Longitudinal Assessment of Cytokine Expression and Plasminogen Activation in Hantavirus Cardiopulmonary Syndrome Reveals Immune Regulatory Dysfunction in End-Stage Disease.

Pathogenic New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS), a severe immunopathogenic disease in humans manifested by pulmonary edema and respiratory distress, with case fatality rates approaching 40%. High levels of inflammatory mediators are present in the lungs and systemic circulation of HCPS patients. Previous studies have provided insights into the pathophysiology of HCPS. However, the longitudinal correlations of innate and adaptive immune responses and disease outcomes remain unresolved. This study analyzed serial immune responses in 13 HCPS cases due to Sin Nombre orthohantavirus (SNV), with 11 severe cases requiring extracorporeal membrane oxygenation (ECMO) treatment and two mild cases. We measured viral load, levels of various cytokines, urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). We found significantly elevated levels of proinflammatory cytokines and PAI-1 in five end-stage cases. There was no difference between the expression of active uPA in survivors' and decedents' cases. However, total uPA in decedents' cases was significantly higher compared to survivors'. In some end-stage cases, uPA was refractory to PAI-1 inhibition as measured by zymography, where uPA and PAI-1 were strongly correlated to lymphocyte counts and IFN-γ. We also found bacterial co-infection influencing the etiology and outcome of immune response in two cases. Unsupervised Principal Component Analysis and hierarchical cluster analyses resolved separate waves of correlated immune mediators expressed in one case patient due to a sequential co-infection of bacteria and SNV. Overall, a robust proinflammatory immune response, characterized by an imbalance in T helper 17 (Th17) and regulatory T-cells (Treg) subsets, was correlated with dysregulated inflammation and mortality. Our sample size is small; however, the core differences correlated to survivors and end-stage HCPS are instructive.

Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection.

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.

The multifaceted role of plasminogen in inflammation.

A fine-tuned activation and deactivation of proteases and their inhibitors are involved in the execution of the inflammatory response. The zymogen/proenzyme plasminogen is converted to the serine protease plasmin, a key fibrinolytic factor by plasminogen activators including tissue-type plasminogen activator (tPA). Plasmin is part of an intricate protease network controlling proteins of initial hemostasis/coagulation, fibrinolytic and complement system. Activation of these protease cascades is required to mount a proper inflammatory response. Although best known for its ability to dissolve clots and cleave fibrin, recent studies point to the importance of fibrin-independent functions of plasmin during acute inflammation and inflammation resolution. In this review, we provide an up-to-date overview of the current knowledge of the enzymatic and cytokine-like effects of tPA and describe the role of tPA and plasminogen receptors in the regulation of the inflammatory response with emphasis on the cytokine storm syndrome such as observed during coronavirus disease 2019 or macrophage activation syndrome. We discuss tPA as a modulator of Toll like receptor signaling, plasmin as an activator of NFkB signaling, and summarize recent studies on the role of plasminogen receptors as controllers of the macrophage conversion into the M2 type and as mediators of efferocytosis during inflammation resolution.

The interaction of two novel putative proteins of Leptospira interrogans with E-cadherin, plasminogen and complement components with potential role in bacterial infection.

