Methylprednisolone improves lung mechanics and reduces the inflammatory response in pulmonary but not in extrapulmonary mild acute lung injury in mice

Objective: Gorticosteroids have been proposed to be effective in modulating the inflammatory response and pulmonary tissue remodeling in acute lung injury (ALI). We hypothesized that steroid treatment might act differently in models of pulmonary (p) or extrapulmonary (exp) ALI with similar mechanical compromise. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: One hundred twenty-eight BALB/c mice (20-25 g). Interventions: Mice were divided into six groups. In control animals sterile saline solution was intratracheally (0.05 mL, Cp) or intraperitoneally (0.5 mL, Gexp) injected, whereas ALI animals received Escherichia coli lipopolysaccharide intratracheally (10 mu g, ALIp) or intraperitoneally (125 mu g, ALIexp). Six hours after lipopolysaccharide administration, ALIp and ALlexp animals were further randomized into subgroups receiving saline (0.1 mL intravenously) or methylprednisolone (2 mg/kg intravenously, Mp and Mexp, respectively). Measurements and Main Results: At 24 hrs, lung state elastance, resistive and viscoelastic pressures, lung morphometry, and collagen fiber content were similar in both ALI groups. KC, interieukin-6, and transforming growth factor (TGF)-beta levels in bronchoatveolar lavage fluid, as well as tumor necrosis factor (TNF)-alpha, migration inhibitory factor (MIF), interferon (IFN)-gamma, TGF-beta 1 and TGF-beta 2 messenger RNA expression in lung tissue were higher in ALIp than in ALIexp animals. Methylprednisolone attenuated mechanical and morphometric changes, cytokine levels, and TNF-alpha, MIF, IFN gamma, and TGF-beta 2 messenger RNA expression only in ALIp animals, but prevented any changes in collagen fiber content in both ALI groups. Conclusions. Methylprednisolone is effective to inhibit fibrogenesis independent of the etiology of ALI, but its ability to attenuate inflammatory responses and lung mechanical changes varies according to the cause of ALI.

Comparative respiratory toxicity of particles produced by traffic and sugar cane burning

The impact of particle emissions by biomass burning is increasing throughout the world. We explored the toxicity of particulate matter produced by sugar cane burning and compared these effects with equivalent mass of traffic-derived particles. For this purpose, BALB/c mice received a single intranasal instillation of either distilled water (C) or total suspended particles (15 mu g) from an urban area (SP group) or biomass burning-derived particles (Bio group). Lung mechanical parameters (total, resistive and viscoelastic pressures, static elastance, and elastic component of viscoelasticity) and histology were analyzed 24h after instillation. Trace elements and polycyclic aromatic hydrocarbons (PAHs) metabolites of the two sources of particles were determined. All mechanical parameters increased similarly in both pollution groups compared with control, except airway resistive pressure, which increased only in Bio. Both exposed groups showed significantly higher fraction area of alveolar collapse, and influx of polymorphonuclear cells in lung parenchyma than C. The composition analysis of total suspended particles showed higher concentrations of PAHs and lower concentration of metals in traffic than in biomass burning-derived particles. In conclusion, we demonstrated that a single low dose of ambient particles, produced by traffic and sugar cane burning, induced significant alterations in pulmonary mechanics and lung histology in mice. Parenchymal changes were similar after exposure to both particle sources, whereas airway mechanics was more affected by biomass-derived particles. Our results indicate that biomass particles were at least as toxic as those produced by traffic. (C) 2008 Elsevier Inc. All rights reserved.

Disarrangement of Collagen Fibers in Reinke`s Edema

Objective/Hypothesis: To describe the arrangement of collagen fibers in the superficial layer of the lamina propria of the vocal folds with Reinke` edema. Study Design: Cross sectional analysis of the lamina propria of the vocal folds with Reinke`s edema (RE). Method: The picrosirius polarization method was used to study the arrangement of collagen fiber. Findings of collagen disarrangement were categorized semiquantitatively and correlated with RE severity, age, cigarette smoking and duration of dysphonia. Results: Analysis of 20 specimens of vocal folds with RE showed that the intertwined network of collagen fibers resembling a wicker-basket normally observed in vocal folds was disarranged in RE. The collagen fibers were loosely arranged, fragmented and intermixed with varying amounts of myxoid stroma. Moderate and large areas of disarrangement (90% of cases) predominated. Collagen fiber arrangement in the region underneath the epithelium was better preserved when compared with fibers in the deeper region of the superficial layer of the lamina propria. There was a statistical difference in collagen disarrangement between grade II and grade III severity (P = .007) that appeared to be due to the large areas of disarrangement observed in 73% of patients with grade III severity and in 44% of grade II severity. Age was the only variable correlated with collagen fiber disarrangement (r = 0.47, P = .037). Conclusion: Our findings suggest that the flexible framework which maintains the uniformity of the lamina propria was lost in RE caused by the disarrangement of the collagen fibers.

