Bronchial Alveolar Lavage Fluid Research Articles

Expression of intelectin-1 in bronchial epithelial cells of asthma is correlated with T-helper 2 (Type-2) related parameters and its function

BackgroundIntelectin-1 (ITLN-1) is secreted by intestinal goblet cells and detectable in blood. Its expression is increased in IL-13-overexpressing mouse airways. However, its expression and function in human airways is poorly understood.MethodsDistal and proximal bronchial epithelial cells (BECs) were isolated from bronchoscopic brushings of disease control (D-CON), COPD, inhaled corticosteroid-treated asthma (ST-Asthma) and inhaled corticosteroid-naïve asthma (SN-Asthma) patients. ITLN-1 mRNA expression in freshly isolated BECs, primary cultured BECs with or without IL-13 and inhibition effects of mometasone furoate (MF) were investigated by quantitative real-time PCR (qPCR). Correlations between ITLN-1 mRNA and Type-2 related parameters (e.g. FeNO, IgE, iNOS, CCL26, periostin and DPP4 mRNA) were analyzed. ITLN-1 protein distribution in asthmatic airway tissue was assessed by immunohistochemistry. Bronchial alveolar lavage (BAL) and serum ITLN-1 protein were measured by ELISA. The effect of recombinant human (rh) ITLN-1 on stimulated production of CXCL10 and phospho(p)-STAT1 expression examined in lung fibroblasts.ResultsITLN-1 mRNA was expressed in freshly isolated BECs and was correlated with Type-2 related parameters. ITLN-1 protein was increased in goblet cells in SN-Asthmatics and increased in SN-Asthmatic BAL fluid. There were no any differences in serum ITLN-1 concentration between ST and SN-Asthma. IL-13 enhanced ITLN-1 expression and inhibited by MF from BECs in vitro, while rhITLN-1 inhibited CXCL10 production and p-STAT1 expression in HFL-1 cells.ConclusionITLN-1 is induced by IL-13 and expressed mainly in goblet cells in untreated asthma where its levels correlate with known Type-2 related parameters. Further, ITLN-1 inhibits Type-1 chemokine expression.

Effect of water-soluble C(60) fullerenes on pulmonary fibrosis induced by bleomycin in mice

Objective: To evaluate the role of water-soluble C(60) fullerenes in mice model of lung injury and fibrosis that induced by bleomycin. Methods: A total of 20 healthy C57BL/6J mice were randomly divided into normal control group, bleomycin group, high dose C(60) group, low dose C(60) group, each group with 5 mice. Mice were induced pulmonary fibrosis by intratracheal injection of bleomycin except the normal control group, which was induced by saline instead. In low dose C(60) group and high dose C(60) group, 1 mg·kg(-1)·d(-1) and 10 mg·kg(-1)·d(-1) water-soluble C(60) fullerenes was injected into mice intraperitoneally every day, which began from one day before intratracheal instillation of bleomycin until the end of observation. Saline was given to mice in the same way in normal control and bleomycin group. This study investigated the variation of weight and survival rate of mice for 14 d. HE-staining and Masson's trichrome staining were used to assess the severity of fibrosis according to the method proposed by Ashcroft at 14th day. Total lung collagen content was determined by hydroxyproline assay. The changes of transforming growth factor-β(1) (TGF-β(1)) and tumor necrosis factor α (TNF-α) in plasma, bronchial alveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay (ELISA). And, the amount of reactive oxygen species (ROS) was tested by 2, 7-dichlorofuorescin diacetate (DCFH-DA), and determined by the ratio of fluorescence intensity and protein content (OD/μg). Results: C(60) can protect mice that injured by bleomycin from weight loss. According the method proposed by Ashcroft et al.HE and Masson's trichrome staining showed that collagen deposition in lung tissue were markedly attenuated in C(60) (1 mg·kg(-1)·d(-1) and 10 mg·kg(-1)·d(-1)) treated mice compared with bleomycin model mice[(4.08±0.52), (3.00±0.41) vs (6.75±0.75) points, both P<0.01]. In low dose C(60) group and high dose C(60) group, the content of hydroxyproline in lung tissue were significantly lower than that in bleomycin group[(0.36±0.06), (0.35±0.08) vs (0.55±0.16) μg/mg, both P<0.05]. The level of TGF-β(1) in BALF and lung tissue were also decreased in mice treated with C(60) (10 mg·kg(-1)·d(-1)) compared with bleomycin model mice, but the difference had no statistical significance[(9.38±5.32) vs (23.60±8.96) pg/ml, (2.89±0.35) vs (6.44±2.95) pg/mg, both P>0.05]. Also, in high dose C(60) group, the content of TNF-α in plasma, BALF and lung tissue were significantly lower than those in bleomycin group[(4.56±0.73) vs (7.21±2.26) pg/ml, (34.58±23.30) vs (151.00±27.34) pg/ml, (22.99±5.83) vs (122.90±22.04) pg/mg, all P<0.05]. In addition, Compared with bleomycin group, ROS in lung tissue was significantly decreased after treatment with C(60) (10 mg·kg(-1)·d(-1))[(19.68±0.91) vs (22.92±1.71) OD/μg, P<0.05]. Conclusion: Water-soluble C(60) fullerenes reduce the severity of pulmonary fibrosis induced by bleomycin in mice.

