The contact system proteases play disparate roles in streptococcal sepsis.

Sepsis causes an activation of the human contact system, an inflammatory response mechanism against foreign surfaces, proteins and pathogens. The serine proteases of the contact system, factor XII and plasma kallikrein, are decreased in plasma of septic patients, which was previously associated with an unfavorable outcome. However, the precise mechanisms and roles of contact system factors in bacterial sepsis are poorly understood. We, therefore, studied the physiological relevance of factor XII and plasma kallikrein in a mouse model of experimental sepsis. We show that decreased plasma kallikrein concentration in septic mice is a result of reduced mRNA expression plasma prekallikrein gene, indicating that plasma kallikrein belong to negative acute phase proteins. Investigations regarding the pathophysiological function of contact system proteases during sepsis revealed different roles for factor XII and plasma kallikrein. In vitro, factor XII decelerated bacteria induced fibrinolysis, whereas plasma kallikrein supported it. Remarkably, depletion of plasma kallikrein (but not factor XII) by treatment with antisense-oligonucleotides, dampens bacterial dissemination and growth in multiple organs in the mouse sepsis model. These findings identify plasma kallikrein as a novel host pathogenicity factor in Streptococcus pyogenes sepsis.

Prothrombin, alone or in complex concentrates or plasma, reduces bleeding in a mouse model of blood exchange-induced coagulopathy.

Prothrombin complex concentrates (PCC) are fractionated plasma protein drugs that reverse warfarin anticoagulation. PCC may control more general bleeding. We sought to identify the dominant procoagulant factor in PCC in vivo. We tested PCC or coagulation factor (F) treatment in CD1 mice made coagulopathic by exchange of whole blood for washed red cells. Anesthetized mice were transfused with murine fresh-frozen plasma (mFFP), PCC, mixtures of human vitamin K-dependent proteins (VKDP) (prothrombin, FVII, FIX, or FX), or purified single human VKDP, immediately prior to tail transection (TT), liver laceration (LL), or intravascular laser injury (ILI). Plasma donor mice were treated with vehicle or control antisense oligonucleotide (ASO-CON) or ASO specific for prothrombin (FII) (ASO-FII) to yield mFFP or ASO-CON mFFP or ASO-FII mFFP. Blood losses were determined spectrophotometrically (TT) or gravimetrically (LL). Thrombus formation was quantified by intravital microscopy of laser-injured arterioles. PCC or four factor- (4F-) VKDP or prothrombin significantly reduced bleeding from TT or LL. Omission of prothrombin from 4F-VKDP significantly reduced its ability to limit bleeding. Mice transfused with ASO-FII mFFP demonstrated inferior haemostasis versus those transfused with ASO-FII following TT, LL, or ILI. Prothrombin is the dominant procoagulant component of PCC and could limit bleeding in trauma.

In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides.

BACKGROUND & AIMS: Current treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting. METHODS: A lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively. RESULTS: ASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro. CONCLUSION: Our results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.

Inhibition of vascular permeability by antisense-mediated inhibition of plasma kallikrein and coagulation factor 12.

Hereditary angioedema (HAE) is a rare disorder characterized by recurrent, acute, and painful episodes of swelling involving multiple tissues. Deficiency or malfunction of the serine protease inhibitor C1 esterase inhibitor (C1-INH) results in HAE types 1 and 2, respectively, whereas mutations in coagulation factor 12 (f12) have been associated with HAE type 3. C1-INH is the primary inhibitor of multiple plasma cascade pathways known to be altered in HAE patients, including the complement, fibrinolytic, coagulation, and kinin-kallikrein pathways. We have selectively inhibited several components of both the kinin-kallikrein system and the coagulation cascades with potent and selective antisense oligonucleotides (ASOs) to investigate their relative contributions to vascular permeability. We have also developed ASO inhibitors of C1-INH and characterized their effects on vascular permeability in mice as an inducible model of HAE. Our studies demonstrate that ASO-mediated reduction in C1-INH plasma levels results in increased vascular permeability and that inhibition of proteases of the kinin-kallikrein system, either f12 or prekallikrein (PKK) reverse the effects of C1-INH depletion with similar effects on both basal and angiotensin converting enzyme (ACE) inhibitor-induced permeability. In contrast, inhibition of coagulation factors 11 (f11) or 7 (f7) had no effect. These results suggest that the vascular defects observed in C1-INH deficiency are dependent on the kinin-kallikrein system proteases f12 and PKK, and not mediated through the coagulation pathways. In addition, our results highlight a novel therapeutic modality that can potentially be employed prophylactically to prevent attacks in HAE patients.

Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates.

OBJECTIVE: During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. APPROACH AND RESULTS: We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. CONCLUSIONS: These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.

Inhibition of the intrinsic coagulation pathway factor XI by antisense oligonucleotides: a novel antithrombotic strategy with lowered bleeding risk.

Existing anticoagulants effectively inhibit the activity of coagulation factors of the extrinsic and common pathway but have substantial limitations and can cause severe bleeding complications. Here we describe a novel therapeutic approach to thrombosis treatment. We have developed and characterized the efficacy and safety of selective second-generation antisense oligonucleotides (ASOs) targeting coagulation factor XI (FXI), a member of the intrinsic coagulation pathway. Systemic treatment of mice with FXI ASO led to a potent, specific, and dose-dependent reduction of FXI mRNA levels in the liver with corresponding reductions in plasma levels of FXI protein and activity. FXIASO treatment produced potent, dose-dependent antithrombotic activity in various venous and arterial thrombosis models, comparable with warfarin or enoxaparin. However, unlike warfarin or enoxaparin, FXI inhibition did not cause bleeding. Coadministration of FXI ASO with enoxaparin or the antiplatelet drug clopidogrel produced improved antithrombotic activity without increased bleeding. Finally, plasma-derived FXI concentrate was shown to effectively and rapidly reverse the anticoagulant effect of FXI antisense therapy. These results support the concept that inhibition of FXI through antisense therapy might serve as a new and effective strategy for the treatment and prevention of venous thromboembolism with improved specificity and safety.

Inhaled CD86 antisense oligonucleotide suppresses pulmonary inflammation and airway hyper-responsiveness in allergic mice.

The B7-family molecule CD86, expressed on the surface of pulmonary and thoracic lymph node antigen-presenting cells, delivers essential costimulatory signals for T-cell activation in response to inhaled allergens. CD86-CD28 signaling is involved in priming allergen-specific T cells, but it is unclear whether these interactions play a role in coordinating memory T-helper 2 cell responses. In the ovalbumin (OVA)-induced mouse model of asthma, administration of CD86-specific antibody before systemic sensitization suppresses inhaled OVA-induced pulmonary inflammation and airway hyper-responsiveness (AHR). In previously OVA-sensitized mice, systemic and intranasal coadministration of CD86 antibody is required to produce these effects. To directly assess the importance of pulmonary CD86 expression in secondary immune responses to inhaled allergens, mice were sensitized and locally challenged with nebulized OVA before treatment with an inhaled aerosolized CD86 antisense oligonucleotide (ASO). CD86 ASO treatment suppressed OVA-induced up-regulation of CD86 protein expression on pulmonary dendritic cells and macrophages as well as on recruited eosinophils. Suppression of CD86 protein expression correlated with decreased methacholine-induced AHR, airway inflammation, and mucus production following rechallenge with inhaled OVA. CD86 ASO treatment reduced BAL eotaxin levels, but it did not reduce CD86 protein on cells in the draining lymph nodes of the lung, and it had no effect on serum IgE levels, suggesting a local and not a systemic effect. These results demonstrate that CD86 expression on pulmonary antigen-presenting cells plays a vital role in regulating pulmonary secondary immune responses and suggest that treatment with an inhaled CD86 ASO may have utility in asthma and other chronic inflammatory lung conditions.

Anti-inflammatory activity of inhaled IL-4 receptor-alpha antisense oligonucleotide in mice.

The Th2 cytokines IL-4 and IL-13 mediate allergic pulmonary inflammation and airways hyperreactivity (AHR) in asthma models through signaling dependent upon the IL-4 receptor-alpha chain (IL-4Ralpha). IL-13 has been further implicated in the overproduction of mucus by the airway epithelium and in lung remodeling that commonly accompanies chronic inflammation. IL-4Ralpha-deficient mice are resistant to allergen-induced asthma, highlighting the therapeutic promise of selective molecular inhibitors of IL-4Ralpha. We designed a chemically modified IL-4Ralpha antisense oligonucleotide (IL-4Ralpha ASO) that specifically inhibits IL-4Ralpha protein expression in lung eosinophils, macrophages, dendritic cells, and airway epithelium after inhalation in allergen-challenged mice. Inhalation of IL-4Ralpha ASO attenuated allergen-induced AHR, suppressed airway eosinophilia and neutrophilia, and inhibited production of airway Th2 cytokines and chemokines in previously allergen-primed and -challenged mice. Histologic analysis of lungs from these animals demonstrated reduced goblet cell metaplasia and mucus staining that correlated with inhibition of Muc5AC gene expression in lung tissue. Therapeutic administration of inhaled IL-4Ralpha ASO in chronically allergen-challenged mice produced a spectrum of anti-inflammatory activity similar to that of systemically administered Dexamethasone with the added benefit of reduced airway neutrophilia. These data support the potential utility of a dual IL-4 and IL-13 oligonucleotide inhibitor in allergy/asthma, and suggest that local inhibition of IL-4Ralpha in the lung is sufficient to suppress allergen-induced pulmonary inflammation and AHR.

