Protein Levels In Bronchoalveolar Lavage Fluid Research Articles

ATP-Binding Cassette Transporter 1 Attenuates Ovalbumin-Induced Neutrophilic Airway Inflammation

Apolipoprotein A-I (apoA-I) is an important component of high-density lipoprotein particles that mediates reverse cholesterol transport out of cells by interacting with the ATP-binding cassette transporter 1 (ABCA1). apoA-I has also been shown to attenuate neutrophilic airway inflammation in experimental ovalbumin (OVA)-induced asthma by reducing the expression of granulocyte colony-stimulating factor (G-CSF). Here, we hypothesized that overexpression of the ABCA1 transporter might similarly attenuate OVA-induced neutrophilic airway inflammation. Tie2-human ABCA1 (hABCA1) mice expressing human ABCA1 under the control of the Tie2 promoter, which is primarily expressed by vascular endothelial cells, but can also be expressed by macrophages, received daily intranasal OVA challenges, 5 d/wk for 5 weeks. OVA-challenged Tie2-hABCA1 mice had significant reductions in total bronchoalveolar lavage fluid (BALF) cells that reflected a decrease in neutrophils, as well as reductions in peribronchial inflammation, OVA-specific IgE levels, and airway epithelial thickness. The reduced airway neutrophilia in OVA-challenged Tie2-hABCA1 mice was associated with significant decreases in G-CSF protein levels in pulmonary vascular endothelial cells, alveolar macrophages, and BALF. Intranasal administration of recombinant murine G-CSF to OVA-challenged Tie2-hABCA1 mice for 5 days increased BALF neutrophils to a level comparable to that of OVA-challenged wild-type mice. We conclude that ABCA1 suppresses OVA-induced airway neutrophilia by reducing G-CSF production by vascular endothelial cells and alveolar macrophages. These findings suggest that ABCA1 expressed by vascular endothelial cells and alveolar macrophages may play important roles in attenuating the severity of neutrophilic airway inflammation in asthma.

Improvement of pulmonary absorptions of poorly absorbable drugs using Gelucire 44/14 as an absorption enhancer.

This study aims to evaluate the absorption-enhancing effects of Gelucire 44/14 on the pulmonary absorption of different poorly absorbable drugs and relative mechanism of action. Absorption-enhancing effect of Gelucire 44/14 were examined by an in-vivo pulmonary absorption experiment in rats, and the membrane toxicity of Gelucire 44/14 was evaluated by measuring levels of protein and dehydrogenase (LDH) in the bronchoalveolar lavage fluid (BALF) and morphological observation. Pulmonary absorptions of fluorescein isothiocyanate-dextrans, insulin and calcitonin were enhanced by Gelucire 44/14 (0.1-2.0%, w/v) in a concentration-dependent manner, and the maximal absorption-enhancing effect was obtained when the concentration of Gelucire 44/14 increased to 2.0% (w/v). Furthermore, Gelucire 44/14 neither increase the levels of protein and LDH in BALF nor change morphology of lung compared with control group. In addition, a well correlation between the absorption-enhancing effect and surface tension of insulin solution in the presence of Gelucire 44/14 was observed, suggesting Gelucire 44/14-mediated decrease in the surface tension of the gas-liquid interface in alveolar tissue was possible one of the improving mechanisms of Gelucire 44/14. Gelucire 44/14 was a potential and safe absorption enhancer for improving the absorption of poorly absorbable drugs including insulin and calcitonin by pulmonary delivery.

The effect of C1‐inhibitor in a murine model of transfusion‐related acute lung injury

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related morbidity and mortality. Specific therapy is lacking. We assessed whether C1-inhibitor attenuates lung injury in a 'two-hit' TRALI model. Mice were primed with lipopolysaccharide, subsequently TRALI was induced by MHC-I antibodies. In the intervention group, C1-inhibitor was infused concomitantly. Mice were supported with mechanical ventilation. After 2h, mice were killed, lungs were removed and bronchoalveolar lavage fluid (BALF) was obtained. Injection of MHC-I antibodies induced TRALI, illustrated by an increase in wet-to-dry ratio of the lungs, in BALF protein levels and in lung injury scores. TRALI was further characterized by complement activation, demonstrated by increased BALF levels of C3a and C5a. Administration of C1-inhibitor resulted in increased pulmonary C1-inhibitor levels with high activity. C1-inhibitor reduced pulmonary levels of complement C3a associated with improved lung injury scores. However, levels of pro-inflammatory mediators were unaffected. In a murine model of TRALI, C1-inhibitor attenuated pulmonary levels of C3a associated with improved lung injury scores, but with persistent high levels of inflammatory cytokines.

