In vitro characterization of a purified NS2/3 protease variant of hepatitis C virus.

The cleavage of the hepatitis C virus polyprotein between the nonstructural proteins NS2 and NS3 is mediated by the NS2/3 protease, whereas the NS3 protease is responsible for the cleavage of the downstream proteins. Purification and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature. NS2/3 protease activity could only be detected in cells or in in vitro translation assays with the addition of microsomal membranes or detergent. To facilitate purification of this poorly characterized protease, we truncated the N-terminal hydrophobic domain, resulting in an active enzyme with improved biophysical properties. We define a minimal catalytic region of NS2/3 protease retaining autocleavage activity that spans residues 904-1206 and includes the C-terminal half of NS2 and the N-terminal NS3 protease domain. The NS2/3 (904-1206) variant was purified from Escherichia coli inclusion bodies and refolded by gel filtration chromatography. The purified inactive form of NS2/3 (904-1206) was activated by the addition of glycerol and detergent to induce autocleavage at the predicted site between Leu(1026) and Ala(1027). NS2/3 (904-1206) activity was dependent on zinc ions and could be inhibited by NS4A peptides, peptides that span the cleavage site, or an N-terminal peptidic cleavage product. This NS2/3 variant will facilitate the development of an assay suitable for identifying inhibitors of HCV replication.

Monocyte chemoattractant protein-1 and its receptor CCR-2 in piglet lungs exposed to inhaled nitric oxide and hyperoxia.

Monocyte chemoattractant protein-1 (MCP-1), acting through its C-C chemokine receptor 2 (CCR-2), has important roles in inflammation, angiogenesis, and wound repair. The individual and combined effects of inhaled nitric oxide (NO) and hyperoxia on lung MCP-1 and CCR-2 in relation to lung leukocyte dynamics are unknown. Because MCP-1 gene is up-regulated by oxidants, we hypothesized that inhaled NO with hyperoxia will increase MCP-1 production and CCR-2 expression more than either gas alone. We randomly assigned young piglets to breathe room air (RA), RA+50 ppm NO (RA+NO), O(2), or O(2)+NO for 1 or 5 d before sacrifice. Lungs were lavaged and tissues preserved for hybridization studies, Western blotting, histology, and immunohistochemistry. The results show that lung MCP-1 production and alveolar macrophage count were significantly elevated in the 5-d O(2) and O(2)+NO groups relative to the RA group (p < or = 0.05). In contrast, lung CCR-2 abundance was diminished in the O(2) group (p

Neonatal complications of preterm premature rupture of membranes. Pathophysiology and management.

Midtrimester PROM is an infrequent, yet potentially disastrous complication of pregnancy. The most likely neonatal complication is preterm delivery with associated morbidity and mortality risks. Unique neonatal complications following PPROM include skeletal deformations and pulmonary hypoplasia related to prolonged oligohydramnios exposure. A systematic approach to an infant with respiratory insufficiency following PPROM delivery is possible with an understanding of the pathophysiology of pulmonary hypoplasia. Neonatal management should include immediate resuscitation including surfactant replacement, with careful attention to techniques of mechanical ventilation to avoid early volutrauma. Adjunctive therapies directed at pulmonary hypertension may now permit survival of some infants with less severe forms of pulmonary hypoplasia.

The precarious antioxidant defenses of the preterm infant.

Oxygen radicals are considered to be major causative factors in many illnesses of preterm infants. This article reviews the antioxidant defenses in immature animals and preterm infants, and attempts to quantitate their vulnerabilities to oxidants. Sources of oxidants, including hyperoxia, iron, parenteral nutrition, nitric oxide, and prooxidants, and their impact on immature antioxidant defenses are discussed. Genetic manipulations of antioxidant enzymes such as knockout and transgenic mice models are reviewed. The various clinical and investigational antioxidant therapies in animals and humans and difficulties in the design of antioxidant therapy studies are explored.

High-dose inhaled nitric oxide and hyperoxia increases lung collagen accumulation in piglets.

