined cellular expressions of IAIPs in vitro in cultured cortical mouse neurons, in cultured rat neurons, microglia, and astrocytes, and in vivo on brain sections by immunohistochemistry from embryonic (E) day 18 mice and postnatal (P) day 10 rats. Cultured cortical mouse neurons expressed the light chain gene Ambp and heavy chain genes Itih-1, 2, 3, 4, and 5 mRNA transcripts and IAIP proteins. IAIP proteins were detected by immunohistochemistry in cultured cells as well as brain sections from E18 mice and P10 rats. Immunoreactivity was found in neurons, microglia, astrocytes and oligodendroglia in multiple brain regions including cortex and hippocampus, as well as within both the ependyma and choroid plexus. Our findings suggest that IAIPs are endogenous proteins expressed in a wide variety of cell types and regions both in vitro and in vivo in rodent CNS. We speculate that endogenous IAIPs may represent endogenous neuroprotective immunomodulatory proteins within the CNS. © 2016 IBRO.

Chen X.,Brown University | Sadowska G.B.,Brown University | Zhang J.,Brown University | Kim J.-E.,Brown University | And 11 more authors.
Neurobiology of Disease | Year: 2015

We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1β monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1β protein. This antibody also neutralizes the effects of interleukin-1β protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1β monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127. days of gestation were studied after 30. min of carotid occlusion and 24. h of reperfusion. Groups were sham operated placebo-control- (n. =. 5), ischemia-placebo- (n. =. 6), ischemia-anti-IL-1β antibody- (n. =. 7), and sham-control antibody- (n. =. 2) treated animals. Systemic infusions of placebo (0.154. M NaCl) or anti-interleukin-1β monoclonal antibody (5.1. ±. 0.6. mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15. min and 4. h after ischemia. Concentrations of interleukin-1β protein and anti-interleukin-1β monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (. Ki) with α-aminoisobutyric acid in multiple brain regions. Interleukin-1β protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1β protein increased (. P<. 0.001) after ischemia-reperfusion. After anti-interleukin-1β monoclonal antibody infusions, plasma anti-interleukin-1β monoclonal antibody was elevated (. P<. 0.001), brain anti-interleukin-1β monoclonal antibody levels were higher (. P<. 0.03), and interleukin-1β protein concentrations (. P<. 0.03) and protein expressions (. P<. 0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion-related increases in Ki across the brain regions (. P<. 0.04), and Ki showed an inverse linear correlation (r. = -. 0.65, P<. 0.02) with anti-interleukin-1β monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1β monoclonal antibody infusions after ischemia result in brain anti-interleukin-1β antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1β protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1β, contributes to impaired blood-brain barrier function after ischemia in the fetus. © 2014 Elsevier Inc.

Gaudet C.M.,Rhode Island College | Lim Y.-P.,Prothera Biologics, Llc | Stonestreet B.S.,Brown University | Threlkeld S.W.,Rhode Island College
Behavioural Brain Research | Year: 2016

Neonatal cerebral hypoxia-ischemia (HI) commonly results in cognitive and sensory impairments. Early behavioral experience has been suggested to improve cognitive and sensory outcomes in children and animal models with perinatal neuropathology. In parallel, we previously showed that treatment with immunomodulator Inter-alpha Inhibitor Proteins (IAIPs) improves cellular and behavioral outcomes in neonatal HI injured rats. The purpose of the current study was to evaluate the influences of early experience and typical maturation in combination with IAIPs treatment on spatial working and reference memory after neonatal HI injury. A second aim was to determine the effects of these variables on hippocampal CA1 neuronal morphology. Subjects were divided into two groups that differed with respect to the time when exposed to eight arm radial water maze testing: Group one was tested as juveniles (early experience, Postnatal day (P) 36-61) and adults (P88-113), and Group two was tested in adulthood only (P88-113; without early experience). Three treatment conditions were included in each experience group (HI + Vehicle, HI + IAIPs, and Sham subjects). Incorrect arm entries (errors) were compared between treatment and experience groups across three error types (reference memory (RM), working memory incorrect (WMI), working memory correct (WMC)). Early experience led to improved working memory performance regardless of treatment. Combining IAIPs intervention with early experience provided a long-term behavioral advantage on the WMI component of the task in HI animals. Anatomically, early experience led to a decrease in the average number of basal dendrites per CA1 pyramidal neuron for IAIP treated subjects and a significant reduction in basal dendritic length in control subjects, highlighting the importance of pruning in typical early life learning. Our results support the hypothesis that early behavioral experience combined with IAIPs improve outcome on a relativity demanding cognitive task, beyond that of a single intervention strategy, and appears to facilitate neuronal plasticity following neonatal brain injury. © 2016 Elsevier B.V.

Spasova M.S.,Brown University | Chen X.,Brown University | Sadowska G.B.,Brown University | Horton E.R.,Brown University | And 2 more authors.
Developmental Neurobiology | Year: 2017

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. © 2016 Wiley Periodicals, Inc.