Cytotoxic and proinflammatory effects of PVP-coated silver nanoparticles after intratracheal instillation in rats

Silver nanoparticles (AgNP) are among the most promising nanomaterials, and their usage in medical applications and consumer products is growing rapidly. To evaluate possible adverse health effects, especially to the lungs, the current study focused on the cytotoxic and proinflammatory effects of AgNP after the intratracheal instillation in rats. Monodisperse, PVP-coated AgNP (70 nm) showing little agglomeration in aqueous suspension were instilled intratracheally. After 24 hours, the lungs were lavaged, and lactate dehydrogenase (LDH), total protein, and cytokine levels as well as total and differential cell counts were measured in the bronchoalveolar lavage fluid (BALF). Instillation of 50 µg PVP-AgNP did not result in elevated LDH, total protein, or cytokine levels in BALF compared to the control, whereas instillation of 250 µg PVP-AgNP caused a significant increase in LDH (1.9-fold) and total protein (1.3-fold) levels as well as in neutrophil numbers (60-fold) of BALF. Furthermore, while there was no change in BALF cytokine levels after the instillation of 50 µg PVP-AgNP, instillation of 250 µg PVP-AgNP resulted in significantly increased levels of seven out of eleven measured cytokines. These finding suggest that exposure to inhaled AgNP can induce moderate pulmonary toxicity, but only at rather high concentrations.

Endothelial cell adhesion molecule CD146: implications for its role in the pathogenesis of COPD

CD146 is an adhesion molecule localized at endothelial cell junctions and facilitates cell-cell interactions. The circulating soluble form (sCD146) lacks both the intracellular and the transmembrane domains. In this study we show that CD146 expression was significantly decreased in the lung tissue of smokers with chronic obstructive pulmonary disease (COPD) and also in rats exposed to second-hand smoke (SHS). Concurrently, levels of sCD146 were increased in both the plasma and bronchoalveolar lavage fluid (BALF) of COPD patients as well as in BALF from rats exposed to SHS. Decreased or abolished CD146 protein expression in rat pulmonary micro- and macrovascular endothelial cells was found after treatment with cigarette smoke extract (CSE), proinflammatory cytokine interleukin 18 (IL-18) or after silencing CD146 expression with siRNA. The decrease in CD146 protein was accompanied by increased endothelial monolayer permeability and enhanced macrophage infiltration in vitro. In CD146 knockout (KO) mice, distinct perivascular oedema was seen and increased numbers of inflammatory cells, along with increased protein levels in BALF. Increased sCD146 was found in BALF and plasma from patients with COPD. The circulating plasma levels of sCD146 correlated positively with the presence of anti-endothelial cell antibodies (AECAs). sCD146 in combination with AECAs may be useful markers for early detection of COPD. Our study indicates that loss of CD146 function damages pulmonary endothelial integrity. This damage may represent part of the pathophysiological processes that are involved in the basic aetiology of COPD/emphysema.

Linezolid Exerts Greater Bacterial Clearance but No Modification of Host Lung Gene Expression Profiling: A Mouse MRSA Pneumonia Model

BackgroundLinezolid (LZD) is beneficial to patients with MRSA pneumonia, but whether and how LZD influences global host lung immune responses at the mRNA level during MRSA-mediated pneumonia is still unknown.MethodsA lethal mouse model of MRSA pneumonia mediated by USA300 was employed to study the influence of LZD on survival, while the sublethal mouse model was used to examine the effect of LZD on bacterial clearance and lung gene expression during MRSA pneumonia. LZD (100mg/kg/day, IP) was given to C57Bl6 mice for three days. On Day 1 and Day 3 post infection, bronchoalveolar lavage fluid (BALF) protein concentration and levels of cytokines including IL6, TNFα, IL1β, Interferon-γ and IL17 were measured. In the sublethal model, left lungs were used to determine bacterial clearance and right lungs for whole-genome transcriptional profiling of lung immune responses.ResultsLZD therapy significantly improved survival and bacterial clearance. It also significantly decreased BALF protein concentration and levels of cytokines including IL6, IL1β, Interferon-γ and IL17. No significant gene expression changes in the mouse lungs were associated with LZD therapy.ConclusionLZD is beneficial to MRSA pneumonia, but it does not modulate host lung immune responses at the transcriptional level.