Nitric oxide (NO), a pro-oxidant gas, is used with hyperoxia (O(2)) to treat neonatal pulmonary hypertension and recently bronchopulmonary dysplasia, but great concerns remain regarding NO's potential toxicity. Based on reports that exposure to oxidant gases results in pulmonary extracellular matrix injury associated with elevated lavage fluid levels of extracellular matrix components, we hypothesized that inhaled NO with or without hyperoxia will have the same effect. We measured alveolar septal width, lung collagen content, lavage fluid hydroxyproline, hyaluronan and laminin levels in neonatal piglets after 5 days' exposure to room air (RA), RA + 50 ppm NO (RA + NO), O(2) (FiO(2) > 0.96) or O(2) + NO. Matrix metalloproteinase (MMP) activity and MMP-2 mRNA were also measured. In recovery experiments, we measured lung collagen content in piglets exposed to RA + NO or O(2) + NO and then allowed to recover for 3 days. The results show that lung collagen increased 4-fold in the RA + NO piglets, the O(2) and O(2) + NO groups had only a 2-fold elevation relative to RA controls. Unlike the RA + NO piglets, the O(2) and O(2) + NO groups had more than 20-fold elevation in lung lavage fluid hydroxyproline compared to the RA group. O(2) and O(2) + NO also had increased lung MMP activity, extravascular water, and lavage fluid proteins. MMP-2 mRNA levels were unchanged. After 3 days' recovery in room air, the RA + NO groups' lung collagen had declined from 4-fold to 2-fold above the RA group values. The O(2) + NO group did not decline. Alveolar septal width increased significantly only in the O(2) and O(2) + NO groups. We conclude that 5 days' exposure to NO does not result in pulmonary matrix degradation but instead significantly increases lung collagen content. This effect appears potentially reversible. In contrast, hyperoxia exposure with or without NO results in pulmonary matrix degradation and increased lung collagen content. The observation that NO increased lung collagen content represents a new finding and suggests NO could potentially induce pulmonary fibrosis.

Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease: towards smaller inhibitors.

Structure-activity studies on a hexapeptide N-terminal cleavage product of a dodecamer substrate led to the identification of very potent and highly specific inhibitors of the HCV NS3 protease/NS4A cofactor peptide complex. The largest increase in potency was accomplished by the introduction of a (4R)-naphthalen-1-yl-4-methoxy substituent to the P2 proline. N-Terminal truncation resulted in tetrapeptides containing a C-terminal carboxylic acid, which exhibited low micromolar activity against the HCV serine protease.

2',6'-Dimethylphenoxyacetyl: a new achiral high affinity P(3)-P(2) ligand for peptidomimetic-based HIV protease inhibitors.

Starting from palinavir (1), our lead HIV protease inhibitor, we have discovered a new series of truncated analogues in which the P(3)-P(2) quinaldic-valine portion of 1 was replaced by 2', 6'-dimethylphenoxyacetyl. With EC(50)'s in the 1-2 nM range, some of these compounds are among the most potent inhibitors of HIV replication in vitro, reported to date. One of the most promising members in this series (compound 27, BILA 2185 BS) exhibited a favorable overall pharmacokinetic profile, with 61% apparent oral bioavailability in rat. X-ray crystal structures and molecular modeling were used to rationalize the high potency resulting from incorporation of this structurally simple, achiral ligand into the P(3)-P(2) position of hydroxyethylamine-based HIV protease inhibitors.

Independent and combined effects of prolonged inhaled nitric oxide and oxygen on lung inflammation in newborn piglets.

Clinical use of nitric oxide (NO) is usually in conjunction with high oxygen concentrations, the effects of which may include lung neutrophil accumulation, apoptosis and upregulation of antioxidant enzyme activity. To define the effects of NO on neutrophils from young piglets and its relationship to lung neutrophil dynamics during hyperoxia we exposed thirty piglets to room air (RA), RA+NO (50 ppm NO), O2 (FiO2> or =0.96) or O2+NO for 5 days. Ten additional animals breathed RA+NO or O2+NO, then recovered in RA for 3 days before sacrifice. Neutrophil CD18 and intracellular oxidant production were measured by flow cytometry. Lung apoptosis were assessed by TUNEL assay. Lung myeloperoxidase, SOD and catalase were measured biochemically. When compared to RA group, there was significant reduction in neutrophil CD18 and intracellular oxidant production in the RA+NO group, but lung MPO was unchanged. The O2 and O2+NO groups did not differ in CD18 expression or in intracellular oxidant production, but had significant increase in lung myeloperoxidase compared to the RA group. Apoptosis increased significantly only in the O2+NO group. The O2 group showed significantly increased lung SOD and catalase activity compared to the RA group, whereas the RA+NO and O2+NO groups did not. We conclude that inhaled NO at 50 ppm decreases neutrophil CD18 expression as well as intracellular oxidant production. However, this effect does not impact lung neutrophil accumulation during concurrent hyperoxia. The combination of NO and O2 exposure produces an increase in lung apoptosis. Finally, NO may prevent upregulation of SOD and catalase activity during hyperoxia, potentially increasing injury.