Integrin αvβ5 inhibition protects against ischemia-reperfusion-induced lung injury in an autophagy-dependent manner.

Integrin αvβ5 mediates pulmonary endothelial barrier function and acute lung injury (LI), but its roles in cell apoptosis and autophagy are unclear. Thus, the aims of this study were to investigate the significance of αvβ5 in ischemia-reperfusion (I/R)-induced apoptosis and LI and to explore the relationship between αvβ5 and autophagy. Human pulmonary microvascular endothelial cells (HPMVECs) were pretreated with an αvβ5-blocking antibody (ALULA) and challenged with oxygen-glucose deprivation/oxygen-glucose restoration, which mimics I/R; then, cellular autophagy and apoptosis were detected, and cell permeability was assessed. In vivo, mice were pretreated with the autophagy inhibitor chloroquine (CLQ), followed by treatment with ALULA. The mice then underwent operative lung I/R. LI was assessed by performing a pathological examination, calculating the wet/dry lung weight ratio and detecting the bronchial alveolar lavage fluid (BALF) protein concentration. αvβ5 inhibition promoted HPMVEC autophagy under I/R in vitro, alleviated cell permeability, decreased the apoptosis ratio, and activated caspase-3 expression. These outcomes were significantly diminished when autophagy was inhibited with a small-interfering RNA construct targeting autophagy-related gene 7 (siATG7). Moreover, ALULA pretreatment alleviated I/R-induced LI (I/R-LI), which manifested as a decreased wet/dry lung weight ratio, an altered BALF protein concentration, and lung edema. Preinhibiting autophagy with CLQ, however, eliminated the protective effects of ALULA on I/R-LI. Therefore, inhibiting αvβ5 effectively ameliorated I/R-induced endothelial cell apoptosis and I/R-LI. This process was dependent on improved autophagy and its inhibitory effects on activated caspase-3.

Anti-inflammatory effect of dexmedetomidine combined with hypothermia on acute respiratory distress syndrome in rats

BackgroundTo investigate the protective effect of combination of dexmedetomidine and hypothermia on lipopolysaccharide (LPS) induced acute respiratory distress syndrome in rats. MethodsFifty male Wistar rats were randomly divided into five groups, with 10 rats in each group. The acute respiratory distress syndrome model was reproduced by LPS injected into the right external jugular vein (L group); only saline was injected into the right external jugular vein for control group (C group). In hypothermia group (T group), the body temperature was lowered to 32.5°C-33.0°C after 1 h of LPS injection, and 10 rats were sacrificed at 8 h. Group dexmedetomidine (D group) and dexmedetomidine combined with hypothermia group (DT group) received intraperitoneal dexmedetomidine 30 min before LPS was injected. The arterial blood gas was determined in all the groups before and 8 h after instillation of saline or LPS, and the oxygenation index (PaO2/FiO2) was calculated. The pro-inflammatory cytokines TNF-alpha (TNF-α) and interleukin- 6 (IL-6) levels were determined by enzyme-linked immunosorbent assay. The expression of inflammatory signaling proteins in bronchial alveolar lavage fluid was determined by Western blot. ResultsCompared with group L, TNF-α and IL-6 levels in serum of rats were significantly lower (P < 0.05), the expression of toll-like receptors 4 and phosphorylated c-Jun N-terminal kinase was significantly lower (P < 0.05), and the p-Akt level was significantly higher (P < 0.05). Moreover, the dexmedetomidine combined with hypothermia treated was superior to the single method. ConclusionsThe combination of dexmedetomidine and hypothermia could alleviate acute lung injury in rats.

Hydrostatin-SN1, a Sea Snake-Derived Bioactive Peptide, Reduces Inflammation in a Mouse Model of Acute Lung Injury.