Extracellular matrix components and regulators in the airway smooth muscle in asthma

There is an intimate relationship between the extracellular matrix (ECM) and smooth muscle cells within the airways. Few studies have comprehensively assessed the composition of different ECM components and its regulators within the airway smooth muscle (ASM) in asthma. With the aid of image analysis, the fractional areas of total collagen and elastic fibres were quantified within the ASM of 35 subjects with fatal asthma (FA) and compared with 10 nonfatal asthma (NFA) patients and 22 nonasthmatic control cases. Expression of collagen I and III, fibronectin, versican, matrix metalloproteinase (MMP)-1, -2, -9 and -12 and tissue inhibitor of metalloproteinase-1 and -2 was quantified within the ASM in 22 FA and 10 control cases. In the large airways of FA cases, the fractional area of elastic fibres within the ASM was increased compared with NFA and controls. Similarly, fibronectin, MMP-9 and MMP-12 were increased within the ASM in large airways of FA cases compared with controls. Elastic fibres were increased in small airways in FA only in comparison with NFA cases. There is altered extracellular matrix composition and a degradative environment within the airway smooth muscle in fatal asthma patients, which may have important consequences for the mechanical and synthetic functions of airway smooth muscle.

Post-transplant recurrent hepatitis C: immunohistochemical detection of hepatitis C virus core antigen and possible pathogenic implications

Introduction: The mechanisms by which severe cholestatic hepatitis develops after liver transplantation are not fully understood. Reports on immunohistochemical distribution of hepatitis C virus (HCV) antigens are still scarce, but recently, HCV immunostaining was suggested for early diagnosis of cholestatic forms of recurrent hepatitis C in liver grafts. After purification, Rb246 pab anticore (aa1-68) yielded specific, granular cytoplasmic staining in hepatocytes. Signal amplification through the Envision-Alkaline Phosphatase System avoided endogenous biotin and peroxidase. Aims/Methods: Rb246 was applied to liver samples of explants of 12 transplant recipients, six with the most severe form of post-transplantation recurrence, severe cholestatic hepatitis (group 1) and six with mild recurrence (group 2). We also assessed immuno-reactivity at two time-points post-transplantation (median 4 and 22 months) in both groups. HCV-core Ag was semiquantified from 0 to 3+ in each time point. Serum HCV-RNA was also measured on the different time points by branched DNA. Results: In the early post-transplant time point, one patient had a mild staining (1+), two patients had a moderate staining (2+) and the other three had no staining in group 1, compared with five patients with no staining (0) and one patient with mild staining (1+) in group 2. Late post-transplant liver samples were available in nine patients, and two out of four samples in group 1 showed a mild staining, compared with no staining patients in five patients in group 2. Strikingly, on the explant samples, HCV immunostaining was strongly positive in group 1, and mildly positive in group 2. Two out of five samples showed 3+ staining, and three samples showed 2+ staining in group 1; two out of five samples showed no staining, two samples showed 1+ staining and one sample showed 2+ staining in group 2. Serum HCV-RNA was significantly higher in group 1, on both time-points post-transplantation. HCV-core Ag was not directly associated with serum HCV-RNA on the different time points. Conclusion: These preliminary results suggest that strong HCV immunostaining in the explant is predictive of more severe disease recurrence.

Pulmonary mechanic and lung histology injury induced by Crotalus durissus terrificus snake venom

In the present work we investigated the effects of Crotalus durissus terrificus venom (CdtV) on the pulmonary mechanic events [static and dynamic elastance, resistive (Delta P1) and viscoelastic pressures (Delta P2)] and histology after intramuscular injection of saline solution (control) or venom (0.6 mu g/g). The static and dynamic elastance values were increased significantly after 3 It of venom inoculation, but were reduced at control values in the other periods studied. The Delta P1 values that correspond to the resistive properties of lung tissue presented a significant increase after 6 h of CdtV injection, reducing to basal levels 12 h after the venom injection. In Delta P2 analysis, correspondent to viscoelastic components, an increase occurred 12 h after the venom injection, returning to control values at 24 h. CdtV also caused an increase of leukocytes recruitment (3-24 h) to the airways wall as well as to the lung parenchyma. In conclusion, C. durissus terrificus rattlesnake venom leads to lung injury which is reverted, after 24 h of inoculation. (c) 2008 Elsevier Ltd. All rights reserved.