Lung elastic tissue maturation and perturbations during the evolution of chronic lung disease.

BACKGROUND: Infants <30 weeks' gestation have difficulty maintaining adequate functional residual capacity after the first week of life without positive end-expiratory pressure. We hypothesized that this is caused, in part, by increased lung elastic recoil. Our aims were to quantitate parenchymal elastic tissue during normal fetal development and in infants born at 23 to 30 weeks' gestation with prolonged survival at risk for chronic lung disease (CLD). METHODS: The controls were 22 to 42 weeks' gestation (n = 71), received ventilator care, and died within 48 hours of birth, plus 7 term infants who died at 43 to 50 weeks' postconceptional age from nonpulmonary causes. Infants who were 23 to 30 weeks' gestation, at risk for CLD, and who lived 5 to 59 days (n = 44), were separated into groups based on respiratory score (SCORE; The integrated area under the curve of the average daily fraction of inspired oxygen x mean airway pressure (cm H(2)O) over the number of days lived). The SCORE groups, <20, 21 to 69 and 70 to 200, related clinically to mild to severe lung disease. The lungs were tracheally perfused and formalin-fixed and total lung volume (TLV) was measured by water displacement. The paraffin-embedded lung blocks were stained with Miller's elastic stain. The parenchyma and parenchymal elastic tissue were point-counted. The absolute elastic tissue was calculated by multiplying TLV by the parenchymal and elastic fractions. Septal width, alveoli and alveolar duct diameters, and internal surface area (ISA) were also measured. RESULTS: In the controls, the volume density of parenchymal elastic tissue and absolute quantity of elastic tissue increased progressively from 22 to 50 weeks. In infants with CLD and SCORE >/=20, the volume density and absolute quantity of elastic tissue increased significantly. Mean absolute elastic tissue in the 20 to 69 group was 0.76 +/- 0.20 cm(3) greater than in the <20 group (0.46 +/- 0.10 cm(3)) who were similar to the controls, and the 70 to 200 group was 1.32 +/- 0.56 cm(3) greater than the 20 to 69 group. Elastic tissue for infants at risk for CLD, as a percent of predicted for same-age controls, rose linearly with increasing SCORE (r = 0.73; r(2) = 0.55). Control TLV and ISA were linearly related to age. Thirty-nine of the 44 CLD-risk infants had TLVs greater than controls. However, 77% with SCORE 20 to 200 had ISAs less than or equal to the control 95% confidence interval. Control septal width decreased sharply from 23 to 30 weeks, then gradually decreased to term. All infants with SCORE 70 to 200 and 80% of those with SCORE 20 to 69 had widths more than the control 95% confidence interval. Control alveolar and duct diameters doubled from 23 to 50 weeks and were significantly greater in infants with SCORES 20 to 200. DISCUSSION: Lung elastic tissue maturation is tightly controlled during fetal development. With increasing SCORE, elastic tissue increased >200%, accounting, in part, for the positive end-expiratory pressure needed to maintain end-expiratory lung volume in infants at risk for CLD. Saccule and duct diameters more than doubled, and septa thickened significantly in CLD. We propose the following sequence to be operative in CLD: at birth, the preterm infant (

The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors.

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 microg x kg(-1) x min(-1)) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47+/-3 mm Hg (1 mm Hg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3+/-0.1, 2.5+/-0.9, and 5.2+/-1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 microg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.

Solution structure of substrate-based ligands when bound to hepatitis C virus NS3 protease domain.

The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors.

Discovery of non-peptidic P2-P3 butanediamide renin inhibitors with high oral efficacy.

A new series of non-peptidic renin inhibitors having a 2-substituted butanediamide moiety at the P2 and P3 positions has been identified. The optimized inhibitors have IC50 values of 0.8 to 1.4 nM and 2.5 to 7.6 nM in plasma renin assays at pH 6.0 and 7.4, respectively. When evaluated in the normotensive cynomolgus monkey model, two of the most potent inhibitors were orally active at a dose as low as 3 mg/kg. These potent renin inhibitors are characterized by oral bioavailabilities of 40 and 89% in the cynomolgus monkey. Inhibitor 3z (BILA 2157 BS) was selected as candidate for pre-development.

