Engineering the HK97 virus-like particle as a nanoplatform for biotechnology applications.

The research described here looks at the development of virus-like particles (VLPs) derived from bacteriophage HK97 as versatile scaffolds for bionanomaterials construction. Based on molecular models, the Prohead I HK97 VLP was engineered to allow attachment of small molecules to the interior by introducing a reactive cysteine into the genetic sequence of the HK97 GP5 protein that self assembles to form the VLP structure. In addition, methods for entrapping large protein macromolecules were evaluated and found to produce high encapsulation numbers of green fluorescent proteins (GFP) in the internal space of the HK97 VLP. A method for modular modification of the external surface was engineered by constructing a plasmid allowing the addition of peptide sequences to the C-terminus of the GP5 protein, which was validated by appending the sortase recognition peptide sequence, LPETG, to the C-terminus of GP5 and showing the attachment of a polyglycine-GFP to the HK97 VLP through sortase mediated ligation. To demonstrate the potential for advanced applications, an HK97 VLP covalently labeled on the interior surface with fluorescein and containing an externally displayed integrin binding peptide sequence (RGD) was evaluated and found to be preferentially localized at C2C12 cells relative to the HK97 VLP lacking the RGD peptide. Together, these results support the potential of the HK97 VLP as a versatile nanoparticle platform that can be modified internally and externally in a modular fashion for the purpose of programming the VLP for desired applications.

Targeting the PAI-1 Mechanism with a Small Peptide Increases the Efficacy of Alteplase in a Rabbit Model of Chronic Empyema.

The incidence of empyema is increasing and associated with a mortality rate of 20% in patients older than 65 years. Since 30% of patients with advanced empyema have contraindications to surgical treatment, novel, low-dose, pharmacological treatments are needed. A Streptococcus pneumoniae-induced rabbit model of chronic empyema recapitulates the progression, loculation, fibrotic repair, and pleural thickening of human disease. Treatment with single chain (sc) urokinase (scuPA) or tissue type (sctPA) plasminogen activators in doses 1.0-4.0 mg/kg were only partially effective in this model. Docking Site Peptide (DSP; 8.0 mg/kg), which decreased the dose of sctPA for successful fibrinolytic therapy in acute empyema model did not improve efficacy in combination with 2.0 mg/kg scuPA or sctPA. However, a two-fold increase in either sctPA or DSP (4.0 and 8.0 mg/kg or 2.0 and 16.0 mg/kg sctPA and DSP, respectively) resulted in 100% effective outcome. Thus, DSP-based Plasminogen Activator Inhibitor 1-Targeted Fibrinolytic Therapy (PAI-1-TFT) of chronic infectious pleural injury in rabbits increases the efficacy of alteplase rendering ineffective doses of sctPA effective. PAI-1-TFT represents a novel, well-tolerated treatment of empyema that is amenable to clinical introduction. The chronic empyema model recapitulates increased resistance of advanced human empyema to fibrinolytic therapy, thus allowing for studies of muti-injection treatments.

A Novel Rabbit Model of Retained Hemothorax with Pleural Organization.

Retained hemothorax (RH) is a commonly encountered and potentially severe complication of intrapleural bleeding that can organize with lung restriction. Early surgical intervention and intrapleural fibrinolytic therapy have been advocated. However, the lack of a reliable, cost-effective model amenable to interventional testing has hampered our understanding of the role of pharmacological interventions in RH management. Here, we report the development of a new RH model in rabbits. RH was induced by sequential administration of up to three doses of recalcified citrated homologous rabbit donor blood plus thrombin via a chest tube. RH at 4, 7, and 10 days post-induction (RH4, RH7, and RH10, respectively) was characterized by clot retention, intrapleural organization, and increased pleural rind, similar to that of clinical RH. Clinical imaging techniques such as ultrasonography and computed tomography (CT) revealed the dynamic formation and resorption of intrapleural clots over time and the resulting lung restriction. RH7 and RH10 were evaluated in young (3 mo) animals of both sexes. The RH7 recapitulated the most clinically relevant RH attributes; therefore, we used this model further to evaluate the effect of age on RH development. Sanguineous pleural fluids (PFs) in the model were generally small and variably detected among different models. The rabbit model PFs exhibited a proinflammatory response reminiscent of human hemothorax PFs. Overall, RH7 results in the consistent formation of durable intrapleural clots, pleural adhesions, pleural thickening, and lung restriction. Protracted chest tube placement over 7 d was achieved, enabling direct intrapleural access for sampling and treatment. The model, particularly RH7, is amenable to testing new intrapleural pharmacologic interventions, including iterations of currently used empirically dosed agents or new candidates designed to safely and more effectively clear RH.