Recruitment maneuver in pulmonary and extrapulmonary experimental acute lung injury

Objective. The aim of this study is to test the hypothesis that recruitment maneuvers (RMs) might act differently in models of pulmonary (p) and extrapulmonary (exp) acute lung injury (ALI) with similar transpulmonary pressure changes. Design: Prospective, randomized, controlled experimental study. Setting. University research laboratory. Subjects: Wistar rats were randomly divided into four groups. In control groups, sterile saline solution was intratracheally (0.1 mL, Cp) or intraperitoneally (1 mL, Cexp) injected, whereas ALI animals received Escherichia coli lipopolysaccharide intratracheally (100 jig, ALIp) or intraperitoneally (1 mg, ALIexp). After 24 hrs, animals were mechanically ventilated (tidal volume, 6 mL/kg; positive end-expiratory pressure, 5 cm H2O) and three RMs (pressure inflations to 40 cm H2O for 40 secs, 1 min apart) applied. Measurements and Main Results. Pao(2), lung resistive and viscoelastic pressures, static elastance, lung histology (light and electron microscopy), and type III procollagen messenger RNA expression in pulmonary tissue were measured before RMs and at the end of 1 hr of mechanical ventilation. Mechanical variables, gas exchange, and the fraction of area of alveolar collapse were similar in both ALI groups. After RMs, lung resistive and viscoelastic pressures and static elastance decreased more in ALIexp (255%,180%, and 118%, respectively) than in ALIp (103%, 59%, and 89%, respectively). The amount of atelectasis decreased more in ALIexp than in ALIp (from 58% to 19% and from 59% to 33%, respectively). RMs augmented type III procollagen messenger RNA expression only in the ALIp group (19%), associated with worsening in alveolar epithelium injury but no capillary endothelium lesion, whereas the ALIexp group showed a minor detachment of the alveolar capillary membrane. Conclusions. Given the same transpulmonary pressures, RMs are more effective at opening collapsed alveoli in ALIexp than in ALIp, thus improving lung mechanics and oxygenation with limited damage to alveolar epithelium.

Expression of smooth muscle and extracellular matrix proteins in relation to airway function in asthma

Background: Smooth muscle content is increased within the airway wall in patients with asthma and is likely to play a role in airway hyperresponsiveness. However, smooth muscle cells express several contractile and structural proteins, and each of these proteins may influence airway function distinctly. Objective: We examined the expression of contractile and structural proteins of smooth muscle cells, as well as extracellular matrix proteins, in bronchial biopsies of patients with asthma, and related these to lung function, airway hyperresponsiveness, and responses to deep inspiration. Methods: Thirteen patients with asthma (mild persistent, atopic, nonsmoking) participated in this cross-sectional study. FEV1 % predicted, PC20 methacholine, and resistance of the respiratory system by the forced oscillation technique during tidal breathing and deep breath were measured. Within 1 week, a bronchoscopy was performed to obtain 6 bronchial biopsies that were immunuhistochemically stained for alpha-SM-actin, desmin, myosin light chain kinase (MLCK), myosin, calponin, vimentin, elastin, type III collagen, and fibronectin. The level of expression was determined by automated densitometry. Results: PC20 methacholine was inversely related to the expression of alpha-smooth muscle actin (r = -0.62), desmin (r = -0.56), and elastin (r = -0.78). In addition, FEV1% predicted was positively related and deep inspiration-induced bronchodilation inversely related to desmin (r = -0.60), MLCK (r = -0.60), and calponin (r = -0.54) expression. Conclusion: Airway hyperresponsiveness, FEV1% predicted, and airway responses to deep inspiration are associated with selective expression of airway smooth muscle proteins and components of the extracellular matrix.

The effect of phospholipase A2 from Crotalus durissus collilineatus on Leishmania (Leishmania) amazonensis infection

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.

Impact of lung remodelling on respiratory mechanics in a model of severe allergic inflammation

We developed a model of severe allergic inflammation and investigated the impact of airway and lung parenchyma remodelling on in vivo and in vitro respiratory mechanics. BALB/c mice were sensitized and challenged with ovalbumin in severe allergic inflammation (SA) group. The control group (C) received saline using the same protocol. Light and electron microscopy showed eosinophil and neutrophil infiltration and fibrosis in airway and lung parenchyma, mucus gland hyperplasia, and airway smooth muscle hypertrophy and hyperplasia in SA group. These morphological changes led to in vivo (resistive and viscoelastic pressures, and static elastance) and in vitro (tissue elastance and resistance) lung mechanical alterations. Airway responsiveness to methacholine was markedly enhanced in SA as compared with C group. Additionally, IL-4, IL-5, and IL-13 levels in the bronchoalveolar lavage fluid were higher in SA group. In conclusion, this model of severe allergic lung inflammation enabled us to directly assess the role of airway and lung parenchyma inflammation and remodelling on respiratory mechanics. (C) 2007 Elsevier B.V. All rights reserved.