Influence of inhaled nitric oxide and hyperoxia on Na,K-ATPase expression and lung edema in newborn piglets.

This study was undertaken to examine the combined effect of nitric oxide (NO) and hyperoxia on lung edema and Na,K-ATPase expression. Newborn piglets were exposed to room air (FiO2 = 0.21), room air plus 50 ppm NO, hyperoxia (FiO2 >/= 0.96) or to hyperoxia plus 50 ppm NO for 4-5 days. Animals exposed to NO in room air experienced only a slight decrease in Na,K-ATPase alpha subunit protein level. Hyperoxia, in the absence of NO, induced both the mRNA and the protein level of Na,K-ATP-ase alpha subunit and significantly increased wet lung weight, extravascular lung water, and alveolar permeability. NO in hyperoxia decreased the hyperoxic-mediated induction of Na,K-ATPase alpha subunit mRNA and protein while wet lung weight, extravascular lung water, and alveolar permeability remained elevated. These results suggest that 50 ppm of inhaled NO may not improve hyperoxic-induced lung injury and may interfere with the expression of Na,K-ATPase which constitutes a part of the cellular defense mechanism against oxygen toxicity.

Compressed air as a source of inhaled oxidants in intensive care units.

Exhaled gas from mechanically ventilated preterm infants was found to have similar oxidant concentrations, regardless of lung disease, leading to the hypothesis that wall outlet gases were an oxidant source. Oxidants in compressed room air and oxygen from wall outlets were assessed in three hospitals. Samples were collected by flowing wall outlet gas through a heated humidifier and an ice-packed condenser. Nitric oxide (NO) was measured in intensive care room air and in compressed air with and without a charcoal filter using a Sievers NOA280 nitric oxide analyzer (Boulder, CO). Oxidants were measured by spectrophotometry and expressed as nMol equivalents of H2O2/mL. The quantity of oxidant was also expressed as amount of Vitamin C (nMol/mL) added until the oxidant was nondetectable. This quantity of Vitamin C was also expressed in Trolox Equivalent Antioxidant Capacity (TEAC) units (mMol/L). Free and total chlorine were measured with a Chlorine Photometer. Oxidants were not found in compressed oxygen and were only found in compressed air when the compression method used tap water. At a compressed room air gas flow of 1.5 L/min, the total volume of condensate was 20.2 +/- 1 mL/hr. The oxidant concentration was 1.52 +/- 0.09 nMol/mL equivalents of H2O2/mL of sample and 30.8 +/- 1.2 nMol/hr; 17.9% of that found in tap water. Oxidant reduction required 2.05 +/-0.12 nMol/mL vitamin C, (1.78 +/- 0.1 x 10(-3) TEAC units). Free and total chlorine in tap water were 0.3 +/- 0.02 mg/mL and 2.9 +/- 0.002 mg/mL, respectively. Outlet gas contained 0.4 +/- 0.06 mg/mL and 0.07 + 0.01 mg/mL total and free chlorine, respectively; both 14% of tap water. When a charcoal filter was installed in the hospital with oxidants in compressed air, oxidants were completely removed. Nursery room air contained 12.4 +/- 0.5 ppb NO; compressed wall air without a charcoal filter, 8.1 +/- 0.1 ppb and compressed air with a charcoal filter 12.5 +/- 0.5 ppb. A charcoal filter does not remove NO. (Table 3) We recommend that all compressed air methods using tap water have charcoal filters at the compression site and the gases be assessed periodically for oxidants.

Airway muscle in infants with congenital diaphragmatic hernia: response to treatment.