Encapsulation of Pseudomonas aeruginosa elastase inside the P22 virus-like particle for controlling enzyme-substrate interactions.

Controlling interactions between enzymes and interaction partners, such as substrates, is important for applications in cellular biology and molecular biochemistry. A strategy for controlling enzyme access with substrate interaction partners is to exploit encapsulation of enzymes inside nanoparticles to limit the accessibility of the enzymes to large macromolecules, but allow free exchange of small-molecule substrates. The research here evaluates the encapsulation of Pseudomonas aeruginosa elastase inside the bacteriophage P22 virus-like particle (VLP) to examine the ability to allow free soluble substrates access to the enzyme while blocking large macromolecular substrate interactions. The results show that the active elastase protease can be encapsulated inside the P22 VLP, which blocks its ability to disrupt cell monolayers, but allows soluble substrates to be catalytically cleaved, supporting the viability of this approach for future investigations.

Precision targeting of the plasminogen activator inhibitor-1 mechanism increases efficacy of fibrinolytic therapy in empyema.

Plasminogen activator inhibitor-1 (PAI-1) is an endogenous irreversible inhibitor of tissue-type (tPA) and urokinase (uPA) plasminogen activators. PAI-1-targeted fibrinolytic therapy (PAI-1-TFT) is designed to decrease the therapeutic dose of tPA and uPA, attenuating the risk of bleeding and other complications. Docking site peptide (DSP) mimics the part of the PAI-1 reactive center loop that interacts with plasminogen activators, thereby affecting the PAI-1 mechanism. We used DSP for PAI-1-TFT in two rabbit models: chemically induced pleural injury and Streptococcus pneumoniae induced empyema. These models feature different levels of inflammation and PAI-1 expression. PAI-1-TFT with DSP (2.0 mg/kg) converted ineffective doses of single chain (sc) tPA (72.5 µg/kg) and scuPA (62.5 µg/kg) into effective ones in chemically induced pleural injury. DSP (2.0 mg/kg) was ineffective in S. pneumoniae empyema, where the level of PAI-1 is an order of magnitude higher. DSP dose escalation to 8.0 mg/kg resulted in effective PAI-1-TFT with 0.25 mg/kg sctPA (1/8th of the effective dose of sctPA alone) in empyema. There was no increase in the efficacy of scuPA. PAI-1-TFT with DSP increases the efficacy of fibrinolytic therapy up to 8-fold in chemically induced (sctPA and scuPA) and infectious (sctPA) pleural injury in rabbits. PAI-1 is a valid molecular target in our model of S. pneumoniae empyema in rabbits, which closely recapitulates key characteristics of empyema in humans. Low-dose PAI-1-TFT is a novel interventional strategy that offers the potential to improve fibrinolytic therapy for empyema in clinical practice.

Inhibition of Platelet Adhesion from Surface Modified Polyurethane Membranes.

Coronary thrombosis is one of the leading causes of mortality and morbidity in cardiovascular diseases, and patients who received vascular stent treatments are likely to suffer from restenosis due to tissue damage from stenting procedures (extrinsic pathway) and/or presence of unregulated factor XII (intrinsic pathway). Regardless of the pathway, coagulation factors and exposed collagen activate the G-protein-coupled receptors located at the plasma membrane of the resting platelets resulting in the change of their shapes with protrusions of filopodia and lamellipodia for surface adhesion. In this mini review, we discussed the mechanisms involved in platelet activation, adhesion, and aggregation. More importantly, we reviewed the use of polyurethane membranes with modified surface functional groups to down-regulate platelet adhesion and aggregation activities. Polyurethane membranes with hydrophilic and negatively charged surface properties showed a reduced αIIb-ß3 signaling from the activated platelets, resulting in the decrease of platelet adhesion and aggregation. The use of polyurethane membranes with modified surface properties as coatings on vascular stents provides an engineering approach to mitigate blood clotting associated with restenosis.