Inhaled p38alpha mitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice.

The p38 mitogen-activated protein kinase (MAPK) plays a critical role in the activation of inflammatory cells. Therefore, we investigated the antiinflammatory effects of a respirable p38alpha MAPK antisense oligonucleotide (p38alpha-ASO) in a mouse asthma model. A potent and selective p38alpha-ASO was characterized in vitro. Inhalation of aerosolized p38alpha-ASO using an aerosol chamber dosing system produced measurable lung deposition of ASO and significant reduction of ovalbumin (OVA-)-induced increases in total cells, eosinophils, and interleukin 4 (IL-4), IL-5, and IL-13 levels in bronchoalveolar lavage fluid, and dose-dependent inhibition of airway hyperresponsiveness in allergen-challenged mice. Furthermore, inhaled p38alpha-ASO markedly inhibited OVA-induced lung tissue eosinophilia and airway mucus hypersecretion. Quantitative polymerase chain reaction analysis of bronchoalveolar lavage fluid cells and peribronchial lymph node cells showed that p38alpha-ASO significantly reduced p38alpha MAPK mRNA expression. Nose-only aerosol exposure of mice verified the p38alpha-ASO-induced inhibition of OVA-induced pulmonary eosinophilia, mucus hypersecretion, and airway hyperresponsiveness. None of the effects of the p38alpha-ASO were produced by a six-base mismatched control oligonucleotide. These findings demonstrate antisense pharmacodynamic activity in the airways after aerosol delivery and suggest that a p38alpha MAPK ASO approach may have therapeutic potential for asthma and other inflammatory lung diseases.

Chimera analysis supports a predominant role of PDGFRbeta in promoting smooth-muscle cell chemotaxis after arterial injury.

The carotid artery shows a common response to many forms of injury, including a rapid activation of smooth muscle cell (SMC) proliferation in the media and migration of SMCs into the intima to form a neointima. Platelet-derived growth factor (PDGF) is believed to play a role in this response to injury, but it has proven difficult to distinguish whether it is stimulating cell migration or cell proliferation, and whether the action is direct or indirect. To determine this, we created chimeric mice composed of both wild-type (WT) and marked PDGF receptor beta (PDGFRbeta)-deficient cells, and determined the consequences of PDGFRbeta expression for SMC participation in response to ligation of the left common carotid artery. The proportion of PDGFRbeta-/- SMCs increased 4.5-fold in the media and decreased 1.8-fold during formation of the neointima, consistent with migration of WT SMCs out of the media and into the intima, leaving the PDGFRbeta-/- cells behind. The fibrotic reaction in the adventitia, which does not involve cell migration, did not result in any change in relative abundance of WT and PDGFRbeta-deficient fibroblasts. We conclude that the most significant direct role of PDGFRbeta is to mediate responses that involve cell migration rather than proliferation.

A causative relationship exists between eosinophils and the development of allergic pulmonary pathologies in the mouse.

Asthma and mouse models of allergic respiratory inflammation are invariably associated with a pulmonary eosinophilia; however, this association has remained correlative. In this report, a causative relationship between eosinophils and allergen-provoked pathologies was established using eosinophil adoptive transfer. Eosinophils were transferred directly into the lungs of either naive or OVA-treated IL-5(-/-) mice. This strategy resulted in a pulmonary eosinophilia equivalent to that observed in OVA-treated wild-type animals. A concomitant consequence of this eosinophil transfer was an increase in Th2 bronchoalveolar lavage cytokine levels and the restoration of intracellular epithelial mucus in OVA-treated IL-5(-/-) mice equivalent to OVA-treated wild-type levels. Moreover, the transfer also resulted in the development of airway hyperresponsiveness. These pulmonary changes did not occur when eosinophils were transferred into naive IL-5(-/-) mice, eliminating nonspecific consequences of the eosinophil transfer as a possible explanation. Significantly, administration of OVA-treated IL-5(-/-) mice with GK1.5 (anti-CD4) Abs abolished the increases in mucus accumulation and airway hyperresponsiveness following adoptive transfer of eosinophils. Thus, CD4(+) T cell-mediated inflammatory signals as well as signals derived from eosinophils are each necessary, yet alone insufficient, for the development of allergic pulmonary pathology. These data support an expanded view of T cell and eosinophil activities and suggest that eosinophil effector functions impinge directly on lung function.