Cardiopulmonary toxicity of pulmonary exposure to occupationally relevant zinc oxide nanoparticles

Exposure to zinc oxide (ZnO) metal fumes is linked to adverse human health effects; however, the hazards of ZnO nanoparticles (ZnONPs) remain unclear. To determine pulmonary exposure to occupationally relevant ZnONPs cause cardiopulmonary injury, Sprague-Dawley rats were exposed to ZnONPs via intratracheal (IT) instillation and inhalation. The relationship between intrapulmonary zinc levels and pulmonary oxidative-inflammatory responses 72 h after ZnONP instillation was determined in bronchoalveolar lavage fluid (BALF). Instilled ZnONPs altered zinc balance and increased the levels of total cells, neutrophils, lactate dehydrogenase (LDH) and total protein in BALF and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in blood after 72 h. The ZnONPs accumulated predominantly in the lungs over 24 h, and trivial amounts of zinc were determined in the heart, liver, kidneys and blood. Furthermore, the inflammatory-oxidative responses induced by occupationally relevant levels of 1.1 and 4.9 mg/m3 of ZnONP inhalation for 2 weeks were determined in BALF and blood at 1, 7 and 30 days post-exposure. Histopathological examinations of the rat lungs and hearts were performed. Inhalation of ZnONP caused an inflammatory cytological profile. The total cell, neutrophil, LDH and total protein levels were acutely increased in the BALF, and there was an inflammatory pathology in the lungs. There were subchronic levels of white blood cells, granulocytes and 8-OHdG in the blood. Cardiac inflammation and the development of fibrosis were detected 7 days after exposure. Degeneration and necrosis of the myocardium were detected 30 days after exposure. The results demonstrate that ZnONPs cause cardiopulmonary impairments. These findings highlight the occupational health effects for ZnONP-exposed workers.

The roles of cytokines and water sodium channel proteins in acute kidney injury-induced acute lung injury rats

Objective To observe the physiopathologic changes of lung in rats with acute ischemic kidney injury,and to study the roles of cytokine,epithelial sodium channel protein (ENaC) and aquaporin 1 (AQP1) in acute lung injury brought on by acute ischemic kidney injury in rats.Methods A total of 60 healthy male Wistar rats (300-320 g) were randomly (random number) divided into control groups (group A,n =30) and acute kidney injury group (group B,n =30).The model of acute ischemic kidney injury in rats was made by bilateral renal arteiovenous blockage with clamps.Six rats of each group were sacrificed at 0,2,4,6 and 8 hours after modeling.Lung tissue of rats was harvested and stained with hematoxylin-eosin (HE) staining method,and the pathological changes of lung were observed under microscope.The ratio of wet and dry weight (W/D) of lung was calculated.The levels of protein in bronchoalveolar lavage fluid (BALF) were measured.The levels of IL-6 and TNF-α both in serum and BALF were tested.The concentrations of AQP1 and α-ENaC in lung were measured.Results At six hours after modeling,the pH value of arterial blood of rats in group B began to get lowered compared to group A.There was no difference in partial pressure of oxygen in arterial blood between two groups during entire period of experiment (P ＞0.05).Protein level in BALF and W/D of lung increased significantly two hours after modeling in rats of group B (P ＜ 0.05).The histopathological changes of acute lung injury including swollen aleolar epithelium,widened interalveolar septum,edema of alveoli and alveolar interstitium,alveolar neurophil sequestration,erythrocytes and protein in exudates were observed.The levels of TNF-α and IL-6 in serum and in BALF began to increase at two hours after modeling.The levels of AQP1 and α-ENaC of lung in rats with acute kidney injury decreased gradually and were lower than those in rats of group A (P ＜0.05).Conclusions Aleolar epithelial-endothelial barrier function was already compromised at the beginning of AKI,suggesting the acute lung injury was already brought on.The levels of TNF-α,IL-6 in serum and in BALF increased after the occurrence of acute kidney injury.The decreases in lung AQP1 and α-ENaC might contribute to the lung injury caused by early acute kidney injury. Key words: Acute kidney injury; Acute lung injury; Cytokine; Aquaporin 1; Epithelial sodium channel protein

