TRANSIENT LIPOPOLYSACCHARIDE-INDUCED CYTOKINE RESPONSES IN THE MATERNAL AND FETAL GUINEA PIG

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study.

A Quantitative Investigation of Selected Reactions in the Fibrinolytic Cascade

Previous work has shown that thrombin activatable fibrinolysis inhibitor (TAFI) was unable to prolong lysis of purified clots in the presence of Lys-plasminogen (Lys-Pg), indicating a possible mechanism for fibrinolysis to circumvent prolongation mediated by activated TAFI (TAFIa). Therefore, the effects of TAFIa on Lys-Pg activation and Lys-plasmin (Lys-Pn) inhibition by antiplasmin (AP) were quantitatively investigated using a fluorescently labeled recombinant Pg mutant which does not produce active Pn. High molecular weight fibrin degradation products (HMW-FDPs), a soluble fibrin surrogate that models Pn modified fibrin, treated with TAFIa decreased the catalytic efficiency (kcat/Km) of 5IAF-Glu-Pg cleavage by 417-fold and of 5IAF-Lys-Pg cleavage by 55-fold. A previously devised intact clot system was used to measure the apparent second order rate constant (k2) for Pn inhibition by AP over time. While TAFIa was able to abolish the protection associated with Pn modified fibrin in clots formed with Glu-Pg, it was not able to abolish the protection in clots formed with Lys-Pg. However, TAFIa was still able to prolong the lysis of clots formed with Lys-Pg. TAFIa prolongs clot lysis by removing the positive feedback loop for Pn generation. The effect of TAFIa modification of the HMW-FDPs on the rate of tissue type plasminogen activator (tPA) inhibition by plasminogen activator inhibitor type 1 (PAI-1) was investigated using a previously devised end point assay. HMW-FDPs decreased the k2 for tPA inhibition rate by 3-fold. Thus, HMW-FDPs protect tPA from PAI-1. TAFIa treatment of the HMW-FDPs resulted in no change in protection. Vitronectin also did not appreciably affect tPA inhibition by PAI-1. Pg, in conjunction with HMW-FDPs, decreased the k2 for tPA inhibition by 30-fold. Hence, Pg, when bound to HMW-FDPs, protects tPA by an additional 10-fold. TAFIa treatment of the HMW-FDPs completely removed this additional protection provided by Pg. In conclusion, an additional mechanism was identified whereby TAFIa can prolong clot lysis by increasing the rate of tPA inhibition by PAI-1 by eliminating the protective effects of Pn-modified fibrin and Pg. Because TAFIa can suppress Lys-Pg activation but cannot attenuate Lys-Pn inhibition by AP, the Glu- to Lys-Pg/Pn conversion is able to act as a fibrinolytic switch to ultimately lyse the clot.

COVALENT LIPOPROTEIN(A) ASSEMBLY: Characterization of Oxidase Activity Responsible for Catalyzing Covalent Lipoprotein(a) Assembly

Lipoprotein(a) (Lp(a)) has been identified as an emerging risk factor for the development of vascular diseases. The Lp(a) particle is assembled in a 2-step process upon secretion of the LDL and apo(a) components from hepatocytes. Work done by the Koschinsky group has identified an oxidase-like activity present in the conditioned medium (CM) harvested from human hepatoma (HepG2), as well as HEK 293 (human endothelian kidney) cells that catalyzes the rate of covalent Lp(a) formation. We have taken a candidate enzyme approach to identifying this oxidase activity. Specifically, we have proposed that the QSOX (Quiescin/sulfhydryl oxidase) is responsible for catalysis of covalent Lp(a) assembly. An oxidase activity assay developed by Dr. Thorpe (University of Delaware) was used to detect QSOX1 in CM harvested from cultured cell lines that catalyze covalent Lp(a) assembly. In addition, the QSOX1 transcript was identified in each cell line and quantified with the use of Real-Time RT-PCR. Quantitative assays of covalent Lp(a) assembly were performed to study some characteristics of the unkwown oxidase activity. First, conditioned medium was dialyzed through a 5 kDa cutoff, as this has previously been shown to reduce the aforementioned oxidase activity. Purified QSOX was then added back to the reaction and the rate of catalysis was observed. The addition of QSOX appeared to enhance the rate of covalent Lp(a) assembly in a dose-dependent manner. Additional covalent Lp(a) assembly assays were performed where various chemicals were added to determine whether Lp(a) assembly was affected. The addition of EDTA did not affect covalent assembly, suggesting that the oxidase activity may not be metallo-dependent. Moreover, dose-dependent addition of Calcium, DTT, Copper and glutathione to dialyzed medium also did not affect the rate of Lp(a) assembly. Taken together, these studies will aid in identifying the nature of the oxidase activity that catalyzes covalent Lp(a) assembly. This will provide us with valuable information on how Lp(a) particles are assembled, and may lead to the development of drugs inhibiting Lp(a) formation.