Enhancement of fibrinogen-triggered pro-coagulant activation of monocytes in vitro by matrix metalloproteinase-9

<p>Abstract</p> <p>Background</p> <p>Interaction of fibrinogen with specific leukocyte integrins of monocytes may link coagulation and inflammation, however, the precise mechanism of fibrinogen leading to the pro-inflammatory and pro-coagulatory response on monocytes is yet unknown.</p> <p>Results</p> <p>Fibrinogen and its digestion fragment D induced pro-coagulant activation of monocytes as assessed in a cellular coagulation assay by reductions in clotting times. Pro-coagulant activation was reversed by blocking antibodies against Mac-1 or LFA-1. Pre-exposure of monocytes to the p38 MAPK inhibitor SB 202190 and the MEK1.2 inhibitor U0126 led to significant increasees in coagulation times whereas blocking JNKII with its inhibitor had no such effect. Blocking NFκB with MG-132 also inhibited pro-coagulant activation of monocytes by fibrinogen. A selective inhibitor of matrix metalloproteinase-9 increased times to clot formation whereas other matrix metalloproteinase inhibitors did not significantly interfere with fibrinogen-augmented clot formation in this assay. Treatment of monocytes with fibrinogen increased concentrations of matrix metalloproteinase-9 immunoreactivity in their supernatants.</p> <p>Conclusions</p> <p>Fibrinogen induces monocyte pro-coagulant activation in an integrin-, nuclear factor κB-, p38 MAPK-, and MEK1.2-dependent manner. Activation of monocytes by fibrinogen increases metalloproteinase-9 secretion, metalloproteinase-9 itself enhances monocyte coagulation by an autocrine mechanism. Results provide further evidence that mediators of hemostasis have a profound impact on cells of the immune system and are closely related to inflammatory pathways.</p

The militarisation of English schools: Troops to Teaching and the implications for Initial Teacher Education and race equality

This article considers the implications of the Troops to Teaching (TtT) programme, to be introduced in England in autumn 2013, for Initial Teacher Education (ITE) and race equality. TtT will fast-track ex-armed service members to teach in schools, without necessarily the requirement of a university degree. Employing theories of white supremacy, and Althusser’s (1971) concept of Ideological and Repressive State Apparatus, I argue that this initiative both stems from, and contributes to, a system of social privilege and oppression in education. Despite appearing to be aimed at all young people, the planned TtT initiative is actually aimed at poor and racially subordinated youth. This is likely to further entrench polarisation in a system which already provides two tier educational provision: TtT will be a programme for the inner-city disadvantaged, whilst wealthier, whiter schools will mostly continue to get highly qualified teachers. Moreover, TtT contributes to a wider devaluing of current ITE; ITE itself is rendered virtually irrelevant, as it seems TtT teachers will not be subject specialists, rather will be expected to provide military-style discipline, the skills for which they will be expected to bring with them. More sinister, I argue that TtT is part of the wider militarisation of education. This military-industrial-education complex seeks to contain and police young people who are marginalised along lines of race and class, and contributes to a wider move to increase ideological support for foreign wars - both aims ultimately in the service of neoliberal objectives which will feed social inequalities

A phase I study evaluating the pharmacokinetics, safety and tolerability of an antibody-based tissue factor antagonist in subjects with acute lung injury or acute respiratory distress syndrome

<p>Abstract</p> <p>Background</p> <p>The tissue factor (TF)-dependent extrinsic pathway has been suggested to be a central mechanism by which the coagulation cascade is locally activated in the lungs of patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) and thus represents an attractive target for therapeutic intervention. This study was designed to determine the pharmacokinetic and safety profiles of ALT-836, an anti-TF antibody, in patients with ALI/ARDS.</p> <p>Methods</p> <p>This was a prospective, randomized, placebo-controlled, dose-escalation Phase I clinical trial in adult patients who had suspected or proven infection, were receiving mechanical ventilation and had ALI/ARDS (PaO₂/FiO₂≤ 300 mm). Eighteen patients (6 per cohort) were randomized in a 5:1 ratio to receive ALT-836 or placebo, and were treated within 48 hours after meeting screening criteria. Cohorts of patients were administered a single intravenously dose of 0.06, 0.08 or 0.1 mg/kg ALT-836 or placebo. Blood samples were taken for pharmacokinetic and immunogenicity measurements. Safety was assessed by adverse events, vital signs, ECGs, laboratory, coagulation and pulmonary function parameters.</p> <p>Results</p> <p>Pharmacokinetic analysis showed a dose dependent exposure to ALT-836 across the infusion range of 0.06 to 0.1 mg/kg. No anti-ALT-836 antibody response was observed in the study population during the trial. No major bleeding episodes were reported in the ALT-836 treated patients. The most frequent adverse events were anemia, observed in both placebo and ALT-836 treated patients, and ALT-836 dose dependent, self-resolved hematuria, which suggested 0.08 mg/kg as an acceptable dose level of ALT-836 in this patient population.</p> <p>Conclusions</p> <p>Overall, this study showed that ALT-836 could be safely administered to patients with sepsis-induced ALI/ARDS.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01438853">NCT01438853</a></p