Targeting plasminogen activator inhibitor-1 in tetracycline-induced pleural injury in rabbits.

Elevated active plasminogen activator inhibitor-1 (PAI-1) has an adverse effect on the outcomes of intrapleural fibrinolytic therapy (IPFT) in tetracycline-induced pleural injury in rabbits. To enhance IPFT with prourokinase (scuPA), two mechanistically distinct approaches to targeting PAI-1 were tested: slowing its reaction with urokinase (uPA) and monoclonal antibody (mAb)-mediated PAI-1 inactivation. Removing positively charged residues at the "PAI-1 docking site" (179RHRGGS184→179AAAAAA184) of uPA results in a 60-fold decrease in the rate of inhibition by PAI-1. Mutant prourokinase (0.0625-0.5 mg/kg; n = 12) showed efficacy comparable to wild-type scuPA and did not change IPFT outcomes ( P > 0.05). Notably, the rate of PAI-1-independent intrapleural inactivation of mutant uPA was 2 times higher ( P < 0.05) than that of the wild-type enzyme. Trapping PAI-1 in a "molecular sandwich"-type complex with catalytically inactive two-chain urokinase with Ser195Ala substitution (S195A-tcuPA; 0.1 and 0.5 mg/kg) did not improve the efficacy of IPFT with scuPA (0.0625-0.5 mg/kg; n = 11). IPFT failed in the presence of MA-56A7C10 (0.5 mg/kg; n = 2), which forms a stable intrapleural molecular sandwich complex, allowing active PAI-1 to accumulate by blocking its transition to a latent form. In contrast, inactivation of PAI-1 by accelerating the active-to-latent transition mediated by mAb MA-33B8 (0.5 mg/kg; n = 2) improved the efficacy of IPFT with scuPA (0.25 mg/kg). Thus, under conditions of slow (4-8 h) fibrinolysis in tetracycline-induced pleural injury in rabbits, only the inactivation of PAI-1, but not a decrease in the rate of its reaction with uPA, enhances IPFT. Therefore the rate of fibrinolysis, which varies in different pathologic states, could affect the selection of PAI-1 inhibitors to enhance fibrinolytic therapy.

Precision-guided, Personalized Intrapleural Fibrinolytic Therapy for Empyema and Complicated Parapneumonic Pleural Effusions: The Case for the Fibrinolytic Potential.

Complicated pleural effusions and empyema with loculation and failed drainage are common clinical problems. In adults, intrapleural fibrinolytic therapy is commonly used with variable results and therapy remains empiric. Despite the intrapleural use of various plasminogen activators; fibrinolysins, for about sixty years, there is no clear consensus about which agent is most effective. Emerging evidence demonstrates that intrapleural administration of plasminogen activators is subject to rapid inhibition by plasminogen activator inhibitor-1 and that processing of fibrinolysins is importantly influenced by other factors including the levels and quality of pleural fluid DNA. Current therapy for loculation that accompanies pleural infections also includes surgery, which is invasive and for which patient selection can be problematic. Most of the clinical literature published to date has used flat dosing of intrapleural fibrinolytic therapy in all subjects but little is known about how that strategy influences the processing of the administered fibrinolysin or how this influences outcomes. We developed a new test of pleural fluids ex vivo, which is called the Fibrinolytic Potential or FP, in which a dose of a fibrinolysin is added to pleural fluids ex vivo after which the fibrinolytic activity is measured and normalized to baseline levels. Testing in preclinical and clinical empyema fluids reveals a wide range of responses, indicating that individual patients will likely respond differently to flat dosing of fibrinolysins. The test remains under development but is envisioned as a guide for dosing of these agents, representing a novel candidate approach to personalization of intrapleural fibrinolytic therapy.

Efficacy of neutral and negatively charged liposome-loaded gentamicin on planktonic bacteria and biofilm communities.