STAT3 modulates cigarette smoke-induced inflammation and protease expression

Signal transducer and activator of transcription-3 (STAT3) regulates inflammation, apoptosis, and protease expression, which are critical processes associated with airway injury and lung tissue destruction. However, the precise role of STAT3 in the development of airway diseases such as chronic obstructive pulmonary disease (COPD) has not been established. This study shows that cigarette smoke activates STAT3 in the lungs of mice. Since cigarette smoke activated STAT3 in the lung, we then evaluated how the loss of STAT3 would impact on smoke-mediated lung inflammation, protease expression, and apoptosis. STAT3+/+ and STAT3−/− mice were exposed to 8 days of cigarette smoke. Compared to the STAT3+/+ mice bronchoalveolar lavage fluid (BALF) cellularity was significantly elevated in the STAT3−/− mice both before and after cigarette smoke exposure, with the increase in cells primarily macrophages. In addition, smoke exposure induced significantly higher BALF protein levels of Interleukin-1α (IL-1α), and monocyte chemotactic protein-1 (MCP-1) and higher tissue expression of keratinocyte chemoattractant (KC) in the STAT3−/− mice. Lung mRNA expression of MMP-12 was increased in STAT3−/− at baseline. However, the smoke-induced increase in MMP-10 expression seen in the STAT3+/+ mice was not observed in the STAT3−/− mice. Moreover, lung protein levels of the anti-inflammatory proteins SOCS3 and IL-10 were markedly lower in the STAT3−/− mice compared to the STAT3+/+ mice. Lastly, apoptosis, as determined by caspase 3/7 activity assay, was increased in the STAT3−/− at baseline to levels comparable to those observed in the smoke-exposed STAT3+/+ mice. Together, these results indicate that the smoke-mediated induction of lung STAT3 activity may play a critical role in maintaining normal lung homeostasis and function.

Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1d–28d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS+ and cyclooxygenase-2+) and alternatively activated profibrotic (YM-1+ and galectin-3+) macrophages appeared in the lung following NM administration; this was evident within 1d, and persisted for 28d. AG administration (50mg/kg, 2×/day, 1d–3d) abrogated NM-induced injury, oxidative stress and inflammation at 1d and 3d post exposure, with no effects at 7d or 28d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis.

Induced hypothermia is protective in a rat model of pneumococcal pneumonia associated with increased adenosine triphosphate availability and turnover*

To determine the effect of induced hypothermia on bacterial growth, lung injury, and mitochondrial function in a rat model of pneumococcal pneumosepsis. Animal study. University research laboratory. Male Sprague-Dawley rats. Subjects were inoculated intratracheally with Streptococcus pneumoniae and controls received saline. After the development of pneumonia, mechanical ventilation was started with or without induced mild hypothermia (32 °C). Bacterial growth and inflammatory markers were determined in bronchoalveolar lavage fluid, blood, and organs. Oxidative phosphorylation and adenosine triphosphate contents were measured in mitochondria isolated from the liver and soleus muscle. Inoculation with S. pneumoniae resulted in severe pneumonia with bacterial dissemination, distal organ injury, and blunted peripheral oxygen consumption on mechanical ventilation. Hypothermia did not affect bacterial growth in bronchoalveolar lavage fluid and in homogenized lungs compared with normothermic controls but was associated with reduced bacterial dissemination to the spleen with a trend toward reduced bacterial load in blood and liver. Hypothermia reduced lung injury, exemplified by reductions in pulmonary cell influx and bronchoalveolar lavage fluid protein levels compared with controls. Hypothermia reduced bronchoalveolar lavage fluid levels of interleukin-1β, tended to reduce bronchoalveolar lavage fluid CINC-3 levels, but no effect was observed on bronchoalveolar lavage fluid tumor necrosis factor-α and interleukin-6 levels. Induced hypothermia restored the fall in oxygen consumption and adenosine triphosphate levels in the liver, whereas adenosine triphosphate/adenosine diphosphate ratios remained low. In muscle, induced hypothermia also reversed low oxygen consumption as a result of pneumonia, but with an increase in adenosine triphosphate levels, whereas adenosine triphosphate/adenosine diphosphate ratios were low. Hypothermia did not adversely affect bacterial growth, but rather reduced bacterial dissemination in a rat model of pneumococcal pneumosepsis. Furthermore, hypothermia reduced lung injury associated with restored adenosine triphosphate availability and turnover. These findings suggest that hypothermia may reduce organ injury by preventing sepsis-related mitochondrial dysfunction.