Leptospirosis is a worldwide zoonosis caused by pathogenic species of Leptospira. Leptospires are able to adhere to exposed extracellular matrix in injured tissues and, once in the bloodstream, can survive the attack of the immune system and spread to colonize target organs. In this work, we report that two novel putative proteins, coded by the genes LIC11711 and LIC12587 of L. interrogans serovar Copenhageni are conserved among pathogenic strains, and probably exposed in the bacterial surface. Soluble recombinant proteins were expressed in Escherichia coli, purified and characterized. Both recombinant proteins bound to laminin and E-cadherin, suggesting an initial adhesion function in host epithelial cells. The recombinant protein LIC11711 (rLIC11711) was able to capture plasminogen (PLG) from normal human serum and convert to enzymatically active plasmin (PLA), in the presence of PLG activator. rLIC12587 (recombinant protein LIC12587) displayed a dose dependent and saturable interaction with components C7, C8, and C9 of the complement system, reducing the bactericidal effect of the complement. Binding to C9 may have consequences such as C9 polymerization inhibition, interfering with the membrane attack complex formation. Blocking LIC11711 and LIC12587 on bacterial cells by the respective antiserum reduced leptospiral cell viability when exposed to normal human serum (NHS). Both recombinant proteins could be recognized by serum samples of confirmed leptospirosis, but not of unrelated diseases, suggesting that the native proteins are immunogenic and expressed during leptospirosis. Taken together, our data suggest that these proteins may have a role in leptospiral pathogenesis, participating in immune evasion strategies.

MASP-1 of the complement system alters fibrinolytic behaviour of blood clots.

BACKGROUND: The lectin pathway serine protease mannan-binding lectin-associated serine protease 1 (MASP-1) has been demonstrated to be a major link between complement and coagulation, yet little is known about its interactions with the fibrinolytic system. The aim of this work was to assess the effects of MASP-1 on fibrin clot lysis in different experimental settings. METHODS: Rotational thrombelastometry was used to evaluate the effect of MASP-1 on the lysis of clots formed in whole blood under static conditions. Whole blood clots were also formed in the presence and absence of MASP-1 under flow conditions in the Chandler loop and their lysis was analysed separately by fluorescence release of incorporated labelled fibrin. Real-time observation by laser scanning confocal microscopy was used to investigate the lysis of plasma clots where MASP-1 was present either during clot formation or lysis. Cleavage of tPA or plasminogen by MASP-1 was analysed by gel electrophoresis. We performed a turbidimetric clot lysis assay in the presence and absence of the MASP-1 inhibitor SGMI-1 (Schistocerca gregaria protease inhibitor (SGPI)-based MASP inhibitor-1) to evaluate the effect of endogenous MASP-1 in normal plasma and plasma samples from sepsis patients. RESULTS: In the thrombelastometric experiments, where MASP-1 was present during the entire clotting and lysis process, MASP-1 had a significant profibrinolytic effect and accelerated clot lysis. When clots were formed in the presence of MASP-1 under flow in the Chandler loop, the effects on fibrinolysis were heterogenous with impaired fibrinolysis in some individuals (n = 5) and no (n = 3) or even the opposite effect (n = 2) in others. In plasma clot lysis observed by confocal microscopy, lysis was prolonged when MASP-1 was added to the lysis solution, yet there was no difference in lysis time when MASP-1 was present during clot formation. When MASP-1 was incubated with tPA or plasminogen, respectively, cleavage of single-chain tPA into two-chain tPA and a slight reduction of plasminogen were observed. SGMI-1 significantly prolonged clot lysis in the turbidimetric clot lysis assay suggesting that MASP-1 accelerated lysis in plasma samples. CONCLUSION: MASP-1 is able to alter the susceptibility of blood clots to the fibrinolytic system. MASP-1 has complex, mostly promoting effects on fibrinolysis with high inter-individual variation. Interactions of MASP-1 with the fibrinolytic system may be relevant in the development and therapy of cardiovascular and thrombotic diseases.

Plasminogen and the Plasminogen Receptor, Plg-RKT, Regulate Macrophage Phenotypic, and Functional Changes.