We investigated the efficacy of liposomal gentamicin formulations of different surface charges against Pseudomonas aeruginosa and Klebsiella oxytoca. The liposomal gentamicin formulations were prepared by the dehydration-rehydration method, and their sizes and zeta potential were measured. Gentamicin encapsulation efficiency inside the liposomal formulations was determined by microbiologic assay, and stability of the formulations in biologic fluid was evaluated for a period of 48 h. The minimum inhibitory concentration and the minimum bactericidal concentration were determined, and the in vitro time kill studies of the free form of gentamicin and liposomal gentamicin formulations were performed. The activities of liposomal gentamicin in preventing and reducing biofilm-forming P. aeruginosa and K. oxytoca were compared to those of free antibiotic. The sizes of the liposomal formulations ranged from 625 to 806.6 nm in diameter, with the zeta potential ranging from -0.22 to -31.7 mV. Gentamicin encapsulation efficiency inside the liposomal formulation ranged from 1.8% to 43.6%. The liposomes retained >60% of their gentamicin content during the 48 h time period. The minimum inhibitory concentration of neutral formulation was lower than that of free gentamicin (0.25 versus 1 mg/L for P. aeruginosa and 0.5 versus 1 mg/L for K. oxytoca). The negatively charged formulation exhibited the same bacteriostatic concentration as that of free gentamicin. The minimum bactericidal concentration of neutral liposomes on planktonic bacterial culture was twofold lower than that of free gentamicin, whereas the negatively charged formulations were comparable to free gentamicin. The killing time curve values for the neutral negatively charged formulation against planktonic P. aeruginosa and K. oxytoca were better than those of free gentamicin. Furthermore, liposomal formulations prevent the biofilm-formation ability of these strains better than free gentamicin. In summary, liposomal formulations could be an effective lipid nanoparticle to combat acute infections where planktonic bacteria are predominant.

Dose dependency of outcomes of intrapleural fibrinolytic therapy in new rabbit empyema models.

The incidence of empyema (EMP) is increasing worldwide; EMP generally occurs with pleural loculation and impaired drainage is often treated with intrapleural fibrinolytic therapy (IPFT) or surgery. A number of IPFT options are used clinically with empiric dosing and variable outcomes in adults. To evaluate mechanisms governing intrapleural fibrinolysis and disease outcomes, models of Pasteurella multocida and Streptococcus pneumoniae were generated in rabbits and the animals were treated with either human tissue (tPA) plasminogen activator or prourokinase (scuPA). Rabbit EMP was characterized by the development of pleural adhesions detectable by chest ultrasonography and fibrinous coating of the pleura. Similar to human EMP, rabbits with EMP accumulated sizable, 20- to 40-ml fibrinopurulent pleural effusions associated with extensive intrapleural organization, significantly increased pleural thickness, suppression of fibrinolytic and plasminogen-activating activities, and accumulation of high levels of plasminogen activator inhibitor 1, plasminogen, and extracellular DNA. IPFT with tPA (0.145 mg/kg) or scuPA (0.5 mg/kg) was ineffective in rabbit EMP (n = 9 and 3 for P. multocida and S. pneumoniae, respectively); 2 mg/kg tPA or scuPA IPFT (n = 5) effectively cleared S. pneumoniae-induced EMP collections in 24 h with no bleeding observed. Although intrapleural fibrinolytic activity for up to 40 min after IPFT was similar for effective and ineffective doses of fibrinolysin, it was lower for tPA than for scuPA treatments. These results demonstrate similarities between rabbit and human EMP, the importance of pleural fluid PAI-1 activity, and levels of plasminogen in the regulation of intrapleural fibrinolysis and illustrate the dose dependency of IPFT outcomes in EMP.

The time course of resolution of adhesions during fibrinolytic therapy in tetracycline-induced pleural injury in rabbits.