Effect of maternal exposure to ozone on reproductive outcome and immune, inflammatory, and allergic responses in the offspring

There is growing concern that exposure to air pollutants during pregnancy affects health outcomes in the offspring due to alterations in the development of immune and other homeostatic processes. To assess the risks of maternal inhalation exposure to ozone (O3), timed pregnant BALB/c mice were exposed to different concentrations of O3 (0, 0.4, 0.8, and 1.2 ppm) for 4 h/day for 10 days during gestation (GD9–GD18), and pulmonary inflammation and immune responses were assessed in the offspring at 6 weeks-of-age. Maternal O3 exposure reduced the number of productive dams by 25% at the highest O3 concentration (1.2 ppm) and decreased the rate of weight gain in the offspring. Delayed-type hypersensitivity responses to bovine serum albumin were suppressed in the female offspring by maternal exposure to the two highest concentrations of O3, whereas humoral immune responses to sheep red blood cells were not altered in either sex. Maternal exposure to 1.2 ppm O3 increased lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluid (BALF) of the offspring but did not affect the number of inflammatory cells or levels of total protein, IFN-γ, IL-17, and IL-4 cytokines in BALF, or CD4+, CD8+, CD25+, and TCRβ+CD1d+ T-cells in the spleen. Offspring born from air-exposed dams sensitized early in life (postnatal day [PND] 3) to ovalbumin (OVA) antigen and then challenged as adults developed eosinophilia, elevated levels of LDH activity and total protein in BALF, and increased pulmonary responsiveness to methacholine, compared with animals sensitized at PND42. Maternal O3 exposure in the 1.2 ppm O3 group decreased BALF eosinophilia and serum OVA-specific IgE in the female offspring sensitized early in life but did not affect development of allergic airway inflammation by offspring sensitized late in life. In summary, maternal exposure to O3 affected reproductive outcome and produced modest decreases in immune function and indicators of allergic lung disease in surviving offspring.

Effect of spontaneous breathing on ventilator-induced lung injury in mechanically ventilated healthy rabbits: a randomized, controlled, experimental study

IntroductionVentilator-induced lung injury (VILI), one of the most serious complications of mechanical ventilation (MV), can impact patients' clinical prognoses. Compared to control ventilation, preserving spontaneous breathing can improve many physiological features in ventilated patients, such as gas distribution, cardiac performance, and ventilation-perfusion matching. However, the effect of spontaneous breathing on VILI is unknown. The goal of this study was to compare the effects of spontaneous breathing and control ventilation on lung injury in mechanically-ventilated healthy rabbits.MethodsSixteen healthy New Zealand white rabbits were randomly placed into a spontaneous breathing group (SB Group) and a control ventilation group (CV Group). Both groups were ventilated for eight hours using biphasic positive airway pressure (BIPAP) with similar ventilator parameters: inspiration pressure (PI) resulting in a tidal volume (VT) of 10 to 15 ml/kg, inspiratory-to-expiratory ratio of 1:1, positive end-expiration pressure (PEEP) of 2 cmH2O, and FiO2 of 0.5. Inflammatory markers in blood serum, lung homogenates and bronchoalveolar lavage fluid (BALF), total protein levels in BALF, mRNA expressions of selected cytokines in lung tissue, and lung injury histopathology scores were determined.ResultsAnimals remained hemodynamically stable throughout the entire experiment. After eight hours of MV, compared to the CV Group, the SB Group had lower PaCO2 values and ratios of dead space to tidal volume, and higher lung compliance. The levels of cytokines in blood serum and BALF in both groups were similar, but spontaneous breathing led to significantly lower cytokine mRNA expressions in lung tissues and lower lung injury histological scores.ConclusionsPreserving spontaneous breathing can not only improve ventilatory function, but can also attenuate selected markers of VILI in the mechanically-ventilated healthy lung.