Snake venom has been used for centuries as a traditional Chinese medicine. Hydrostatin-SN1 (H-SN1), a bioactive peptide extracted from the Hydrophis cyanocinctus venom gland T7 phage display library, was reported to have the ability to reduce inflammation in a dextran sulfate sodium-induced murine colitis model. In this study, we sought to investigate the inhibitory potential of H-SN1 on inflammation in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI), and elucidate the anti-inflammatory mechanism in LPS-stimulated RAW 264.7 cells. In vivo, C57BL/6 male mice were intratracheally instilled with LPS or physiological saline with concurrent intraperitoneal injection of H-SN1 or saline alone. Lung histopathologic changes, lung wet-to-dry weight ratio, and myeloperoxidase activity in lung tissues were assessed. Total cell number, the protein concentration, and cytokine levels were determined in the bronchial alveolar lavage fluid. In vitro, RAW 264.7 cells were treated with various concentrations of H-SN1 for 2 h followed by incubation with or without 1 μg/ml LPS for 0.5 or 24 h. The mRNA expression of inflammatory cytokines was determined via RT-PCR and protein levels in the supernatants were measured via ELISA. Extracellular-signal related kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) pathways were analyzed via western blot. H-SN1 improved pulmonary edema status, decreased vascular permeability, suppressed pro-inflammatory cytokine production, and lessened lung morphological injury. H-SN1 also dose-dependently inhibited the mRNA expression and release of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW 264.7 cells. Moreover, H-SN1 inhibited the LPS-induced phosphorylation of ERK1/2 and the nuclear translocation of NF-κB. Our results suggest that H-SN1 could attenuate LPS-induced ALI in mice, which is associated with the anti-inflammatory effect of H-SN1. The mechanism might involve inhibiting the production of inflammatory cytokines by, at least in part, interfering with the ERK1/2 and NF-κB signaling pathways.

β-glucan in the lymph nodes in sarcoidosis and in Kveim-Siltzbach test reagent.

Background: Previous studies have demonstrated a relationship between biomass of fungi exposure in the home and the risk of sarcoidosis. β-glucan was present in the bronchial alveolar lavage fluid (BALF) of patients with sarcoidosis. The Kveim-Siltzbach test reagent (KSTR) induces a sarcoidosis specific, granulomatous, cutaneous response and was used to establish the diagnosis. To date, the granuloma-inducing component of KSTR is still unknown. The present study was undertaken to investigate the presence of β-glucan in the lymph nodes of patients with sarcoidosis and to determine the relationship between the amounts of this agent with disease severity and to investigate the presence of β-glucan in KSTR. Materials and methods: Lymph node aspirations were collected by transbronchial needle aspiration (TBNA) in region R4 or 7 from patients with newly diagnosed sarcoidosis. The samples were treated to isolate β-glucan and analyzed using a Limulus-based assay. Cultures of Propionibacterium ac. and Mycobacterium gordonae as well as samples of Kveim-Siltzbach test reagent were analyzed to determine β-glucan content. Results: A significant relationship was observed between the amount of the β-glucan in the lymph nodes and the extent of granuloma formation in the lung parenchyma, and the size of the lymph nodes in the mediastinum (r=0.787, p=0.0001 and r=0.664, p<0.001 respectively, Spearman's test). The samples of Kveim-Siltzbach test reagent contained high levels of β-glucan. Cultures of Propionibacterium ac. and Mycobacterium gordonae contained β-glucan, the levels of which were lower in the Mycobacterium cultures. Comments: The results support the hypothesis that β-glucan, and thus fungal exposure, are involved in the pathogenesis of sarcoidosis. (Sarcoidosis Vasc Diffuse Lung Dis 2017; 34: 130-135).

Mucosal and Systemic Immune Responses to Influenza H7N9 Antigen HA1-2 Co-Delivered Intranasally with Flagellin or Polyethyleneimine in Mice and Chickens.

Consecutive cases of human infection with H7N9 influenza viruses since 2013 in China have prompted efforts to develop an effective treatment. Subunit vaccines introduced by intranasal administration can block an infection at its primary site; flagellin (fliC) and polyethyleneimine (PEI) have been shown to be potent adjuvants. We previously generated the hemagglutinin (HA)1–2-fliC fusion protein consisting of the globular head domain (HA1–2; amino acids 62–284) of HA fused with Salmonella typhimurium fliC. In the present study, we investigated its effectiveness of both flagellin and PEI as mucosal adjuvants for the H7N9 influenza subunit vaccine. Mice immunized intranasally with HA1–2-fliC and HA1–2-PEI showed higher HA1–2-specific immunoglobulin (Ig)G and IgA titers in serum, nasal wash, and bronchial alveolar lavage fluid. Moreover, splenocyte activation and proliferation and the number of HA1–2-specific interferon (IFN)-γ- and interleukin (IL)-4-producing splenocytes were markedly increased in the fliC and PEI groups; in the latter, there were more cells secreting IL-4 than IFN-γ, suggesting that fliC induced T helper type (Th)1 and Th2 immune responses, and PEI induced Th2-biased responses, consistent with the serum antibody isotype pattern (IgG1/IgG2a ratio). Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving fliC and PEI adjuvant vaccine exhibited robust immune responses leading to a significant reduction in viral loads of throat and cloaca compared to chickens receiving only HA1–2. These findings provide a basis for the development of H7N9 influenza HA1–2 mucosal subunit vaccines.