Inflammation resolution is an active process that functions to restore tissue homeostasis. Clearance of apoptotic leukocytes by efferocytosis at inflammatory sites plays an important role in inflammation resolution and induces remarkable macrophage phenotypic and functional changes. Here, we investigated the effects of deletion of either plasminogen (Plg) or the Plg receptor, Plg-RKT, on the resolution of inflammation. In a murine model of pleurisy, the numbers of total mononuclear cells recruited to the pleural cavity were significantly decreased in both Plg-/- and Plg-RKT-/- mice, a response associated with decreased levels of the chemokine CCL2 in pleural exudates. Increased percentages of M1-like macrophages were determined in pleural lavages of Plg-/- and Plg-RKT-/- mice without significant changes in M2-like macrophage percentages. In vitro, Plg and plasmin (Pla) increased CD206/Arginase-1 expression and the levels of IL-10/TGF-ß (M2 markers) while decreasing IFN/LPS-induced M1 markers in murine bone-marrow-derived macrophages (BMDMs) and human macrophages. Furthermore, IL4-induced M2-like polarization was defective in BMDMs from both Plg-/- and Plg-RKT-/- mice. Mechanistically, Plg and Pla induced transient STAT3 phosphorylation, which was decreased in Plg-/- and Plg-RKT-/- BMDMs after IL-4 or IL-10 stimulation. The extents of expression of CD206 and Annexin A1 (important for clearance of apoptotic cells) were reduced in Plg-/- and Plg-RKT-/- macrophage populations, which exhibited decreased phagocytosis of apoptotic neutrophils (efferocytosis) in vivo and in vitro. Taken together, these results suggest that Plg and its receptor, Plg-RKT, regulate macrophage polarization and efferocytosis, as key contributors to the resolution of inflammation.

Thymic Microenvironment Is Modified by Malnutrition and Leishmania infantum Infection.

Detrimental effects of malnutrition on immune responses to pathogens have long been recognized and it is considered a main risk factor for various infectious diseases, including visceral leishmaniasis (VL). Thymus is a target of both malnutrition and infection, but its role in the immune response to Leishmania infantum in malnourished individuals is barely studied. Because we previously observed thymic atrophy and significant reduction in cellularity and chemokine levels in malnourished mice infected with L. infantum, we postulated that the thymic microenvironment is severely compromised in those animals. To test this, we analyzed the microarchitecture of the organ and measured the protein abundance in its interstitial space in malnourished BALB/c mice infected or not with L. infantum. Malnourished-infected animals exhibited a significant reduction of the thymic cortex:medulla ratio and altered abundance of proteins secreted in the thymic interstitial fluid. Eighty-one percent of identified proteins are secreted by exosomes and malnourished-infected mice showed significant decrease in exosomal proteins, suggesting that exosomal carrier system, and therefore intrathymic communication, is dysregulated in those animals. Malnourished-infected mice also exhibited a significant increase in the abundance of proteins involved in lipid metabolism and tricarboxylic acid cycle, suggestive of a non-proliferative microenvironment. Accordingly, flow cytometry analysis revealed decreased proliferation of single positive and double positive T cells in those animals. Together, the reduced cortical area, decreased proliferation, and altered protein abundance suggest a dysfunctional thymic microenvironment where T cell migration, proliferation, and maturation are compromised, contributing for the thymic atrophy observed in malnourished animals. All these alterations could affect the control of the local and systemic infection, resulting in an impaired response to L. infantum infection.

Molecular cloning and expression analysis of coagulation factor VIII and plasminogen involved in immune response to GCRV, and immunity activity comparison of grass carp Ctenopharyngodon idella with different viral resistance.

The grass carp reovirus (GCRV) has been shown to cause lethal infections in the grass carp Ctenopharyngodon idella (C. idella). In order to investigate the immune response to GCRV infection, the full-length cDNA sequences of coagulation factor VIII (CiFVIII) and plasminogen (CiPLG) from C. idella were cloned and their involvement in the immune response was studied. The immunity factor levels in C. idella with different GCRV resistances were also analyzed. The full-length 2478 bp cDNA of CiFVIII contained an open reading frame of 1965 bp and encoded a putative polypeptide of 654 amino acid residues. The full-length 2907 bp cDNA of CiPLG contained an open reading frame of 2133 bp and encoded a putative polypeptide of 710 amino acid residues. CiFVIII was closely clustered with that of Clupea harengus. CiPLG was first clustered with those of Cyprinus carpio and Danio rerio. CiFVIII transcripts were most abundant in the liver and least in the skin. The highest expression level of CiPLG was observed in liver and the lowest in muscle. Expression levels of CiFVIII in gill, head kidney and spleen, and expression levels of CiPLG in gill, intestine and liver all reached the maximum at 72 h post GCRV infection. In spleen, expression levels of CiFVIII and CiPLG were significantly positively correlated. The activities of T-AOC, LSZ and IgM in R♂ were significantly higher than those in O♂. Likewise, T-AOC and LSZ activities in F1 were significantly higher than f1 individuals (P < 0.01). These results indicated that CiFVIII and CiPLG may play important roles in the immune response to GCRV infection. In addition, antioxidant ability and serum immune factor activity may confer a different viral resistance to C. idella.

