Hyperfibrinolysis through gene deletion or pharmacological inhibition of TAFI in mice.

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) and Plasminogen Activator Inhibitor-1 (PAI-1) are important regulators of the fibrinolytic system, albeit acting via distinct mechanisms. Both proteins exert their antifibrinolytic function by reducing the tissue-type plasminogen activator (tPA)-mediated activation of plasminogen into plasmin, the key enzyme responsible for fibrin degradation. TAFI can be proteolytically converted into its active enzyme (TAFIa) by thrombin(/thrombomodulin) or plasmin. TAFIa attenuates fibrinolysis by cleaving carboxyterminal lysines on partially degraded fibrin, thereby hampering efficient plasminogen activation. PAI-1 is the most important physiological inhibitor of tPA leading to reduced plasmin generation.Previous studies on the gene deficiency of TAFI or PAI-1 in mice did not reveal an overt phenotype i.e. mice were viable, developed normally and were fertile. Upon challenge in mouse thrombosis models, TAFI knockout (KO) as well as PAI-1 KO mice have different outcomes depending on the model, suggesting rather subtle roles of TAFI and PAI-1 gene deficiency in fibrinolysis. Therefore, we generated and characterized TAFI and PAI-1 double gene deficient mice in order to unravel if the combined deficiency would lead to a more manifested phenotype, with or without challenge in thrombosis models (Chapter 2). However, the phenotype of double gene deficient mice without challenge did not deviate from that of the single gene deficient mice, except for a higher frequency of death of the female breeding pair partners and their pups. Besides the absence of hemorrhage upon double gene deficiency, fibrinolysis was markedly enhanced upon challenge as observed in ex vivo rotational thromboelastometry and in in vivo mouse thromboembolism. However, the profibrinolytic effect was detected in all mice lacking TAFI, regardless of the presence or the absence of PAI-1. This points to a major contribution of TAFI gene deletion to the enhanced fibrinolytic capacity observed in the double gene deficient mice.Because the increased fibrinolytic capacity of the TAFI and PAI-1 gene deficient mice was mostly ascribed to its TAFI gene deletion, this project further concentrated on the evaluation of pharmacological TAFI inhibition. Subsequent to the in vitro functional characterization of monoclonal antibodies raised toward human TAFI, antibodies with inhibitory properties against mouse TAFI were selected to evaluate in in vitro clot lysis experiments and in an in vivo mouse thromboembolism model. In Chapter 3, we have shown that one antibody (MA-TCK26D6) was able to accelerate fibrinolysis and thus evidence was provided for the use of a TAFI inhibitor as profibrinolytic agent in preclinical settings. Besides the evaluation of their efficacy, the antibodies with discriminating inhibitory properties were also valuable to determine the most likely physiological activator of TAFI in diverse settings. Next to the thrombin/thrombomodulin (T/TM) complex as generally accepted TAFI activator, we have shown that plasmin also plays an important role in mediating TAFI activation. First, the profibrinolytic efficacy of MA-TCK26D6, a monoclonal antibody that mainly inhibits the plasmin-mediated TAFI activation, supported our view. Second, using a TAFI variant (TAFI-TI-K133A) that is mainly activated by T/TM and not by plasmin, it was shown that the activatability of TAFI by plasmin is essential for a full effect of TAFI on clot lysis prolongation (Chapter 3). Third, enhanced fibrinolysis was observed in a similar in vitro clot lysis study using a monoclonal antibody selectively inhibiting the plasmin-mediated TAFI activation (MA-TCK11A9; Mishra, Vercauteren et al., 2011). And fourth, in a human whole blood thrombus lysis model, antibodies that inhibit the plasmin-mediated TAFI activation (MA-TCK11A9 and MA-TCK27A4) gave faster lysis than an antibody that inhibits exclusively the T/TM-mediated TAFI activation (MA-T12D11) when the antibodies were added prior to thrombus formation (Chapter 4). Thus, from our studies it is obvious that plasmin is able to regulate its own generation through TAFI activation. In conclusion, T/TM as well as plasmin seems to be important in mediating TAFI activation. Because of the limited availability of MAs directed toward human TAFI that show cross-reactivity with mouse TAFI, we developed mice transiently expressing human TAFI in order to evaluate these antibodies in vivo in mouse thrombosis models (Chapter 5). Despite the promising results using the hydrodynamic gene delivery technique to express other target genes, hardly normal plasma TAFI levels were reached and the transient increase was not sustainable for more than one day. In the future, these mice will facilitate further studies on the in vivo evaluation of antibodies and derivatives generated toward human TAFI. Moreover, the hydrodynamic gene delivery approach would be of major interest to evaluate the effect of increased TAFI expression in hemophilia mice. To conclude, this study describes the major contribution of TAFI gene deficiency on the hyperfibrinolytic state observed in TAFI and PAI-1 double gene deficient mice. Furthermore, preclinical evidence is provided for the use of TAFI inhibiting monoclonal antibodies as profibrinolytics. The evaluation of these antibodies with discriminating inhibitory properties toward TAFI further revealed that plasmin is an important mediator of TAFI activation. In addition, mice expressing human TAFI were generated to facilitate additional studies on the evaluation of antibodies toward human TAFI.status: publishe