Acute and Subchronic Airway Inflammation after Intratracheal Instillation of Quartz and Titanium Dioxide Agglomerates in Mice

This study investigated the acute and subchronic inflammatory effects of micrometer-size (micro-size) and nanometer-size (nano-size) particles after intratracheal (i.t.) installation in mice. The role of the type of compound, polymorphism, and size of the particles was investigated. Studied compounds were the two micro-size reference quartzes, SRM1878a and DQ12, a micro- and nano-size rutile titanium dioxide (TiO2), a nano-size anatase, and an amorphous TiO2. Particles were administered by a single i.t. instillation in mice at a fixed dose of 5, 50, and 500 μg, respectively. Inflammation was evaluated from the bronchoalveolar lavage fluid (BALF) content of inflammatory cells, the cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), as well as from lung histology. Evaluations were at 24 h (acute effects) and 3 months (subchronic effects) after instillations. Both types of quartz induced a dose-dependent acute increase of neutrophils, IL-6, and total protein in BALF. Limited subchronic inflammation was observed. All types of TiO2 induced a dose-dependent acute increase of neutrophils in BALF. In the acute phase, micro- and nano-size rutile and nano-size amorphous TiO2 induced elevated levels of IL-6 and total protein in BALF at the highest dose. At the nano-size rutile and amorphous TiO2, subchronic lung inflammation was apparent from a dose-dependent increase in BALF macrophages. Histology showed little inflammation overall. The two types of quartz showed virtually similar inflammatory effects. Nearly similar effects were observed for two sizes of rutile TiO2. Differences were seen between the different polymorphs of nano-size TiO2, with rutile being the most inflammogenic and amorphous being the most potent in regard to acute tissue damage.

Translational Research Efforts in Biomarkers and Biology of Early Transplant-Related Complications

In the time since the first allogeneic hematopoietic stem cell transplantation (HSCT) was performed over 40 years ago, this life-saving procedure has been used increasingly for patients with hematologic, metabolic, and malignant diseases. Despite major advances in our understanding of the immunologic processes (both beneficial and injurious) that are associated with HSCT and improvements in supportive and critical care medicine, successful outcomes are still limited by several serious complications. As such, the establishment of effective therapeutic strategies for these complications will be crucial as increasing numbers of high-risk transplants are performed each year. The development of such approaches is fundamentally dependent upon a basic understanding of pathophysiologic mechanisms of disease and also on our ability to successfully translate these insights back to the bedside. This brief review will highlight breakthroughs in translational research endeavors that have paved the way for the development of novel strategies intended to change the standard of care and optimize outcomes for patients in whom allogeneic HSCT offers the only hope for a cure.

UVB Light Exposed Human Platelets Mediate Acute Lung Injury In a Two-Event Mouse Model of Transfusion