Serpin Induced Antiviral Activity of Prostaglandin Synthetase-2 against HIV-1 Replication

The serine protease inhibitors (serpins) are anti-inflammatory proteins that have various functions. By screening a diverse panel of viruses, we demonstrate that the serpin antithrombin III (ATIII) has a broad-spectrum anti-viral activity for HIV-1, HCV and HSV. To investigate the mechanism of action in more detail we investigated the HIV-1 inhibition. Using gene-expression arrays we found that multiple host cell signal transduction pathways were activated by ATIII in HIV-1 infected cells but not in uninfected controls. Moreover, the signal pathways initiated by ATIII treatment, were more than 200-fold increased by the use of heparin-activated ATIII. The most up-regulated transcript in HIV-1 infected cells was prostaglandin synthetase-2 (PTGS2). Furthermore, we found that over-expression of PTGS2 reduced levels of HIV-1 replication in human PBMC. These findings suggest a central role for serpins in the host innate anti-viral response. Host factors such as PTGS2 elicited by ATIII treatment could be exploited in the development of novel anti-viral interventions

Thrombin Induces Macrophage Migration Inhibitory Factor Release and Upregulation in Urothelium: A Possible Contribution to Bladder Inflammation

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.MIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.UROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.Urothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression

Collaborative Enhancement of Antibody Binding to Distinct PECAM-1 Epitopes Modulates Endothelial Targeting

Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) facilitate targeted drug delivery to endothelial cells by “vascular immunotargeting.” To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs) to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The “collaborative enhancement” of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The “collaborative enhancement” phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents

Protein C anticoagulant system—anti-inflammatory effects

Activated protein C (APC) plays active roles in preventing progression of a number of disease processes. These include thrombosis due to its direct anticoagulant activity which is likely augmented by its cytoprotective activity, thereby limiting exposure of procoagulant cellular membrane surfaces on cells. Beyond that, the pathway signals the cells to prevent apoptosis, to dampen inflammation, to increase endothelial barrier function, and to selectively downregulate some genes implicated in disease progression. Most of these functions are manifested to APC binding to endothelial protein C receptor (EPCR) allowing PAR1 activation, but activation of other PARS is also implicated in some cases. In addition to EPCR orchestrating these changes, CD11b is also capable of supporting APC signaling. Selective control of these pathways offers potential in new therapeutic approaches to disease

Systemic versus localized coagulation activation contributing to organ failure in critically ill patients

In the pathogenesis of sepsis, inflammation and coagulation play a pivotal role. Increasing evidence points to an extensive cross-talk between these two systems, whereby inflammation not only leads to activation of coagulation but coagulation also considerably affects inflammatory activity. The intricate relationship between inflammation and coagulation may not only be relevant for vascular atherothrombotic disease in general but has in certain clinical settings considerable consequences, for example in the pathogenesis of microvascular failure and subsequent multiple organ failure, as a result of severe infection and the associated systemic inflammatory response. Molecular pathways that contribute to inflammation-induced activation of coagulation have been precisely identified. Pro-inflammatory cytokines and other mediators are capable of activating the coagulation system and downregulating important physiological anticoagulant pathways. Activation of the coagulation system and ensuing thrombin generation is dependent on an interleukin-6-induced expression of tissue factor on activated mononuclear cells and endothelial cells and is insufficiently counteracted by physiological anticoagulant mechanisms and endogenous fibrinolysis. Interestingly, apart from the overall systemic responses, a differential local response in various vascular beds related to specific organs may occur