Experimental chronic kidney disease attenuates ischemia-reperfusion injury in an ex vivo rat lung model.

Lung ischemia reperfusion injury (LIRI) is one of important complications following lung transplant and cardiopulmonary bypass. Although patients on hemodialysis are still excluded as lung transplant donors because of the possible effects of renal failure on the lungs, increased organ demand has led us to evaluate the influence of chronic kidney disease (CKD) on LIRI. A CKD model was induced by feeding Sprague-Dawley rats an adenine-rich (0.75%) diet for 2, 4 and 6 weeks, and an isolated rat lung in situ model was used to evaluate ischemia reperfusion (IR)-induced acute lung injury. The clinicopathological parameters of LIRI, including pulmonary edema, lipid peroxidation, histopathological changes, immunohistochemistry changes, chemokine CXCL1, inducible nitric oxide synthase (iNOS), proinflammatory and anti-inflammatory cytokines, heat shock protein expression, and nuclear factor-κB (NF-κB) activation were determined. Our results indicated that adenine-fed rats developed CKD as characterized by increased blood urea nitrogen and creatinine levels and the deposition of crystals in the renal tubules and interstitium. IR induced a significant increase in the pulmonary arterial pressure, lung edema, lung injury scores, the expression of CXCL1 mRNA, iNOS level, and protein concentration of the bronchial alveolar lavage fluid (BALF). The tumor necrosis factor-α levels in the BALF and perfusate; the interleukin-10 level in the perfusate; and the malondialdehyde levels in the lung tissue and perfusate were also significantly increased by LIRI. Counterintuitively, adenine-induced CKD significantly attenuated the severity of lung injury induced by IR. CKD rats exhibited increased heat shock protein 70 expression and decreased activation of NF-κB signaling. In conclusion, adenine-induced CKD attenuated LIRI by inhibiting the NF-κB pathway.

Maternal high-fat diet in mice leads to innate airway hyperresponsiveness in the adult offspring.

Maternal obesity prior to and during pregnancy has been associated with an increased incidence of childhood asthma. As diets rich in saturated fat are linked to obesity and inflammation, we created a murine model to investigate the effect of maternal high‐fat diet (HFD) on adult offspring airway hyperreactivity (AHR), a cardinal feature of asthma. Balb/cByJ dams were fed a HFD (60% fat Calories) or normal‐fat diet (NFD) (10% fat Calories) from 8 weeks prior to first breeding through their pregnancies. Pups were weaned to either a HFD or NFD (at 4 weeks of age). AHR was measured in the 10‐week‐old offspring following inhaled methacholine challenge by end‐inflation technique. Bronchial alveolar lavage fluid (BALF) was analyzed for cell count, total protein, and IL‐6. Offspring of HFD dams weaned to NFD had increased AHR compared to offspring of NFD dams weaned to NFD. Offspring of HFD dams that remained on HFDs had increased AHR compared to offspring of NFD dams weaned to HFDs. Offspring of HFD dams had higher BALF cell counts, higher neutrophil percentage, greater total protein, and IL‐6 in the BALF. These results demonstrate that a maternal diet high in saturated fat through pregnancy and lactation plays a key role in programming adult offspring AHR.

Antibacterial Loaded Spray Dried Chitosan Polyelectrolyte Complexes as Dry Powder Aerosol for the Treatment of Lung Infections.

Inhalation delivery of aerosolized antibacterials is preferred over conventional methods of delivery for targeting lung infection. The present study is concerned with the development and characterization of a novel, spray dried, aerosolized, chitosan polyelectrolyte complex (PEC) based microparticles containing antibacterials for the treatment of lung infections. Chitosan polyelectrolyte complex microparticles were formulated by spray drying process. Prepared spray dried chitosan PEC microparticles were studied for surface morphology, drug encapsulation efficiency, moisture content, Carr's index, solid state interaction by XRD, aerosolization behaviour and in-vitro drug release. In-vitro cytotoxicity studies of microparticles were carried out on H1299 alveolar cell lines. Antibacterial efficacy of microparticles was assessed on the basis of determination of pharmacokinetic parameters in bronchial alveolar lavage (BAL) of rats using PK/PD analysis. The PEC microparticles were mostly spherical and exhibited high drug encapsulation efficiency. Release profiles showed an initial burst phase followed by a secondary sustained release phase. Good aerosolization behaviour as dry powder inhaler was demonstrated by microparticles with high values of recovered dose, emitted dose, and fine particle fraction. No overt cytotoxicity of microparticles was detected against H1299 alveolar cell line. More than 8 to 9 folds higher Cmax values were obtained in BAL fluid with microparticles as compared to intravenously administered antibacterial solution. The findings of the study suggest that chitosan polyelectrolyte complex based microparticles as dry powder inhaler can be an efficient antibacterial delivery system for sustained and effective management of lung infection.