AbstractAbstract 3354Transfusion related acute lung injury (TRALI) has occurred in patients whose underlying condition led to an inflamed endothelium, and who were transfused with a transfusion product that contained either HLA or HNA antibodies or biologic modifiers such as lipids or antigens from stored cells. Similar two-event reactions can be modeled in animals pretreated with lipopolysaccharide (LPS) and infused with similar types of antibodies or media from stored transfusion products. The first event induces activation of the endothelium and sequestration of neutrophils in the lungs while the second event activates neutrophils to cause local tissue damage. In some animal models of acute lung damage platelet depletion reduces the lung damage while in other models infusion of activated platelets potentiates it. Ultraviolet B (UVB) light has been used on platelet transfusion products to prevent alloimmunization or with chemical sensitizers to reduce pathogens. Such processing may damage platelets and potentiate their storage lesion. UVB exposed human platelets (HPs) were evaluated in a two-event animal model of acute lung injury (ALI) where the sensitizing event was LPS and the second event was infusion of HPs or UVB HPs (2.4 J/cm2). Immunodeficient (SCID) mice were used to minimize the species difference (Piper et al., Transfusion 47:1540-9, 2007). UVB exposure of HPs increased their p-selectin expression (control 17.8±0.3% vs. UVB 35.9±3.2%) and reduced their JC-1 dye ratio indicating mitochondrial damage (8.9±0.7 control vs 1.9±1.2 UVB). Internal organ distribution of intravenous (IV) infused HPs was followed with whole animal imaging, confocal microscopy and with pathophysiological changes in bronchoalveolar lavage fluid (BALF). In LPS-treated mice, UVB HPs labeled with fluorescent dye had more accumulation in lungs compared to untreated HPs (29±12% vs 15±5% respectively; % of total fluorescence recovered), while the accumulation in lungs of healthy animals was equivalent for both UVB treated and untreated HPs. In separate experiments, LPS pretreated mice were infused with UVB exposed HPs or control HPs, and lungs were examined by histology and with confocal microscopy for fluorescent staining for CD41 and CD62 expression. Histology sections revealed extensive changes in lungs, such as thickening of the alveolar septa and obliteration of lung architecture in LPS animals infused with UVB HPs as compared to healthy animals infused with control HPs or with HPs treated with UVB. Confocal microscopy with specific antibodies identified HP accumulation in lungs of LPS treated animals infused with UVB exposed HPs. However, HP accumulation in lungs did not occur with control HPs or in healthy mice. Accumulation of UVB HPs in lungs of LPS pretreated mice was associated with increased (3-4 fold compared to control HPs) protein concentration and leukocyte accumulation in BALF. Increased alveolar permeability to protein and leukocyte accumulation in alveoli is associated with acute lung damage. To examine whether lung damage occurred due to presence of UVB HPs in the lungs or due to biologic mediators released into plasma from UVB exposed HPs we separated plasma from HPs post UVB treatment and infused the UVB plasma alone. Plasma isolated from UVB HPs did not alter BALF protein levels or leukocyte counts even though the UVB HPs did. In conclusion, UVB HPs can accumulate in lungs of LPS primed animals and are associated with lung damage as indicated by histological changes and increased protein and WBCs in BALF fluid. The ALI is associated with direct platelet accumulation in the lungs but not with biologic modifiers released into plasma. Our animal model of ALI suggests that HPs exposed to high doses of UVB could mediate similar effects in patients predisposed to TRALI with sepsis or other causes of endothelial cell inflammation. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures:No relevant conflicts of interest to declare.

Low intensity laser therapy (LILT) in vivo acts on the neutrophils recruitment and chemokines/cytokines levels in a model of acute pulmonary inflammation induced by aerosol of lipopolysaccharide from Escherichia coli in rat

It has been suggested that low intensity laser therapy (LILT) acts on pulmonary inflammation. Thus, we investigate in this work if LILT (650 nm, 2.5 mW, 31.2 mW/cm 2, 1.3 J/cm 2, laser spot size of 0.08 cm 2 and irradiation time of 42 s) can attenuate edema, neutrophil recruitment and inflammatory mediators in acute lung inflammation. Thirty-five male Wistar rats ( n = 7 per group) were distributed in the following experimental groups: control, laser, LPS, LPS + laser and dexamethasone + LPS. Airway inflammation was measured 4 h post-LPS challenge. Pulmonary microvascular leakage was used for measuring pulmonary edema. Bronchoalveolar lavage fluid (BALF) cellularity and myeloperoxidase (MPO) were used for measuring neutrophil recruitment and activation. RT-PCR was performed in lung tissue to assess mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin (IL-10), cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage inflammatory protein-2 (MIP-2) and intercellular adhesion molecule-1 (ICAM-1). Protein levels in both BALF and lung were determined by ELISA. LILT inhibited pulmonary edema and endothelial cytoskeleton damage, as well as neutrophil influx and activation. Similarly, the LILT reduced the TNF-α and IL-1β, in lung and BALF. LILT prevented lung ICAM-1 up-regulation. The rise of CINC-1 and MIP-2 protein levels in both lung and BALF, and the lung mRNA expressions for IL-10, were unaffected. Data suggest that the LILT effect is due to the inhibition of ICAM-1 via the inhibition of TNF-α and IL-1β.

Lung Damage in Mice after Inhalation of Nanofilm Spray Products: The Role of Perfluorination and Free Hydroxyl Groups