Threlkeld S.W.,Rhode Island College | Gaudet C.M.,Rhode Island College | La Rue M.E.,Brown University | Dugas E.,Rhode Island College | And 3 more authors.
Experimental Neurology | Year: 2014

Hypoxic-ischemic (HI) brain injury is frequently associated with premature and/or full term birth related complications. HI injury often results in learning and processing deficits that reflect widespread damage to an extensive range of cortical and sub-cortical brain structures. Further, inflammation has been implicated in the long-term progression and severity of HI injury. Recently, inter-alpha inhibitor proteins (IAIPs) have been shown to attenuate inflammation in models of systemic infection. Importantly, preclinical studies of neonatal HI injury and neuroprotection often focus on single time windows of assessment or single behavioral domains. This approach limits translational validity, given evidence for a diverse spectrum of neurobehavioral deficits that may change across developmental windows following neonatal brain injury. Therefore, the aims of this research were to assess the effects of human IAIPs on early neocortical cell death (72. h post-insult), adult regional brain volume measurements (cerebral cortex, hippocampus, striatum, corpus callosum) and long-term behavioral outcomes in juvenile (P38-50) and adult (P80. +) periods across two independent learning domains (spatial and non-spatial learning), after postnatal day 7 HI injury in rats. Here, for the first time, we show that IAIPs reduce acute neocortical neuronal cell death and improve brain weight outcome 72. h following HI injury in the neonatal rat. Further, these longitudinal studies are the first to show age, task and treatment dependent improvements in behavioral outcome for both spatial and non-spatial learning following systemic administration of IAIPs in neonatal HI injured rats. Finally, results also show sparing of brain regions critical for spatial and non-spatial learning in adult animals treated with IAIPs at the time of injury onset. These data support the proposal that inter-alpha inhibitor proteins may serve as novel therapeutics for brain injury associated with premature birth and/or neonatal brain injury and highlight the importance of assessing multiple ages, brain regions and behavioral domains when investigating experimental treatment efficacy. © 2014 Elsevier Inc.

Chaaban H.,Women and Infants Hospital | Shin M.,Prothera Biologics, Llc | Sirya E.,Prothera Biologics, Llc | Lim Y.-P.,Prothera Biologics, Llc | And 2 more authors.
Journal of Pediatrics | Year: 2010

Objectives: To compare inter-alpha inhibitor protein (IaIp) levels in neonates with proven necrotizing enterocolitis (NEC) and neonates with other, nonspecific abdominal disorders. Study design: This was a prospective observational study of neonates in the neonatal intensive care unit. NEC was diagnosed according to Bell's staging criteria. The nNeonates in the control group had a nonspecific abdominal disorder, but no radiographic evidence of NEC and no disease progression. All neonates with radiographically confirmed NEC were included. Plasma IaIp levels were quantitated by enzyme-linked immunosorbent assay. Results: Seventeen neonates had confirmed NEC, and 34 neonates had nonspecific abdominal disorders that improved rapidly. Gestational age, postnatal age, weight, sex, maternal obstetric variables, rupture of membranes, and mode of delivery did not differ between the two groups. Mean IaIp level was significantly lower in the NEC group compared with the control group (137 ± 38 mg/L; 95% confidence interval [CI], 118-157 mg/L vs 258 ± 53 mg/L; 95% CI, 238-277 mg/L; P <.0001). Conclusions: The finding of significantly lower IaIp levels in neonates with NEC suggests that IaIp might be a useful, sensitive biomarker, allowing initiation of appropriate therapy and reducing antibiotic overuse in neonates with suspected but unproven NEC. Administration of IaIp may significantly reduce the severity of systemic inflammation and associated tissue injury. © 2010 Mosby Inc. All rights reserved.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 498.06K | Year: 2012

DESCRIPTION (provided by applicant): The primary goal of this proposed research is to develop a rapid point-of-care (POC) test that can be used to identify neonatal sepsis in high-risk infants in a simple, user-friendly and portable device suitable for usein the NICU setting. Since sepsis presents a very serious threat to neonates, there is an urgent need to obtain confirmation as soon as possible. Currently, there are no reliable, POC markers for this life threatening disease. Blood culture results are considered the gold standard for bacterial sepsis, but confirmatory results may not be available until at least 48 hours. In this proposal, we will attemp to develop a quantitative rapid test based on Inter-alpha Inhibitor Proteins (IAIP) that has been recently demonstrated as a valuable biomarker with high sensitivity and specificity (89.5% and 99%, respectively) and a high positive and negative predictive value (85% and 98% respectively) for neonatal sepsis. We hypothesize that endogenous IAIP, as part of the innate immune response, protect against the damaging effects of proteases released during acute systemic inflammation following severe infections, burn, trauma and injury. As a consequence, these proteins are rapidly consumed and excreted in the urine,leading to a rapid decrease in plasma levels. Furthermore, the IAIP level seems to inversely correlate with disease severity and progression, thus, a rapid IAIP test with result that can be obtained within 10- 15 min would be useful as a diagnostic and/ortheranostic marker in neonatal sepsis. The proposed specific aims of the study are: 1) Validation and comparison studies of the predictive value of IAIP alone and with other biomarkers in detecting infants with sepsis and systemic inflammation and 2) Prototype development of a quantitative lateral flow immunoassay (LFIA) for IAIP that can be used to diagnose neonatal sepsis and NEC. As a replacement therapy with plasma derived IAIP has been demonstrated to be effective in experimental models of sepsis and clinical trials in adult septic patients are currently underway, our long-term objective is to study the efficacy of concomitant IAIP treatment in critically ill infants due to suspected sepsis or NEC with low levels of IAIP as revealed by the rapid test.By combining the predictive test and therapeutic replacement of plasma derived IAIP, this novel approach may offer a rational, targeted solution for reducing the morbidity and mortality associated with neonatal sepsis and NEC in infants. Furthermore, an IAIP-based rapid test will provide an objective means for reducing antibiotic overuse in infants with suspected but unproven systemic infection. The potential impact of the proposed research is immense when one considers the serious unmet medical need for infants who suffer from the devastating effects of neonatal sepsis and NEC. PUBLIC HEALTH RELEVANCE: The goal of this proposed research is to develop a rapid test that can be used to detect life-threatening conditions such as whole body infection (sepsis and necrotizing enterocolitis) in infants using a simple, user-friendly and portable device suitable fo a bedside testing in the neonatal intensive care unit. The test is based on the level of blood proteins called Inter-alpha Inhibitors that are consumed during the disease. The faster the patient is diagnosed and treated, the better the prognosis and a chance of fewer complications. The potential impact of the proposed research is immense as it will reduce the devastating effects of these diseases.