Culture supernatant of adipose stem cells can ameliorate allergic airway inflammation via recruitment of CD4+CD25+Foxp3 T cells

BackgroundIn a previous study, we demonstrated that intravenous administration of adipose tissue stem cells (ASCs) could significantly reduce allergic symptoms and suppress eosinophilic inflammation.MethodsTo evaluate the secretome of ASCs, we administrated culture supernatant of ASCs (ASC sup, which contains the ASC secretome) and uncultured fresh medium (con sup) into a mouse model of allergic airway inflammation. Subsequently we observed the mice for signs of inflammation and investigated Th1-, Th2-, and Treg-related cytokine levels as well as recruitment of Treg cells into the airway.ResultsWe found that ASC sup could ameliorate allergic airway inflammation in this model; the value of airway hyperresponsiveness, and the occurrence of inflammatory cell infiltration in the lung, as well as the number of eosinophils, and goblet cells in the lung epithelium were all significantly decreased by ASC sup treatment. In addition, ASC sup treatment significantly decreased the levels of IL-4, IL-5, and IL-13 in the bronchial alveolar lavage fluid and in culture medium of lung-draining lymph node cells of the animal model of acute asthma. We detected numerous CTLA-4 and Foxp3-expressing cells in the lung after ASC sup treatment. ASC sup was found to have a higher concentration of IL-10 and TGF-β compared to con sup.ConclusionsStem cells have powerful potential for therapeutic functions in various diseases, but they also have many drawbacks. In this study, we found strong immunosuppressive ability of ASC sup in an allergic airway mouse model. It may be possible to use ASC sup for treatment of many immunological diseases in the near future.

Erratum: "Polygonum multiflorum Decreases Airway Allergic Symptoms in a Murine Model of Asthma".

The root of Polygonum multiflorum (also called He-Shou-Wu in Chinese) is a common herb and medicinal food in Asia used for its anti-aging properties. Our study investigated the therapeutic potential of an extract of the root of Polygonum multiflorum (PME) in allergic asthma by using a mouse model. Feeding of 0.5 and 1 mg/mouse PME inhibited ovalbumin (OVA)-induced allergic asthma symptoms, including airway inflammation, mucus production, and airway hyper-responsiveness (AHR), in a dose-dependent manner. To discern PME's mechanism of action, we examined the profile and cytokine production of inflammatory cells in bronchial alveolar lavage fluid (BALF). We found that eosinophils, the main inflammatory cell infiltrate in the lung of OVA-immunized mice, significantly decreased after PME treatment. Th2 cytokine levels, including interleukin (IL)-4, IL-5, IL-13, eotaxin, and the proinflammatory cytokine tumor necrosis factor (TNF)-[Formula: see text], decreased in PME-treated mice. Elevated mRNA expression of Th2 transcription factor GATA-3 in the lung tissue was also inhibited after oral feeding of PME in OVA-immunized mice. Thus, we conclude that PME produces anti-asthma activity through the inhibition of Th2 cell activation.

Murine Models of Allergic Asthma.

Allergic asthma is a heterogeneous inflammatory lung disease affecting millions of people worldwide and with a steadily increasing incidence. Mouse models have been of utmost importance in uncovering key inflammatory cell types, cytokines, and pathways in the development and maintenance of allergic asthma. Historically, the mainstay in experimental asthma research was sensitizing rodents to the model protein antigen ovalbumin (OVA) with the pro-Th2 adjuvant aluminum hydroxide, followed by repetitive OVA exposures to the airways to initiate a Th2-skewed adaptive immune response leading to eosinophilic airway inflammation and airway hyperreactivity (AHR). In the last 5 years, OVA is often replaced by naturally occurring allergens such as house dust mite (HDM) or cockroach extracts, but the principle of first sensitizing and then repetitively challenging mice with the same antigen is unchanged. Here, we describe an often used and relevant HDM-based protocol to establish acute allergic asthma, and the methods we have developed to rapidly analyze inflammatory cell infiltration in the bronchalveolar lavage fluid by flow cytometry. Moreover, we explain the methods to restimulate T cells from lung-draining mediastinal lymph nodes with HDM to allow the measurement of cytokine secretion profiles of allergen reactive T cells.