Anti-plasminogen antibodies in ANCA-associated vasculitis: An optimized anti-plasminogen assay.

Anti-plasminogen antibodies (α-PLG) were previously detected in a subpopulation of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients, showing a relation to renal lesions and outcome. Several studies showed different proportions of α-PLG positive AAV patients, possibly due to differences in the assays used. We here present a new, optimized α-PLG Enzyme-Linked Immuno Sorbent Assay (ELISA) and validate the presence of α-PLG in AAV. Different ELISA set-ups were tested regarding plasminogen (PLG) antigen, concentrations, coating buffers, blocking agents, and environmental conditions. Purified lysine-PLG (lys-PLG) showed better differentiation between positive samples and negative samples than glutamic acid-PLG (glu-PLG). Therefore, lys-PLG was used as coating antigen. With the optimized α-PLG ELISA we found α-PLG in 14.3% of the myeloperoxidase (MPO)-ANCA patients, whereas all our proteinase-3 (PR3)-ANCA patients tested in our new assay were negative. Concluding, in this study we have combined important technical findings and methods from previous studies to optimize the α-PLG assay, which can be used for future research purposes and will aid in uniform reporting of α-PLG status of patients.

Plasminogen activation is required for the development of radiation-induced dermatitis.

Skin damage caused by radiation therapy (radiodermatitis) is a severe side effect of radiotherapy in cancer patients, and there is currently a lack of effective strategies to prevent or treat such skin damage. In this work, we show with several lines of evidence that plasminogen, a pro-inflammatory factor, is key for the development of radiodermatitis. After skin irradiation in wild-type (plg+/+) mice, the plasminogen level increased in the irradiated area, leading to severe skin damage such as ulcer formation. However, plasminogen-deficient (plg-/-) mice and mice lacking plasminogen activators were mostly resistant to radiodermatitis. Moreover, treatment with a plasminogen inhibitor, tranexamic acid, decreased radiodermatitis in plg+/+ mice and prevented radiodermatitis in plg+/- mice. Together with studies at the molecular level, we report that plasmin is required for the induction of inflammation after irradiation that leads to radiodermatitis, and we propose that inhibition of plasminogen activation can be a novel treatment strategy to reduce and prevent the occurrence of radiodermatitis in patients.

Aspf2 From Aspergillus fumigatus Recruits Human Immune Regulators for Immune Evasion and Cell Damage.

