Ontogeny of inter-alpha inhibitor proteins in ovine brain and somatic tissues

Abstract

Inter-alpha inhibitor proteins (IAIPs) found in relatively high concentrations in human plasma are important in inflammation. IAIPs attenuate brain damage in young and adult subjects, decrease during sepsis and necrotizing enterocolitis in premature infants, and attenuate sepsis-related inflammation in newborn rats. Although a few studies have reported adult organ-specific IAIP expression, information is not available on age-dependent IAIP expression. Given evidence suggesting IAIPs attenuate brain damage in young and adult subjects, and inflammation in newborns, we examined IAIP expression in plasma, cerebral cortex (CC), choroid plexus (CP), cerebral spinal fluid (CSF), and somatic organs in fetal, newborn, and adult sheep to determine the endogenous expression patterns of these proteins during development. IAIPs (enzyme-linked immunosorbent assay) were higher in newborn and adult than fetal plasma (P<0.05). Western immunoblot detected 125 kDa PaI (Pre-alpha Inhibitor) and 250 kDa IaI (Inter-alpha Inhibitor) in plasma, CNS, and somatic organs. PaI expression in CC and CP was higher in fetuses than newborns and adults, but IaI expression was higher in adults than fetuses and newborns. Both PaI and IaI were higher in fetal than newborn CSF. IAIPs exhibited organ-specific ontogenic patterns in placenta, liver, heart, and kidney. These results provide evidence for the first time that plasma, brain, placenta, liver, heart, and kidney express IAIPs throughout ovine development and that expression patterns are unique to each organ. Although exact functions of IAIPs in CNS and somatic tissues are not known, their presence in relatively high amounts during development suggests their potential importance in brain and organ development.

Effects of inter-alpha inhibitor proteins on neonatal brain injury: Age, task and treatment dependent neurobehavioral outcomes

“…urinary trypsin inhibitor (UTI)) also known as ulinastatin or bikunin, to attenuate inflammation and reduce brain injury, emphasize the great potential for the blood plasma derived IAIP to attenuate neonatal brain injury (Baek, et al, 2003, Chaaban, et al, 2010, Chaaban, et al, 2009, Koga, et al, 2010, Singh, et al, 2010, Wang, et al, 2013, Yano, et al, 2003). We have previously demonstrated high levels of IAIPs in the brain of fetal, newborn, and adult sheep and in cerebral spinal fluid of fetal sheep (Spasova, et al, 2014). Based upon these findings, we speculated that IAIPs represent endogenous immunomodulators that could be important in brain development and represent endogenous neuroprotective agents (Spasova, et al, 2014).…”

“…Based upon these findings, we speculated that IAIPs represent endogenous immunomodulators that could be important in brain development and represent endogenous neuroprotective agents (Spasova, et al, 2014). The present study taken together with our previous work (Spasova, et al, 2014) suggests that IAIPs could have an important role in brain development and in the evolution of brain injury after neonatal HI injury.…”

“…The purification process used a monolithic anion-exchange chromatographic method (CIMmultus, BIA Separation, Villach, Austria) that resulted in highly (>90%) pure biologically active IAIPs (US Patent #7,932,365, 2011) (Lim, 2013, Opal, et al, 2011, Spasova, et al, 2014). Following binding, the column was washed sequentially with a buffer containing 200 mM NaCl and 200 mM acetate buffer, pH 3.0.…”

“…Eluted IAIPs were concentrated and buffer exchanged using a tangential flow filtration system (Labscale, Millipore). Purity analysis was performed by SDS-PAGE, Western immunoblot, competitive IAIP ELISA and standardized in-vitro trypsin inhibition assay (Lim, 2013, Opal, et al, 2011, Spasova, et al, 2014). The biological activity is based on the ability of IAIPs to inhibit the hydrolysis of the substrate N-Benzoyl-L-arginine)- p -nitroaniline HCl (BAPNA, Sigma St. Louis, MO) by trypsin.…”

Ischemic-Reperfusion Injury Increases Matrix Metalloproteinases and Tissue Metalloproteinase Inhibitors in Fetal Sheep Brain

“…Our laboratory has previously described the use of the IC sample as a reference standard for immunoblots to ensure consistent quality of loading and transferring, and to control for variability across gels [4,[34][35][36][37]. We employed this method in our laboratory because our initial studies showed that most of the traditional housekeeping protein standards are affected by development and ischemic injury in sheep, thereby necessitating an alternative normalization method.…”