Report of 14 cases of exogenous lipoid pneumonia in the elderly

Objective To investigate the clinical characteristics of exogenous lipoid and to explore the characteristics of the elderly with the disease. Methods 14 cases of elderly patients with exogenous lipoid in Beijing Hospital were analyzed. Results Among the 14 patients(7 male and 7 female), main symptoms included dyspnea(50.0%), cough(35.7%), and the common signs were crackles(28.6%)and wet rales(21.4%). For exogenous lipoid pneumonia, the most common causes of inhalation of lipoid substances included chronic constipation, nasal cavity diseases such as rhinitis(35.7%). Some of elderly dementia patients may wrong inhale lipoid substances(21.4%). X ray or CT features of exogenous lipoid showed a tumor-like mass or lesions(42.9%)and the exudative plaque(64.3%), both of which were easily misdiagnosed as tumor.All of the 14 cases(100%)were misdiagnosed as pneumonia or , and finally confirmed by pathological diagnosis.Among the 14 cases, 2 cases were diagnosed by cytopathology of bronchial alveolar lavage fluid or by transbronchial lung biopsy, 10 cases were diagnosed by invasive examination methods like lobe resection, and 2 cases were confirmed by autopsy.At present, there is still a lack of effective treatment methods for exogenous lipoid pneumonia. Conclusions Due to the basal disease(such as constipation)action, the elderly may easily have access to exogenous lipoid with atypical symptoms, might have more serious manifestations than younger adults, and may have X ray or CT imaging similar to lung cancer or pneumonia.So it is often misdiagnosed.If the patients cannot be timely diagnosed and the causes cannot be removed, it is likely to cause chronic interstitial fibrosis or death.Clinicians must increase the awareness of, and take into account the possibility of lipoid pneumonia, especially meeting the elderly patients with dry cough and dyspnea, rales or crackles and X-ray or CT tumor-like imaging as main manifestation resembling pulmonary inflammation or tumor without treatment futility after common antibiotic treatment. Key words: Lung disease, interstitial; Lipids

Soluble programmed cell death receptor-1 (sPD-1): a potential biomarker with anti-inflammatory properties in human and experimental acute respiratory distress syndrome (ARDS).

BackgroundAcute respiratory distress syndrome (ARDS) remains a common organ dysfunction in the critically ill patient. Mechanisms for its development have focused on immune mediated causes, aspects of our understanding are not complete, and we lack biomarkers.Design, setting, and subjectsBlood and bronchial alveolar lavage fluid (BAL) from humans (n = 10–13) with ARDS and controls (n = 5–10) as well as a murine model of ARDS (n = 5–6) with controls (n = 6–7) were studied.MethodsARDS was induced in mice by hemorrhagic shock (day 1) followed by poly-microbial sepsis (day 2). Samples were then collected on the third day after the animals were euthanized. Ex vivo experiments used splenocytes from animals with ARDS cultured with and without soluble programmed death receptor-1 (sPD-1).ResultsLevels of sPD-1 are increased in both the serum (11,429.3 pg/mL(SD 2133.3) vs. 8061.4(SD 4187.8), p = 0.036) and bronchial alveolar lavage (BAL) fluid (6,311.1 pg/mL(SD 3758.0) vs. 90.7 pg/mL(SD 202.8), p = 0.002) of humans with ARDS. Similar results are seen in the serum (9396.1 pg/mL(SD 1546.0) vs. 3464.5 pg/mL(SD 2511.8), p = 0.001) and BAL fluid (2891.7 pg/mL(SD 868.1) vs. 1385.9 pg/mL(SD 927.8), p = 0.012) of mice. sPD-1 levels in murine blood (AUC = 1(1–1), p = 0.006), murine BAL fluid (AUC = 0.905(0.717–1.093), p = 0.015), and human BAL (AUC = 1(1–1), p = 0.001) fluid predicted ARDS. To assess the importance of sPD-1 in ARDS, ex vivo experiments were undertaken. BAL fluid from mice with ARDS dampens the TNF-α production compared to cells cultured with BAL lacking sPD-1 (2.7 pg/mL(SD 3.8) vs. 52.38 pg/mL(SD 25.1), p = 0.002).ConclusionsThis suggests sPD-1 is elevated in critical illness and may represent a potential biomarker for ARDS. In addition, sPD-1 has an anti-inflammatory mechanism in conditions of marked stress and aids in the resolution of severe inflammation. sPD-1 could be used to not only diagnose ARDS, but may be a potential therapy.

Abstract B26: Novel pro-host role for insulin receptor substrate-1 in lung adenocarcinoma