Ablation of eosinophils leads to a reduction of allergen-induced pulmonary pathology.

A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.

T(H)2-mediated pulmonary inflammation leads to the differential expression of ribonuclease genes by alveolar macrophages.

The eosinophil-associated ribonuclease (Ear) family in the mouse consists of thirteen genes, eleven of which encode RNases that have physical/functional properties similar to the human Ears, eosinophil-derived neurotoxin and eosinophil cationic protein. The expression of Ear genes in the mouse is confined to sites of known eosinophilopoiesis, with the exception of the lung. Two Ear genes, Ear1 and Ear2, are predominantly expressed in the lungs of naive mice. Total Ear gene expression and RNase activity in bronchoalveolar lavage fluid increases significantly upon the induction of pulmonary inflammation using an ovalbumin (OVA) model of allergic sensitization and challenge. Interestingly, pulmonary Ear11 transcripts, which are absent in naive mice, accumulate as a consequence of OVA-mediated T(H)2 inflammation in the lung. The induction of Ear11 expression is dependent on the presence of T cells, in particular, CD4(+) T lymphocytes. This effect is likely the result of the elaboration of T(H)2 cytokine levels, because pulmonary instillation of interleukin-4 or interleukin-13 induces the accumulation of Ear11 transcripts in naive animals. This study demonstrates that despite an allergen-mediated pulmonary eosinophilia and earlier studies showing that Ears are constituents of eosinophil secondary granules, alveolar macrophages are a significant source of these RNases in lungs of OVA-treated mice.

Early phase bronchoconstriction in the mouse requires allergen-specific IgG.

Allergen provocation of allergic asthma patients is often characterized by an initial period of bronchoconstriction, or early phase reaction (EPR), that leads to maximal airway narrowing within 15-30 min, followed by a recovery period returning airway function to baseline within 1-2 h. In this study, we used a defined OVA provocation model and mice deficient for specific leukocyte populations to investigate the cellular/molecular origins of the EPR. OVA-sensitized/challenged wild-type (C57BL/6J) mice displayed an EPR following OVA provocation. However, this response was absent in gene knockout animals deficient of either B or T cells. Moreover, transfer of OVA-specific IgG, but not IgE, before the OVA provocation, was capable of inducing the EPR in both strains of lymphocyte-deficient mice. Interestingly, an EPR was also observed in sensitized/challenged mast cell-deficient mice following an OVA provocation. These data show that the EPR in the mouse is an immunologically based pathophysiological response that requires allergen-specific IgG but occurs independent of mast cell activities. Thus, in the mouse the initial period of bronchoconstriction following allergen exposure may involve neither mast cells nor IgE-mediated events.

Ectopic expression of IL-5 identifies an additional CD4(+) T cell mechanism of airway eosinophil recruitment.

CD4(+) T cells have a critical role in the development of allergic pulmonary inflammation, including the recruitment of eosinophils to the airway lumen and interstitium. The expression of interleukin (IL)-5 by CD4(+) cells has, in particular, often been lionized as the central link between allergic inflammation and the concomitant expansion or recruitment of eosinophils. The mechanism(s) by which CD4(+) T cells mediates eosinophil recruitment was assessed with gene knockout mice deficient for T cells or T cell subtypes and a unique IL-5 transgenic mouse (line NJ.1726) that constitutively overexpresses this cytokine in the lung epithelium. Pulmonary IL-5 expression is significantly attenuated in T cell- and CD4(+) but not CD8(+) cell-deficient animals, suggesting an obvious explanation for the lack of eosinophils in the lungs of T cell-deficient and CD4(-/-) mice. However, although the constitutive expression of IL-5 in the lung epithelium of NJ.1726 mice elicited an eosinophilia in the airway lumen of both naive and ovalbumin-treated mice, in the absence of CD4(+) cells, allergen-mediated eosinophil recruitment to the bronchoalveolar lavage fluid was abolished. Moreover, intranasal instillation of the potent eosinophil-specific chemokine eotaxin-2 was incapable of eliciting eosinophil recruitment in naive and ovalbumin-treated NJ.1726 CD4(-/-) mice, suggesting that eosinophil trafficking during allergic inflammatory responses is a consequence of a CD4(+) cell-mediated event(s) in addition to IL-5 expression and the establishment of a pulmonary chemokine gradient.