The opportunistic fungal pathogen Aspergillus fumigatus can cause life-threatening infections, particularly in immunocompromised patients. Most pathogenic microbes control host innate immune responses at the earliest time, already before infiltrating host immune cells arrive at the site of infection. Here, we identify Aspf2 as the first A. fumigatus Factor H-binding protein. Aspf2 recruits several human plasma regulators, Factor H, factor-H-like protein 1 (FHL-1), FHR1, and plasminogen. Factor H contacts Aspf2 via two regions located in SCRs6-7 and SCR20. FHL-1 binds via SCRs6-7, and FHR1 via SCRs3-5. Factor H and FHL-1 attached to Aspf2-maintained cofactor activity and assisted in C3b inactivation. A Δaspf2 knockout strain was generated which bound Factor H with 28% and FHL-1 with 42% lower intensity. In agreement with less immune regulator acquisition, when challenged with complement-active normal human serum, Δaspf2 conidia had substantially more C3b (>57%) deposited on their surface. Consequently, Δaspf2 conidia were more efficiently phagocytosed (>20%) and killed (44%) by human neutrophils as wild-type conidia. Furthermore, Aspf2 recruited human plasminogen and, when activated by tissue-type plasminogen activator, newly generated plasmin cleaved the chromogenic substrate S2251 and degraded fibrinogen. Furthermore, plasmin attached to conidia damaged human lung epithelial cells, induced cell retraction, and caused matrix exposure. Thus, Aspf2 is a central immune evasion protein and plasminogen ligand of A. fumigatus. By blocking host innate immune attack and by disrupting human lung epithelial cell layers, Aspf2 assists in early steps of fungal infection and likely allows tissue penetration.

Binding of human plasminogen by the lipoprotein LipL46 of Leptospira interrogans.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira. Bacteria disseminate via the bloodstream and colonize the renal tubules of reservoir hosts. Leptospiral surface-exposed proteins are important targets, because due to their location they can elicit immune response and mediate adhesion and invasion processes. LipL46 has been previously reported to be located at the leptospiral outer membrane and recognized by antibodies present in serum of infected hamsters. In this study, we have confirmed the cellular location of this protein by immunofluorescence and FACS. We have cloned and expressed the recombinant protein LipL46 in its soluble form. LipL46 was recognized by confirmed leptospirosis human serum, suggesting its expression during infection. Binding screening of LipL46 with extracellular matrix (ECM) and plasma components showed that this protein interacts with plasminogen. The binding is dose-dependent on protein concentration, but saturation was not reached with the range of protein concentration used. Kringle domains of plasminogen and lysine residues of the recombinant protein are involved in the binding because the lysine analog, amino caproic acid (ACA) almost totally inhibited the reaction. The interaction of LipL46 with plasminogen generates plasmin in the presence of plasminogen activator uPA. Because plasmin generated at the leptospiral surface can degrade ECM molecules and decrease opsonophagocytosis, we tentatively infer that Lip46 has a role in helping the invasion process of pathogenic Leptospira.

Relevance of the plasminogen system in physiology, pathology, and regeneration of oral tissues - From the perspective of dental specialties.

Plasmin is a proteolytic enzyme that is crucial in fibrinolysis. In oral tissues, the plasminogen system plays an essential role in physiological and pathological processes, which in addition to fibrinolysis include degradation of extracellular matrix, inflammation, immune response, angiogenesis, tissue remodeling, cell migration, and wound healing. Oral tissues reveal a change in the plasminogen system during pathological processes such as periodontitis, peri-implantitis, or pulpitis, as well as in response to mechanical load. The plasminogen system is also a key element in tissue regeneration. The number of studies investigating the plasminogen system in dentistry have grown continuously in recent years, highlighting its increasing relevance in dental medicine. In this review, we present the diverse functions of the plasminogen system in physiology and its importance for dental specialists in pathology and regeneration. We thus provide an overview of the current knowledge on the role of the plasminogen system in the different fields of dentistry, including endodontics, orthodontics, periodontics, and oral surgery.

Co-existing ligneous conjunctivitis and IgG4-related disease.

Herein, we elucidate that ligneous conjunctivitis (LC) was proved as an IgG4-related disease (IgG4-RD) by a series of pathologic studies from primary and recurrent episodes of an LC patient. LC was diagnosed based on clinical presentation and pathological appearance; furthermore, combined with serological examination and immunohistochemical study, the case also conformed to the diagnosis of IgG4-RD. The IgG4-RD, broadly discussed in recent times, is an idiopathic disease entity with tissue fibrosis possibly involving multiple organs. To the best of our knowledge, IgG4-RD has never been reported with LC. By reporting the clinical course and literature review, we should pay attention to the association between these two diseases.