Abstract Lung cancer is the leading cause of cancer deaths worldwide. In the United States alone, lung cancer accounts for ~160,000 deaths per year while the five-year survival rate remains stagnant at ~15%. Lung adenocarcinoma (ADCA) accounts for over 50% of non-small lung cancer cases. Within this subset, K-ras is the most common activating mutation accounting for ~35% of cases. Kras mutant tumors have remained a largely un-targetable subtype and a better understanding of the signaling pathway involved in the different ADCA subtypes will be necessary for the development of therapeutics. Insulin Receptor Substrate-1 (IRS-1) is a signaling adaptor protein that interfaces with many of the pathways that are activated in lung cancer. We have undertaken a controlled study of this protein within the context of lung ADCA. Analysis of a well-annotated human lung ADCA TMA (n=136) revealed that positive IRS-1 staining provided a 75-month median survival advantage among all ADCA cases (p=0.01) and an 81-month median advantage within the K-ras subtype (p=0.006). EGFR and non-K-ras/non-EGFR cases did not display differences. To focus in on the K-ras subtype, we generated LSL-K-ras/IRS-1fl/fl mice and studied them over a time course. LSL-K-ras/IRS-1-/- mice displayed highly significant early mortality (p&amp;lt;0.0001) and increased tumor burden when compared to LSL-K-ras/IRS-1+/+ controls (p&amp;lt;0.01). Interestingly, IRS-1 loss in tumor cells generated a robust immune response. The bronchial alveolar lavage fluid of LSL-K-ras/IRS-1-/- mice showed increased immune infiltration, most prominently neutrophilic (5 times higher). Significant increases in many immune cell-recruiting chemokines were observed in the LSL-K-ras/IRS-1-/- mice compared to controls including CCL2, CCL3, CCL4, CXCL1, CXCL2 and CXCL5. Surprisingly, an array of IRS-1 silenced A549 cells versus controls displayed no change in CC/CXC chemokine production. We decided to investigate which factor could be missing in our in vitro system that is necessary for activation of this chemokine response. IL-17 and IL-22 producing cells, which are present at sites of tumor in human lung cancer cases and LSL-K-ras mice are known to induce CC/CXC chemokine responses. The JAK/STAT pathway is also implicated in this response. We found that loss of IRS-1 increases pSTAT3 production in vivo and in vitro in the presence of IL-22. Finally, we treated LSL-K-ras/IRS-1-/- and LSL-K-ras/IRS-1+/+ controls with a JAK inhibitor (AZD1480). A significant reduction in tumor burden and chemokine response was observed in both genotypes, with the most pronounced response in the LSL-K-ras/IRS-1-/- group. K-ras mutant, IRS-1-low patients represent ~40% of K-ras mutant lung ADCA patients. The phenotype of this group of patients is counter-intuitive in that IRS-1 typically functions in a pro-growth manner. In the setting of certain oncogenic drivers, however, IRS-1 plays an opposing role to this in which its presence actually maintains homeostasis. This work provides evidence that JAK inhibition may be a viable therapeutic option for these patients as well as describes a novel role for IRS-1 as a mediator of immune cell recruitment in lung adenocarcinoma. Citation Format: Heather E. Metz, Julia Kargl, Stephanie Busch, Kyoung-Hee Kim, McGarry Houghton. Novel pro-host role for insulin receptor substrate-1 in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B26.

Electroporation-mediated delivery of the FER gene in the resolution of trauma-related fatal pneumonia.

Injured patients with lung contusion (LC) are at risk of developing bacterial pneumonia (PNA) followed by sepsis and death. A recent Genome-wide Association Study, showed FER gene expression positively correlating with survival rates among individuals with above conditions. We sought to determine if electroporation-mediated (EP) delivery of FER gene could indeed improve survival, in a lethal model of combined LC and PNA. C57BL/6 mice sustained unilateral LC, which preceded a 500 Klebsiella CFU inoculation by 6 hrs. In-between these insults, human FER plasmid (pFER) was introduced into the lungs followed by eight EP pulses applied externally (10ms at 200V/cm). Control groups included EP of empty vector (pcDNA3) or Na+/K+-ATPase genes (pPump) and no treatment (LC + PNA). We recorded survival, histology, lung mechanics, bronchial alveolar fluid (BAL), FER and inflammatory gene expression and bacteriology. The data shows that 7-day survival was significantly improved by pFER compared to control groups. pFER increased BAL monocytes and activated antibacterial response genes (NOS, Fizz). pFER treatment showed decreased lung and blood Klebsiella counts reaching, in some cases, complete sterilization. In conclusion, FER gene delivery promoted survival in LC+PNA mice via recruitment of activated immune cells, improving efficiency of bacterial clearance within contused lung

Development of a minipig model for lung injury induced by a single high-dose radiation exposure and evaluation with thoracic computed tomography.