The time required for the effective clearance of pleural adhesions/organization after intrapleural fibrinolytic therapy (IPFT) is unknown. Chest ultrasonography and computed tomography (CT) were used to assess the efficacy of IPFT in a rabbit model of tetracycline-induced pleural injury, treated with single-chain (sc) urokinase plasminogen activators (scuPAs) or tissue PAs (sctPA). IPFT with sctPA (0.145 mg/kg; n = 10) and scuPA (0.5 mg/kg; n = 12) was monitored by serial ultrasonography alone (n = 12) or alongside CT scanning (n = 10). IPFT efficacy was assessed with gross lung injury scores (GLIS) and ultrasonography scores (USS). Pleural fluids withdrawn at 0-240 min and 24 h after IPFT were assayed for PA and fibrinolytic activities, α-macroglobulin/fibrinolysin complexes, and active PA inhibitor 1 (PAI-1). scuPA and sctPA generated comparable steady-state fibrinolytic activities by 20 min. PA activity in the scuPA group decreased slower than the sctPA group (kobs = 0.016 and 0.042 min(-1)). Significant amounts of bioactive uPA/α-macroglobulin (but not tPA; P < 0.05) complexes accumulated at 0-40 min after IPFT. Despite the differences in intrapleural processing, IPFT with either fibrinolysin was effective (GLIS ≤ 10) in animals imaged with ultrasonography only. USS correlated well with postmortem GLIS (r(2) = 0.85) and confirmed relatively slow intrapleural fibrinolysis after IPFT, which coincided with effective clearance of adhesions/organization at 4-8 h. CT scanning was associated with less effective (GLIS > 10) IPFT and higher levels of active PAI-1 at 24 h following therapy. We concluded that intrapleural fibrinolysis in tetracycline-induced pleural injury in rabbits is relatively slow (4-8 h). In CT-scanned animals, elevated PAI-1 activity (possibly radiation induced) reduced the efficacy of IPFT, buttressing the major impact of active PAI-1 on IPFT outcomes.

Targeting of plasminogen activator inhibitor 1 improves fibrinolytic therapy for tetracycline-induced pleural injury in rabbits.

Endogenous active plasminogen activator inhibitor 1 (PAI-1) was targeted in vivo with monoclonal antibodies (mAbs) that redirect its reaction with proteinases to the substrate branch. mAbs were used as an adjunct to prourokinase (single-chain [sc] urokinase [uPA]) intrapleural fibrinolytic therapy (IPFT) of tetracycline-induced pleural injury in rabbits. Outcomes of scuPA IPFT (0.25 or 0.0625 mg/kg) with 0.5 mg/kg of mouse IgG or mAbs (MA-33H1F7 and MA-8H9D4) were assessed at 24 hours. Pleural fluid (PF) was collected at 0, 10, 20, and 40 minutes and 24 hours after IPFT and analyzed for plasminogen activating (PA), uPA, fibrinolytic activities, levels of total plasmin/plasminogen, α-macroglobulin (αM), mAbs/IgG antigens, free active uPA, and αM/uPA complexes. Anti-PAI-1 mAbs, but not mouse IgG, delivered with an eightfold reduction in the minimal effective dose of scuPA (from 0.5 to 0.0625 mg/kg), improved the outcome of IPFT (P < 0.05). mAbs and IgG were detectable in PFs at 24 hours. Compared with identical doses of scuPA alone or with IgG, treatment with scuPA and anti-PAI-1 mAbs generated higher PF uPA amidolytic and PA activities, faster formation of αM/uPA complexes, and slower uPA inactivation. However, PAI-1 targeting did not significantly affect intrapleural fibrinolytic activity or levels of total plasmin/plasminogen and αM antigens. Targeting PAI-1 did not induce bleeding, and rendered otherwise ineffective doses of scuPA able to improve outcomes in tetracycline-induced pleural injury. PAI-1-neutralizing mAbs improved IPFT by increasing the durability of intrapleural PA activity. These results suggest a novel, well-tolerated IPFT strategy that is tractable for clinical development.

Mechanism of fibroblast inflammatory responses to Pseudomonas aeruginosa elastase.

Receptor tyrosine kinases, including the epidermal growth factor receptors (EGFR), are able to activate the mitogen-activated protein kinases (MAPK) via several adaptor proteins and protein kinases such as Raf. EGFR can be activated by a variety of extracellular stimuli including neutrophil elastase, but we are aware of no report as to whether Pseudomonas aeruginosa produced elastase (PE) could elicit such signalling through EGFR activation. We sought to test the inference that PE modulates inflammatory responses in human lung fibroblasts and that the process occurs by activation of the EGFR/MAPK pathways. We utilized IL-8 cytokine expression as a pathway-specific end point measure of the fibroblast inflammatory response to PE. Western blot analysis was performed to detect phosphorylation of EGFR and signal transduction intermediates. Northern blot, real-time PCR, and ELISA methods were utilized to determine cytokine gene expression levels. We found that PE induces phosphorylation of the EGFR and the extracellular signal-regulated proteins (ERK1/2) of the MAPK pathway, and nuclear translocation of NF-κB. Furthermore, enzymically active PE enhances IL-8 mRNA and protein secretion. Pretreatment of the cells with specific inhibitors of EGFR, MAPK kinase and NF-κB markedly attenuated the PE-induced signal proteins phosphorylation and IL-8 gene expression and protein secretion. Collectively, the data show that PE produced by Pseudomonas aeruginosa can modulate lung inflammation by exploiting the EGFR/ERK signalling cascades and enhancing IL-8 production in the lungs via NF-κB activation.

