Matriptase proteolytically activates influenza virus and promotes multicycle replication in the human airway epithelium.

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Measurement of fractional order model parameters of respiratory mechanical impedance in total liquid ventilation.

This study presents a methodology for applying the forced-oscillation technique in total liquid ventilation. It mainly consists of applying sinusoidal volumetric excitation to the respiratory system, and determining the transfer function between the delivered flow rate and resulting airway pressure. The investigated frequency range was f ∈ [0.05, 4] Hz at a constant flow amplitude of 7.5 mL/s. The five parameters of a fractional order lung model, the existing "5-parameter constant-phase model," were identified based on measured impedance spectra. The identification method was validated in silico on computer-generated datasets and the overall process was validated in vitro on a simplified single-compartment mechanical lung model. In vivo data on ten newborn lambs suggested the appropriateness of a fractional-order compliance term to the mechanical impedance to describe the low-frequency behavior of the lung, but did not demonstrate the relevance of a fractional-order inertance term. Typical respiratory system frequency response is presented together with statistical data of the measured in vivo impedance model parameters. This information will be useful for both the design of a robust pressure controller for total liquid ventilators and the monitoring of the patient's respiratory parameters during total liquid ventilation treatment.

Total liquid ventilation efficacy in an ovine model of severe meconium aspiration syndrome.

OBJECTIVE: To test the hypothesis that total liquid ventilation enables a more effective and better tolerated lavage than a bronchoalveolar lavage performed with diluted surfactant in a newborn ovine model of severe acute meconium aspiration syndrome. DESIGN: Prospective, randomized, interventional study. SETTING: Animal research laboratory at the Faculté de médecine et des sciences de la santé de l'université de Sherbrooke, Sherbrooke, Canada. SUBJECTS: Twenty-three newborn lambs, <4 days, 2.5-4.0 kg in weight. INTERVENTIONS: Animals were intubated, anesthetized, and paralyzed. Catheters were placed in the femoral artery and jugular vein. Severe meconium aspiration syndrome was obtained by instillation of a 25% dilution of human meconium in saline (1 mL/kg × 2). Lambs were then randomized in 12 total liquid ventilation-bronchoalveolar lavage (minute ventilation of 160 mL/kg/min with perfluorodecalin) vs. 11 bronchoalveolar lavage performed with diluted surfactant (conventional ventilation + 30 mL/kg in two aliquots bronchoalveolar lavage with 5 mg/mL BLES surfactant). Surviving lambs were ventilated for a total of 4 hrs and euthanized. MEASUREMENTS AND MAIN RESULTS: Arterial blood gases, systemic and pulmonary hemodynamic parameters using the thermodilution method, percentage of recovered meconium, and lung histologic scores. Total liquid ventilation bronchoalveolar lavage enabled a significantly higher PaO2 throughout the experiment. PaCO2, pH, and hemodynamic parameters were comparable for both groups except for an increase in mean pulmonary arterial pressure during total liquid ventilation. Total liquid ventilation bronchoalveolar lavage allowed for 43 ± 14% of the instilled meconium to be removed vs. 28 ± 10% for bronchoalveolar lavage performed with diluted surfactant (p = .022). Lung histologic analysis showed no difference between total scores. CONCLUSIONS: Total liquid ventilation bronchoalveolar lavage is well tolerated and more effective in terms of meconium washout and gas exchange than bronchoalveolar lavage performed with diluted surfactant in this experimental model of severe meconium aspiration syndrome. These positive results open the way to further experiments in our ovine model, ultimately aiming at a clinical trial with total liquid ventilation bronchoalveolar lavage to treat severe meconium aspiration syndrome.

Neonatal total liquid ventilation: is low-frequency forced oscillation technique suitable for respiratory mechanics assessment?

This study aimed to implement low-frequency forced oscillation technique (LFFOT) in neonatal total liquid ventilation (TLV) and to provide the first insight into respiratory impedance under this new modality of ventilation. Thirteen newborn lambs, weighing 2.5 + or - 0.4 kg (mean + or - SD), were premedicated, intubated, anesthetized, and then placed under TLV using a specially design liquid ventilator and a perfluorocarbon. The respiratory mechanics measurements protocol was started immediately after TLV initiation. Three blocks of measurements were first performed: one during initial respiratory system adaptation to TLV, followed by two other series during steady-state conditions. Lambs were then divided into two groups before undergoing another three blocks of measurements: the first group received a 10-min intravenous infusion of salbutamol (1.5 microg x kg(-1) x min(-1)) after continuous infusion of methacholine (9 microg x kg(-1) x min(-1)), while the second group of lambs was chest strapped. Respiratory impedance was measured using serial single-frequency tests at frequencies ranging between 0.05 and 2 Hz and then fitted with a constant-phase model. Harmonic test signals of 0.2 Hz were also launched every 10 min throughout the measurement protocol. Airway resistance and inertance were starkly increased in TLV compared with gas ventilation, with a resonant frequency < or = 1.2 Hz. Resistance of 0.2 Hz and reactance were sensitive to bronchoconstriction and dilation, as well as during compliance reduction. We report successful implementation of LFFOT to neonatal TLV and present the first insight into respiratory impedance under this new modality of ventilation. We show that LFFOT is an effective tool to track respiratory mechanics under TLV.

A regulator for pressure-controlled total-liquid ventilation.

Total-liquid ventilation (TLV) is an innovative experimental method of mechanical-assisted ventilation in which lungs are totally filled and then ventilated with a tidal volume of perfluorochemical liquid by using a dedicated liquid ventilator. Such a novel medical device must resemble other conventional ventilators: it must be able to conduct controlled-pressure ventilation. The objective was to design a robust controller to perform pressure-regulated expiratory flow and to implement it on our latest liquid-ventilator prototype (Inolivent-4). Numerical simulations, in vitro experiments, and in vivo experiments in five healthy term newborn lambs have demonstrated that it was efficient to generate expiratory flows while avoiding collapses. Moreover, the in vivo results have demonstrated that our liquid ventilator can maintain adequate gas exchange, normal acid-base equilibrium, and achieve greater minute ventilation, better oxygenation and CO2 extraction, while nearing flow limits. Hence, it is our suggestion to perform pressure-controlled ventilation during expiration with minute ventilation equal or superior to 140 mL x min(-1) x kg(-1) in order to ensure PaCO2 below 55 mmHg. From a clinician's point of view, pressure-controlled ventilation greatly simplifies the use of the liquid ventilator, which will certainly facilitate its introduction in intensive care units for clinical applications.