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.69K | Year: 2015

DESCRIPTION provided by applicant Wound healing is an intricately regulated process broadly characterized by phases of inflammation proliferation and remodeling Imbalances in any one of these phases can result in inadequate healing scar formation or in the development of chronic wounds such as diabetic ulcers Diabetic ulcers are responsible for the majority of lower extremity amputations in the world and are among the principal reasons for hospitalization of diabetic patients These chronic wounds are debilitating painful and frequently never completely heal Despite extensive research and product development to improve wound care the clinical standard of care for these lesions has been largely unchanged for decades Inter alpha Inhibitor Proteins IAIP are naturally derived molecules that serve as a crucial component of the bodyandapos s protective defenses in modulating host response to pathological insults Currently these proteins are being developed as a potent therapy to treat acute life threatening diseases such as systemic inflammatory response syndrome SIRS sepsis and Anthrax intoxication and infection in biodefense applications IAIP have been described to play an important role not only in inflammation and angiogenesis but also in the wound healing process Our recent investigations using genetically altered mice deficient in IAIP bikunin knockout mice revealed a dysregulated wound repair process due to disruption of extracellular matrix ECM reorganization Additionally we found that IAIP level is markedly decreased in the wound tissues of diabetic mice compared to non diabetic controls suggesting impaired and depleted IAIP in the diabetic wound IAIP consist of multiple subunit heavy and light chains uniquely linked by glycosaminoglycan While the IAIP light chain also called bikunin inhibits various serine proteases the heavy chains of IAIP form covalent complexes with hyaluronan to allow efficient binding to its receptors such as CD The IAIP heavy chains also known to interact with matrix cellular proteins such as vitronectin fibronectin and tenascin c to promote wound healing In this proposal we would like to develop a novel topical IAIP formulation and obtain proof of concept of efficacy of a localized IAIP treatment approach in three different experimental models of wound healing using IAIP deficient KO monogenetic diabetic and polygenetic TallyHo diabetic mice We hypothesize that IAIP will play a significant role in the wound healing process affecting epidermal regeneration as well as extracellular matrix organization If confirmed this novel topical treatment based on plasma derived IAIP can be translated readily into the clinical use for problematic and chronic wound care The localized immunomodulatory IAIP treatment might also prevent wound complication and super infection making the potential impact of this research immense PUBLIC HEALTH RELEVANCE Problematic wounds such as diabetic ulcers are responsible for the majority of lower extremity amputations in the world and are among the principal reasons for hospitalization of diabetic patients These chronic wounds are debilitating painful and frequently never completely heal In this proposal we will develop a novel topical biological product based on natural proteins extracted from human blood As part of the components in wound healing process these proteins regulate inflammation and promote wound repair process This proposal will advance public health by developing a novel therapeutic strategy for wound care

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 203.23K | Year: 2014

DESCRIPTION (provided by applicant): Neonatal hypoxia-ischemia (HI) remains a major cause of acute perinatal brain injury, leading ultimately to neurologic dysfunction manifesting as cerebral palsy, mental retardation, and epilepsy. Cerebral oxygen deprivation and/or reduced blood flow due to umbilical cord occlusion, prolonged labor, and/or intracranial hemorrhage produce an inflammatory response contributing to neuronal cell death. Unfortunately, current treatment and prevention strategies in newborns are lacking and inadequate. There are no currently available therapies to prevent/treat and/or attenuate brain damage in premature infants and the only available therapeutic intervention for full term infants is hypothermia, which is only partially protective. Inter-alpha Inhibitor Proteins (IAIP) are naturally derived molecules that have been shown to play an important role in modulating inflammatory response by down-regulating pro-inflammatory cytokines in several experimental models of newborn and a

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