BACKGROUND/PURPOSE: Airway muscle hyperactivity and chronic lung disease frequently follow congenital diaphragmatic hernia (CDH) treatment. The aim of this study was to compare the quantity of airway muscle and alveolar ductal artery muscle in CDH infants after various treatments. METHODS: Five groups were studied postmortem: CDH, died within 24 hours, without high ventilatory assistance (n = 3); CDH, various extracorporeal membrane oxgenation (ECMO) durations, without high ventilatory assistance (n = 4); CDH, various ECMO durations, with high ventilatory assistance (n = 7); no CDH, without high ventilatory assistance (n = 12); and no CDH, with high ventilatory assistance and bronchopulmonary dysplasia (BPD) (n = 5). Sections from standardized fixed lungs were immunohistochemically stained for alpha-smooth muscle actin. Muscle surrounding conducting airways from small preterminal bronchioles to bronchi was quantitated in both the ipsilateral and contralateral lungs with computerized image analysis. Similarly, muscle mass was quantitated in alveolar ductal arteries. RESULTS: CDH infants with low ventilatory assistance, regardless of postnatal age, had the same quantity of airway muscle as low ventilatory assistance controls. Infants with CDH and prolonged high ventilatory assistance had significantly more muscle throughout the conducting airways, similar to BPD infants without CDH, even though the CDH infants had significantly less exposure to high ventilatory assistance. With both low and high ventilatory assistance, the quantity of muscle in both the ipsilateral and contralateral lungs was similar. In contrast, small acinar arteries in CDH infants have increased muscle mass at birth. This muscle is decreased by ECMO but persists in CDH infants with high ventilatory assistance. CONCLUSIONS: The authors show that postnatally, CDH infants acquire increased muscle quantity throughout the conducting airways, in both the ipsilateral and contralateral lungs, with relatively short exposure to high ventilatory assistance. The normal decrease in acinar arterial mass that occurs postnatally is delayed in CDH infants with high ventilatory assistance.

Effect of inhaled nitric oxide in endothelin-1-induced pulmonary hypertension.

The interaction of two vasoactive substances, the vasoidilator nitric oxide (NO) and the complex-acting peptide endothelin-1 (ET-1), may help explain the pathophysiology of pulmonary hypertension, an important part of many pulmonary disorders in neonates. To understand better the interactions of inhaled NO and ET-1, we investigated the effects of ET-1 infusions with and without inhaled NO in two groups of piglets, one group pretreated with L-nitro-arginine methylester (L-NAME) and the other not pretreated. Inhaled NO (60 ppm) was administered during infusion of 1.0 microgram/kg or 2.5 micrograms/kg of ET-1. In animals not pretreated with L-NAME, the increase in PVR and in SVR induced by either dose of ET-1 was not reduced with administration of NO. The increase in systemic vascular resistance with ET-1 was greater (mean increase of 50% above baseline with 1.0 microgram/kg ET-1 and 100% with 2.5 micrograms/kg ET-1 by 5 min) than the increase in PVR, but the PVR/SVR ratio did not change during ET-1 administration. In contrast, animals pretreated with L-NAME did demonstrate inhibition of ET-1-induced increase in PVR with NO. No differences in effects on SVR were noted. We conclude that ET-1-induced increases in PVR are not diminished by 60 ppm of inhaled NO unless there has been inhibition of endogenous NO production.

Simplified method for purification of colostrum to obtain secretory component of immunoglobulin A, using secretory component as a reference protein in tracheal aspirate fluid.

Many studies employ bronchoalveolar lavage fluid for assessment of biologically active substances secreted from the lung. However, investigators continue to search for a useful reference standard to correct for the inevitable but variable degree of dilution of this fluid. The glycoprotein, soluble secretory component of IgA, may serve as a valid reference protein. We report a simplified method for the purification of secretory component from colostrum. Soluble secretory component was isolated from human colostrum using serial centrifugation, size-exclusion fractionation and ion-exchange chromatography. Secretory component rich fractions were assayed by enzyme immunoassay. They were also evaluated for total amino acid content and distribution and sequence determination with satisfactory agreement with published results. We then demonstrated that soluble secretory component concentration in tracheal aspirate fluid did not correlate with either albumin or with total protein measured in the same samples. Therefore, we conclude that the secretory component of IgA serves as a useful reference marker because its use may avoid errors resulting from leakage of plasma proteins into epithelial lining fluid. Advantages of this method for establishing a standard for secretory component include ready availability of soluble secretory component, simplicity of the method and relative rapidity of the techniques.

Alveolar capillary dysplasia, with and without misalignment of pulmonary veins: an association of congenital anomalies.

Two new cases of alveolar capillary dysplasia (ACD), one without misalignment of the pulmonary vessels (MLV), are reported. They are unique for their association with complex cardiac malformations and asplenia. By reporting these cases we want to stress that ACD is associated with multiple malformations, and that the absence of MLV does not rule out the diagnosis of ACD.