Exposures to two commercial nanofilm spray products (NFPs), a floor sealant (NFP 1) and a coating product for tiles (NFP 2), were investigated for airway irritation, airway inflammation, and lung damage in a mouse inhalation model. The particle exposure was characterized by particle number, particle size distribution, and gravimetric analysis. BALB/cJ mice were exposed for 60 min to the aerosolized products at 3.3–60 mg/m3 (105–106 fine particles/cm3) measured in the breathing zone of the mice. Lung inflammation and lung damage were assessed by study of bronchoalveolar lavage fluid (BALF) cytology, protein in BALF, and histology. Mass spectral analysis showed that NFP 1 and NFP 2 contained hydrolysates and condensates of a perfluorosilane and alkylsilane, respectively. NFP 1 induced a concentration-dependent decrease of the tidal volume lasting for at least 1 day. Exposure concentrations above 16.1 mg/m3 (2.1 × 106 fine particles/cm3) gave rise to significant increases of protein level in BALF and reduced body weight, and histological examination showed atelectasis, emphysema, and hemorrhages. A narrow interval between the no-effect level (16.1 mg/m3) and the lethal concentrations (18.4 mg/m3) was observed. The alkylsilane-based product (NFP 2) had no effect at the concentrations studied. Experiments with different types of perfluorinated silanes and alkylsiloxanes showed that the toxic effects did not arise solely from the perfluorination. The number of free hydroxyl groups in the silanes/alkylsiloxanes was also critical for the toxicity.

Effects of antisense interleukin‐5 gene transferred by recombinant adeno‐associated virus to allergic rats

The accumulation of eosinophils in airways is an important characteristic of asthma. The process is primarily mediated by interleukin-5 (IL-5) secreted by Th2 lymphocytes. This study explored a new approach to asthma therapy in which allergic rats were transfected with the IL-5 antisense gene delivered by the recombinant adeno-associated virus (rAAV-ASIL-5). The viral vector rAAV-ASIL-5 was constructed and the IL-5 antisense gene transfected into allergic rats. The levels of IL-5, IgE, eotaxin and eosinophilic cationic protein (ECP) in sera and bronchoalveolar lavage fluid (BALF) were measured by ELISA. The inflammatory responses in lung tissues were evaluated by histological study. The levels of IL-5 protein in serum and BALF were significantly decreased in the allergic rats treated with rAAV-ASIL-5 (P < 0.05). Serum ovalbumin-specific IgE was reduced in treated rats compared with untreated rats (P < 0.05). rAAV-ASIL-5 treatment also reduced eosinophils in the peripheral blood and BALF, as well as the ECP and eotaxin levels in serum and BALF (P < 0.05). There was significantly less inflammation in the lungs of rAAV-ASIL-5-treated rats than in those of untreated rats. No obvious pathological damage to the kidneys and livers of the rats treated with rAAV was observed. Treatment with rAAV-ASIL-5 inhibited the accumulation of eosinophils and airway inflammation in the rat model of allergic asthma by suppressing IL-5 production. These results suggest that rAAV-ASIL-5-based gene therapy may be used for the treatment of allergic asthma.

A paradoxical protective role for the proinflammatory peptide substance P receptor (NK1R) in acute hyperoxic lung injury

The neuropeptide substance P manifests its biological functions through ligation of a G protein-coupled receptor, the NK1R. Mice with targeted deletion of this receptor reveal a preponderance of proinflammatory properties resulting from ligand activation, demonstrating a neurogenic component to multiple forms of inflammation and injury. We hypothesized that NK1R deficiency would afford a similar protection from inflammation associated with hyperoxia. Counter to our expectations, however, NK1R-/- animals suffered significantly worse lung injury compared with wild-type mice following exposure to 90% oxygen. Median survival was shortened to 84 h for NK1R-/- mice from 120 h for wild-type animals. Infiltration of inflammatory cells into the lungs was significantly increased; NK1R-/- animals also exhibited increased pulmonary edema, hemorrhage, and bronchoalveolar lavage fluid protein levels. TdT-mediated dUTP nick end labeling (TUNEL) staining was significantly elevated in NK1R-/- animals following hyperoxia. Furthermore, induction of metallothionein and Na(+)-K(+)-ATPase was accelerated in NK1R-/- compared with wild-type mice, consistent with increased oxidative injury and edema. In cultured mouse lung epithelial cells in 95% O(2), however, addition of substance P promoted cell death, suggesting the neurogenic component of hyperoxic lung injury is mediated by additional mechanisms in vivo. Release of bioactive constituents including substance P from sensory neurons results from activation of the vanilloid receptor, TRPV1. In mice with targeted deletion of the TRPV1 gene, acute hyperoxic injury is attenuated relative to NK1R-/- animals. Our findings thus reveal a major neurogenic mechanism in acute hyperoxic lung injury and demonstrate concerted actions of sensory neurotransmitters revealing significant protection for NK1R-mediated functions.