AN INDIVIDUAL-BASED MODEL FOR FERAL HOGS IN GREAT SMOKY MOUNTAINS NATIONAL PARK

The expansion of feral hog (Sus scrofa) populations in the United States has resulted in increased efforts to develop and implement control strategies designed to minimize the impacts done by this invasive species. We describe an individual-based model for feral hogs in Great Smoky Mountains National Park (GSMNP). The objectives of the model are to provide an understanding of the population dynamics of this feral hog population and to determine the efficacy of the annual harvest as a population control method. Results suggest that the dynamics of the population are driven by fall hard mast production and the GSMNP harvests currently limit growth of the population, but these control efforts have not reduced the population

Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

Helicobacter suis (H. suis) is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin), administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC)/lysate and intranasal immunization with Cholera toxin (CT)/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually) is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals

H&E and PAS staining of the fundus showing pronounced inflammation and severe pseudo-pyloric metaplasia.

<p>H&E (A) and PAS staining (B) of the fundus of an animal in the FC subcutaneously immunized and challenged group, showing pronounced inflammation and severe pseudo-pyloric metaplasia (original magnification: 100x).</p

The chemical structure of the CCR4 antagonist.

<p>The CCR4 antagonist AF-399/42018025 is a small chemical molecule with a molecular weight of 565.93. It contains containing six 5 or 6 membered aromatic rings and 3 nitrogen, sulfur, and oxygen atoms. The chemical name of the molecule is 4-(1-benzofuran-2-ylcarbonyl)-1-{5-[4-chlorobenzyl)sulfanyl]-1,3,4-thiadiazol-2-yl}-3-hydroxy-5-(2-thienyl)-1,5-dihydro-2H-pyrrol-2-one [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131364#pone.0131364.ref005" target="_blank">5</a>].</p

Histopathological scoring of inflammation Study 1.

<p>Shown are the median scores (min-max) of pseudo-pyloric metaplasia of the fundus, infiltration of the mucosa of the fundus with granulocytes and mononuclear cells 3 weeks after challenge of the vaccinated mice. Scoring: 0 = normal, 1 = mild, 2 = moderate, 3 = severe. Significant differences between the positive control group and the immunized and challenged groups are noted by</p><p>* (p<0.05). (HBSS: Hank’s balanced salt solution FC: Freund’s complete, FIC: Freund’s incomplete, CT: Cholera toxin, Curd: Curdlan, CpG: CpG-DNA IN: intranasally, SC: subcutaneously, con: uninfected group, inf: infected group)</p><p>Histopathological scoring of inflammation Study 1.</p