Libby amphibole-induced mesothelial cell autoantibodies bind to surface plasminogen and alter collagen matrix remodeling.

Lamellar pleural thickening (LPT) is a fibrotic disease induced by exposure to Libby amphibole (LA) asbestos that causes widespread scarring around the lung, resulting in deterioration of pulmonary function. Investigating the effects of autoantibodies to mesothelial cells (MCAA) present in the study populations has been a major part of the effort to understand the mechanism of pathogenesis. It has been shown in vitro that human mesothelial cells (Met5a) exposed to MCAA increase collagen deposition into the extracellular matrix (ECM). In this study, we sought to further elucidate how MCAA drive increased collagen deposition by identifying the protein targets bound by MCAA on the cellular surface using biotinylation to label and isolate surface proteins. Isolated surface protein fractions were identified as containing MCAA targets using ELISA The fractions that demonstrated binding by MCAA were then analyzed by tandem mass spectrometry (MS/MS) and MASCOT analysis. The most promising result from the MASCOT analysis, plasminogen (PLG), was tested for MCAA binding using purified human PLG in an ELISA We report that serum containing MCAA bound at an optical density (OD) 3 times greater than that of controls, and LA-exposed subjects had a high frequency of positive tests for anti-PLG autoantibodies. This work implicates the involvement of the plasminogen/plasmin system in the mechanism of excess collagen deposition in Met5a cells exposed to MCAA Elucidating this mechanism could contribute to the understanding of LPT.

Interaction of Trypanosoma evansi with the plasminogen-plasmin system.

Trypanosoma evansi is a widely-distributed haemoflagellated parasite of veterinary importance that infects a variety of mammals including horses, mules, camels, buffalos, cattle and deer. It is the causal agent of a trypanosomiasis known as Surra which produces epidemics of great economic importance in Africa, Asia and South America. The main pathology includes an enlarged spleen with hypertrophy of lymphoid follicles, congested lungs, neuronal degeneration and meningoencephalitis, where migration of the parasites from the blood to the tissues is essential. Most cells, including pathogenic cells, use diverse strategies for tissue invasion, such as the expression of surface receptors to bind plasminogen or plasmin. In this work, we show that T. evansi is able to bind plasminogen and plasmin on its surface. The analysis of this binding revealed a high affinity dissociation constant (Kd of 0.080±0.009µM) and 1×10(5) plasminogen binding sites per cell. Also a second population of receptors with a Kd of 0.255±0.070µM and 3.2×10(4) plasminogen binding sites per cell was determined. Several proteins with molecular masses between ∼18 and ∼70kDa are responsible for this binding. This parasite-plasminogen interaction may be important in the establishment of the infection in the vertebrate host, where the physiological concentration of available plasminogen is around 2µM.

Suppression of PKC-α attenuates TNF-α-evoked cerebral barrier breakdown via regulations of MMP-2 and plasminogen-plasmin system.

Ischaemic stroke, accompanied by neuroinflammation, impairs blood-brain barrier integrity through a complex mechanism involving both protein kinase C (PKC) and urokinase. Using an in vitro model of human blood-brain barrier (BBB) composed of brain microvascular endothelial cells (HBMEC) and astrocytes, this study assessed the putative roles of these elements in BBB damage evoked by enhanced availability of pro-inflammatory cytokine, TNF-α. Treatment of HBMEC with TNF-α significantly increased the mRNA and protein expressions of all plasminogen-plasmin system (PPS) components, namely tissue plasminogen activator, urokinase, urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 and also the activities of urokinase, total PKC and extracellular MMP-2. Inhibition of urokinase by amiloride abated the effects of TNF-α on BBB integrity and MMP-2 activity without affecting that of total PKC. Conversely, pharmacological inhibition of conventional PKC isoforms dramatically suppressed TNF-α-induced overactivation of urokinase. Knockdown of PKC-α gene via specific siRNA in HBMEC suppressed the stimulatory effects of TNF-α on protein expression of all PPS components, MMP-2 activity, DNA fragmentation rates and pro-apoptotic caspase-3/7 activities. Establishment of co-cultures with BMEC transfected with PKC-α siRNA attenuated the disruptive effects of TNF-α on BBB integrity and function. This was partly due to elevations observed in expression of a tight junction protein, claudin-5 and partly to prevention of stress fibre formation. In conclusion, specific inhibition of PKC-α in cerebral conditions associated with exaggerated release of pro-inflammatory cytokines, notably TNF-α may be of considerable therapeutic value and help maintain endothelial cell viability, appropriate cytoskeletal structure and basement membrane.