Active α-macroglobulin is a reservoir for urokinase after fibrinolytic therapy in rabbits with tetracycline-induced pleural injury and in human pleural fluids.

Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT), capable of clearing pleural adhesions in this model. Pleural fluid (PF) was withdrawn 0-80 min and 24 h after IPFT with scuPA (0-0.5 mg/kg), and activities of free urokinase (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. The peak of uPA activity (5-40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10-20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2(ß) = 40 ± 10 min) was dose independent and 6.7-fold slower than in blood. Up to 260 ± 70 nM of αM/uPA formed in vivo [second order association rate (kass) = 580 ± 60 M(-1)·s(-1)]. αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Intrapleural scuPA processing differs from that in the bloodstream and includes 1) dose-dependent control of scuPA activation by endogenous active PAI-1; 2) two-step inactivation of free uPA with simultaneous formation of αM/uPA; and 3) slow intrapleural degradation of αM/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications.

Liposomal antibiotics for the treatment of infectious diseases.

INTRODUCTION: Liposomal delivery systems have been utilized in developing effective therapeutics against cancer and targeting microorganisms in and out of host cells and within biofilm community. The most attractive feature of liposome-based drugs are enhancing therapeutic index of the new or existing drugs while minimizing their adverse effects. AREAS COVERED: This communication provides an overview on several aspects of liposomal antibiotics including the most widely used preparation techniques for encapsulating different agents and the most important characteristic parameters applied for examining shape, size and stability of the spherical vesicles. In addition, the routes of administration, liposome-cell interactions and host parameters affecting the biodistribution of liposomes are highlighted. EXPERT OPINION: Liposomes are safe and suitable for delivery of variety of molecules and drugs in biomedical research and medicine. They are known to improve the therapeutic index of encapsulated agents and reduce drug toxicity. Recent studies on liposomal formulation of chemotherapeutic and bioactive agents and their targeted delivery show liposomal antibiotics potential in the treatment of microbial infections.

Intrapleural adenoviral delivery of human plasminogen activator inhibitor-1 exacerbates tetracycline-induced pleural injury in rabbits.

Elevated concentrations of plasminogen activator inhibitor-1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, ß-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial cells were used to evaluate the efficiency and effects of transduction. Transduction was selective and limited to the pleural mesothelial monolayer. The intrapleural expression of both genes was transient, with their peak expression at 4 to 5 days. On Day 5, hPAI-1 (40-80 and 200-400 nM of active and total hPAI-1 in lavages, respectively) caused no overt pleural injury, effusions, or fibrosis. The adenovirus-mediated delivery of hPAI-1 with subsequent tetracycline-induced pleural injury resulted in a significant exacerbation of the pleural fibrosis observed on Day 5 (P = 0.029 and P = 0.021 versus vehicle and adenoviral control samples, respectively). Intrapleural fibrinolytic therapy (IPFT) with plasminogen activators was effective in both animals overexpressing hPAI-1 and control animals with tetracycline injury alone. An increase in intrapleural active PAI-1 (from 10-15 nM in control animals to 20-40 nM in hPAI-1-overexpressing animals) resulted in the increased formation of PAI-1/plasminogen activator complexes in vivo. The decrease in intrapleural plasminogen-activating activity observed at 10 to 40 minutes after IPFT correlates linearly with the initial concentration of active PAI-1. Therefore, active PAI-1 in PFs affects the outcome of IPFT, and may be both a biomarker of pleural injury and a molecular target for its treatment.

Inhibition of protein kinase C attenuates Pseudomonas aeruginosa elastase-induced epithelial barrier disruption.