Pulmonary morphofunctional effects of mechanical ventilation with high inspiratory air flow

Objective: Uncertainties about the numerous degrees of freedom in ventilator settings leave many unanswered questions about the biophysical determinants of lung injury. We investigated whether mechanical ventilation with high air flow could yield lung mechanical stress even in normal animals. Design. Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects. Thirty normal male Wistar rats (180-230 g). Interventions: Rats were ventilated for 2 hrs with tidal volume of 10 mL/kg and either with normal inspiratory air flow (V`) of 10 mL/s (F10) or high V` of 30 mL/s (F30). In the control group, animals did not undergo mechanical ventilation. Because high flow led to elevated respiratory rate (200 breaths/min) and airway peak inspiratory pressure (PIP,aw = 17 cm H2O), two additional groups were established to rule out the potential contribution of these variables: a) normal respiratory rate = 100 breaths/min and V` = 30 mL/sec; and b) PIP,aw = 17 cm H2O and V` 10 mL/sec. Measurements and Main Results: Lung mechanics and histology (light and electron microscopy), arterial blood gas analysis, and type III procollagen messenger RNA expression in lung tissue were analyzed. Ultrastructural microscopy was similar in control and F10 groups. High air flow led to increased lung plateau and peak pressures, hypoxemia, alveolar hyperinflation and collapse, pulmonary neutrophilic infiltration, and augmented type III procollagen messenger RNA expression compared with control rats. The reduction of respiratory rate did not modify the morphofunctional behavior observed in the presence of increased air flow. Even though the increase in peak pressure yielded mechanical and histologic changes, type III procollagen messenger RNA expression remained unaltered. Conclusions: Ventilation with high inspiratory air flow may lead to high tensile and shear stresses resulting in lung functional and morphologic compromise and elevation of type III procollagen messenger RNA expression.

Recruitment maneuver in experimental acute lung injury: The role of alveolar collapse and edema

Objective: In acute lung injury, recruitment maneuvers have been used to open collapsed lungs and set positive end-expiratory pressure, but their effectiveness may depend on the degree of lung injury. This study uses a single experimental model with different degrees of lung injury and tests the hypothesis that recruitment maneuvers may have beneficial or deleterious effects depending on the severity of acute lung injury. We speculated that recruitment maneuvers may worsen lung mechanical stress in the presence of alveolar edema. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: Thirty-six Wistar rats randomly divided into three groups (n = 12 per group). Interventions: In the control group, saline was intraperitoneally injected, whereas moderate and severe acute lung injury animals received paraquat intraperitoneally (20 mg/kg [moderate acute lung injury] and 25 mg/kg [severe acute lung injury]). After 24 hrs, animals were further randomized into subgroups (n = 6/each) to be recruited (recruitment maneuvers: 40 cm H(2)O continuous positive airway pressure for 40 secs) or not, followed by 1 hr of protective mechanical ventilation (tidal volume, 6 mL/kg; positive end-expiratory pressure, 5 cm H(2)O). Measurements and Main Results: Only severe acute lung injury caused alveolar edema. The amounts of alveolar collapse were similar in the acute lung injury groups. Static lung elastance, viscoelastic pressure, hyperinflation, lung, liver, and kidney cell apoptosis, and type 3 procollagen and interleukin-6 mRNA expressions in lung tissue were more elevated in severe acute lung injury than in moderate acute lung injury. After recruitment maneuvers, static lung elastance, viscoelastic pressure, and alveolar collapse were lower in moderate acute lung injury than in severe acute lung injury. Recruitment maneuvers reduced interleukin-6 expression with a minor detachment of the alveolar capillary membrane in moderate acute lung injury. In severe acute lung injury, recruitment maneuvers were associated with hyperinflation, increased apoptosis of lung and kidney, expression of type 3 procollagen, and worsened alveolar capillary injury. Conclusions: In the presence of alveolar edema, regional mechanical heterogeneities, and hyperinflation, recruitment maneuvers promoted a modest but consistent increase in inflammatory and fibrogenic response, which may have worsened lung function and potentiated alveolar and renal epithelial injury. (Crit Care Med 2010; 38: 2207-2214)