Bacterial pathogens activate plasminogen to breach tissue barriers and escape from innate immunity.

Both coagulation and fibrinolysis are tightly connected with the innate immune system. Infection and inflammation cause profound alterations in the otherwise well-controlled balance between coagulation and fibrinolysis. Many pathogenic bacteria directly exploit the host's hemostatic system to increase their virulence. Here, we review the capacity of bacteria to activate plasminogen. The resulting proteolytic activity allows them to breach tissue barriers and evade innate immune defense, thus promoting bacterial spreading. Yersinia pestis, streptococci of group A, C and G and Staphylococcus aureus produce a specific bacterial plasminogen activator. Moreover, surface plasminogen receptors play an established role in pneumococcal, borrelial and group B streptococcal infections. This review summarizes the mechanisms of bacterial activation of host plasminogen and the role of the fibrinolytic system in infections caused by these pathogens.

The plasminogen activation system in neuroinflammation.

The plasminogen activation (PA) system consists in a group of proteases and protease inhibitors regulating the activation of the zymogen plasminogen into its proteolytically active form, plasmin. Here, we give an update of the current knowledge about the role of the PA system on different aspects of neuroinflammation. These include modification in blood-brain barrier integrity, leukocyte diapedesis, removal of fibrin deposits in nervous tissues, microglial activation and neutrophil functions. Furthermore, we focus on the molecular mechanisms (some of them independent of plasmin generation and even of proteolysis) and target receptors responsible for these effects. The description of these mechanisms of action may help designing new therapeutic strategies targeting the expression, activity and molecular mediators of the PA system in neurological disorders involving neuroinflammatory processes. This article is part of a Special Issue entitled: Neuro Inflammation edited by Helga E. de Vries and Markus Schwaninger.

Dengue Virus Nonstructural Protein 1-Induced Antibodies Cross-React with Human Plasminogen and Enhance Its Activation.

Dengue virus (DENV) infection is the most common mosquito-borne viral disease, and it can cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks of DHF/DSS. However, the mechanism underlying hemorrhage in DHF/DSS remains elusive. In previous studies, plasminogen (Plg) cross-reactive Abs, which can recognize DENV nonstructural protein (NS) 1, have been found in dengue patients. However, it is unclear whether these Abs are indeed induced by DENV NS1. Thus, we immunized mice with recombinant NS1 from both bacteria and drosophila to determine whether NS1 can induce Plg cross-reactive Abs. The results from the NS1-immunized mouse sera indicated that NS1 immunization induced Abs that could cross-react with Plg. To study the effects of these NS1-induced Plg cross-reactive Abs on fibrinolysis, we isolated several Plg cross-reactive anti-NS1 mAbs from these mice and found that some of them could enhance Plg activation. In addition, epitope mapping with a phage-displayed random peptide library revealed that one of these mAbs (2A5) could recognize NS1 C-terminal residues 305-311, which share sequence homology with Plg residues 590-597. A synthetic peptide of NS1 residues 305-311 could inhibit the binding of both 2A5 and its Fab to Plg and its enhanced activation. Thus, our results suggest that DENV NS1 can induce Plg cross-reactive Abs through molecular mimicry, which can enhance Plg activation and may contribute to the pathogenesis of DHF/DSS.