Pseudomonas aeruginosa pulmonary infection compromises the human airway epithelium, and can be especially devastating to immunocompromised or debilitated individuals. We have reported earlier that P. aeruginosa elastase (PE) increases paracellular permeability in epithelial cell monolayers by mechanisms involving tight junction (TJ) disruption and cytoskeletal reorganization, leading to destruction of epithelial barrier function. The aim of this study was to investigate putative TJ targets and potential mechanisms by which PE induces barrier disruption. We found that PE decreased localization of TJ proteins, occludin and zonula occludens (ZO)-1, in membrane fractions, and induced reorganization of F-actin within 1 hour. Although inhibition of protein kinase (PK) C α/ß signaling modestly altered the extent of cytoskeletal disruption and ZO-1 translocation, we found PKC signaling to play a significant role in decreased occludin functionality during PE exposure. Furthermore, elevated PKC levels correlated with decreased levels of TJ proteins in membrane fractions, and increased paracellular permeability in a time-dependent manner. Therefore, we conclude that PKC signaling is involved during PE-induced epithelial barrier disruption via TJ translocation and cytoskeletal reorganization. Specifically, occludin, as well as associated ZO-1 and F-actin, may be early targets of PE pathogenesis occurring via a PKC-dependent pathway.

Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.

BACKGROUND: To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA). METHODOLOGY: Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated. RESULTS: The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold. CONCLUSIONS: Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections.

Bismuth-thiol incorporation enhances biological activities of liposomal tobramycin against bacterial biofilm and quorum sensing molecules production by Pseudomonas aeruginosa.

Recurrent pulmonary infection and inflammation are major risk factors for high morbidity and mortality in patients with cystic fibrosis (CF). As such, frequent antibiotic use and drug resistant bacterial strains are main concerns in individuals with CF. Bacterial virulence and resistance are influenced by unique CF airways fluid lining and Pseudomonas aeruginosa quorum sensing (QS) and biofilm formation. We have developed a novel liposome formulation consist of bismuth-thiol and tobramycin (LipoBiEDT-TOB) that is non-toxic and highly effective against planktonic bacteria. In this study, we examined the effect of LipoBiEDT-TOB on QS molecule N-acyl homoserine lactone (AHL) secretion by P. aeruginosa isolates in the presence of Agrobacterium tumefaciens reporter strain (A136). LipoBiEDT-TOB activity against biofilm forming P. aeruginosa was compared to free tobramycin using the Calgary Biofilm Device (CBD). Our data indicate that LipoBiEDT-TOB prevents AHL production at low tobramycin concentration (as low as 0.012 mg/l) and stops biofilm forming P. aeruginosa growth at 64 mg/l. The formulation is stable in different biological environments (biofilm, sputum, and bronchoalveolar lavage) and is able to penetrate CF sputum. Taken together, co-encapsulation of bismuth-thiol metal with tobramycin in liposome improves its antimicrobial activities in vitro.

Liposomal bismuth-ethanedithiol formulation enhances antimicrobial activity of tobramycin.

Pseudomonas aeruginosa and Burkholderia cenocepacia (formally, genomovar III genotype of Burkholderia cepacia complex) have emerged as serious opportunistic resistant pathogens in patients with cystic fibrosis (CF). We have developed a liposomal formulation containing bismuth-ethanedithiol (BiEDT) and tobramycin to overcome bacterial resistance. The stability of liposomal BiEDT-tobramycin (LipoBiEDT-TOB) was studied in phosphate buffered saline (PBS) and human pooled plasma at 4 and 37 degrees C. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) for free tobramycin and LipoBiEDT-TOB against clinical isolates of P. aeruginosa and B. cenocepacia were determined by the broth dilution method. The toxicity profile and the influence on bacterial adhesion of LipoBiEDT-TOB formulation were determined using a human lung carcinoma cell line (A549). LipoBiEDT-TOB exhibited lower MICs than the conventional antibiotic (0.25mg/L vs. 1024 mg/L) and eradicated this highly resistant bacterial strain of P. aeruginosa (PA-48913) at very low concentrations (4 mg/L vs. 4096 mg/L). LipoBiEDT-TOB was significantly less toxic when compared to the free BiEDT, as evaluated by the MTT and LDH assay. The LipoBiEDT-TOB formulation suppressed bacterial adhesion (B. cenocepacia M13642R) to A549 cells. These data suggest that the novel LipoBiEDT-TOB drug delivery system could be utilized as a new strategy to enhance the efficacy of existing antibiotics against resistant organisms that commonly affect individuals with chronic lung infections.