Bone Marrow Mononuclear Cell Therapy Led to Alveolar-Capillary Membrane Repair, Improving Lung Mechanics in Endotoxin-Induced Acute Lung Injury

The aim of this study was to test the hypothesis that bone marrow mononuclear cell (BMDMC) therapy led an improvement in lung mechanics and histology in endotoxin-induced lung injury. Twenty-four C57BL/6 mice were randomly divided into four groups (n = 6 each). In the acute lung injur;y (ALI) group, Escherichia coli lipopolysaccharide (LPS) was instilled intratracheally (40 mu g, IT), and control (C) mice received saline (0.05 ml, IT). One hour after the administration of saline or LPS, BMDMC (2 x 10(7) cells) was intravenously injected. At day 28, animals were anesthetized and lung mechanics [static elastance (E(st)), resistive (Delta P(1)), and viscoelastic (Delta P(2)) pressures] and histology (light and electron microscopy) were analyzed. Immunogold electron microscopy was used to evaluate if multinucleate cells were type II epithelial cells. BMDMC therapy prevented endotoxin-induced lung inflammation, alveolar collapse, and interstitial edema. In addition, BMDMC administration led to epithelial and endothelial repair with multinucleated type II pneumocytes. These histological changes yielded a reduction in lung E(st), Delta P(1), and Delta P(2) compared to ALI. In the present experimental ALI model, the administration of BMDMC yielded a reduction in the inflammatory process and a repair of epithelium and endothelium, reducing the amount of alveolar collapse, thus leading to an improvement in lung mechanics.

Airway smooth muscle thickness in asthma is related to severity but not duration of asthma

Asthma is characterised by an increased airway smooth muscle (ASM) area (ASMarea) within the airway wall. The present study examined the relationship of factors including severity and duration of asthma to ASMarea. The perimeter of the basement membrane (PBM) and ASMarea were measured on transverse sections of large and small airways from post mortem cases of fatal (n=107) and nonfatal asthma (n=37) and from control subjects (n=69). The thickness of ASM (ASMarea/PBM) was compared between asthma groups using multivariate linear regression. When all airways were considered together, ASMarea/PBM (in millimetres) was increased in nonfatal (median 0.04; interquartile range 0.013-0.051; p=0.034) and fatal cases of asthma (0.048; 0.025-0.078; p<0.001) compared with controls (0.036; 0.024-0.042). Compared with cases of nonfatal asthma, ASMarea/PBM was greater in cases of fatal asthma in large (p<0.001) and medium (p<0.001), but not small, airways. ASMarea/PBM was not related to duration of asthma, age of onset of asthma, sex or smoking. No effect due to study centre, other than that due to sampling strategy, was found. The thickness of the ASM layer is increased in asthma and is related to the severity of asthma but not its duration.

Effect of Exercise Training on Plasma Levels and Functional Properties of High-Density Lipoprotein Cholesterol in the Metabolic Syndrome

Intense lifestyle modifications can change the high-density lipoprotein (HDL) cholesterol concentration. The aim of the present study was to analyze the early effects of short-term exercise training, without any specific diet, on the HDL cholesterol plasma levels and HDL functional characteristics in patients with the metabolic syndrome (MS). We studied 30 sedentary subjects, 20 with and 10 without the MS. The patients with the MS underwent moderate intensity exercise training for 3 months on bicycle ergometers. Blood was sampled before and after training for biochemical analysis, paraoxonase-1 activity, and HDL subfraction composition and antioxidative capacity. Lipid transfer to HDL was assayed in vitro using a labeled nanoemulsion as the lipid donor. At baseline, the MS group had greater triglyceride levels and a lower HDL cholesterol concentration and lower paraoxonase-1 activity than did the controls. Training decreased the plasma triglycerides but did not change the low-density lipoprotein or HDL cholesterol levels. Nonetheless, exercise training increased the HDL subfractions` antioxidative capacity and paraoxonase-1 activity. After training, the MS group had compositional changes in the smallest HDL subfractions associated with increased free cholesterol and cholesterol ester transfers to HDL, reaching normal values. In conclusion, the present investigation has added relevant information about the dissociation between the quantitative and qualitative aspects of HDL after short-term exercise training without any specific diet in those with the MS, highlighting the importance of evaluating the functional aspects of the lipoproteins, in addition to their plasma levels. (C) 2011 Elsevier Inc. All rights reserved. (Am J Cardiol 2011;107:1168-1172)