Radiation-induced lung injury (RILI) due to nuclear or radiological exposure remains difficult to treat because of insufficient clinical data. The goal of this study was to establish an appropriate and efficient minipig model and introduce a thoracic computed tomography (CT)-based method to measure the progression of RILI. Göttingen minipigs were allocated to control and irradiation groups. The most obvious changes in the CT images after irradiation were peribronchial opacification, interlobular septal thickening, and lung volume loss. Hounsfield units (HU) in the irradiation group reached a maximum level at 6 weeks and decreased thereafter, but remained higher than those of the control group. Both lung area and cardiac right lateral shift showed significant changes at 22 weeks post irradiation. The white blood cell (WBC) count, a marker of pneumonitis, increased and reached a maximum at 6 weeks in both peripheral blood and bronchial alveolar lavage fluid. Microscopic findings at 22 weeks post irradiation were characterized by widening of the interlobular septum, with dense fibrosis and an increase in the radiation dose–dependent fibrotic score. Our results also showed that WBC counts and microscopic findings were positively correlated with the three CT parameters. In conclusion, the minipig model can provide useful clinical data regarding RILI caused by the adverse effects of high-dose radiotherapy. Peribronchial opacification, interlobular septal thickening, and lung volume loss are three quantifiable CT parameters that can be used as a simple method for monitoring the progression of RILI.

Classically Activated Macrophages Protect against Lipopolysaccharide-induced Acute Lung Injury by Expressing Amphiregulin in Mice.

Alveolar macrophages (AMs) activated into M1 phenotype are involved in the development of lipopolysaccharide-induced acute lung injury (ALI). However, whether AMs express amphiregulin and what roles amphiregulin plays in lipopolysaccharide-induced ALI remain poorly understood. Acute lung injury was induced by intratracheal instillation of lipopolysaccharide in male C57BL/6 mice. Lung injury scores, level of protein, and level of neutrophils in bronchial alveolar lavage fluid of lipopolysaccharide-induced ALI mice were compared with those in mice challenged with recombinant exogenous amphiregulin and antiamphiregulin antibody. Amphiregulin expression in macrophages and neutrophils in bronchial alveolar lavage fluid of lipopolysaccharide-induced ALI mice was determined by using immunofluorescence technique and further detected in M0, M1, and M2 phenotypes of both peritoneal macrophages and AMs. The effect of amphiregulin on apoptosis of MLE12 cells and activation of epithelial growth factor receptor-AKT pathway were, respectively, examined by using flow cytometry and western blotting. Alveolar macrophages were found to highly express amphiregulin in ALI mice. Amphiregulin neutralization aggravated, whereas recombinant exogenous amphiregulin attenuated lipopolysaccharide-induced ALI in mice (n = 6). In cultured AMs and peritoneal macrophages, amphiregulin was mainly generated by M1, rather than M0 or M2 phenotype (n = 5). Apoptosis ratio of lipopolysaccharide-challenged MLE12 cells was significantly reduced by recombinant exogenous amphiregulin from 16.60 ± 1.82 to 9.47 ± 1.67% (n = 5) but significantly increased from 17.45 ± 1.13 to 21.67 ± 1.10% (n = 5) after stimulation with supernatant of M1-polarized AM media conditioned with amphiregulin-neutrolizing antibody. Western blotting revealed that amphiregulin activated epithelial growth factor receptor and AKT in the lung tissues and MLE12 cells (n = 5). Different from the common notion that classically activated AMs have just a detrimental effect on the lung tissues, the results of this study showed that classically activated AMs also exerted a protective effect on the lung tissues by producing high-level amphiregulin in lipopolysaccharide-induced ALI.

Ozone-induced IL-17A and neutrophilic airway inflammation is orchestrated by the caspase-1-IL-1 cascade.

Ozone is a common environmental air pollutant leading to respiratory illness. The mechanisms regulating ozone-induced airway inflammation remain poorly understood. We hypothesize that ozone-triggered inflammasome activation and interleukin (IL)-1 production regulate neutrophilic airway inflammation through IL-17A. Pulmonary neutrophilic inflammation was induced by extended (72 h) low-dose (0.7 ppm) exposure to ozone. IL-1 receptor 1 (Il1r1)−/−, Il17a−/− mice and the caspase-1 inhibitor acetyl-YVAD-chloromethylketone (Ac-YVAD-cmk) were used for in vivo studies. Cellular inflammation and protein levels in bronchial alveolar lavage fluid (BALF), cytokines, and IL-17A-producing γδT-cells, as well as mitochondrial reactive oxygen species (ROS), mitochondrial DNA (mtDNA) release, and inflammasome activation in lung macrophages were analyzed. Ozone-induced neutrophilic airway inflammation, accompanied an increased production of IL-1β, IL-18, IL-17A, Granulocyte-colony stimulating factor (G-CSF), Interferon-γ inducible protein 10 (IP-10) and BALF protein in the lung. Ozone-induced IL-17A production was predominantly in γδT-cells, and Il17a-knockout mice exhibited reduced airway inflammation. Lung macrophages from ozone-exposed mice exhibited higher levels of mitochondrial ROS, enhanced cytosolic mtDNA, increased caspase-1 activation, and higher production of IL-1β. Il1r1-knockout mice or treatment with Ac-YVAD-cmk decreased the IL-17A production and subsequent airway inflammation. Taken together, we demonstrate that ozone-induced IL-17A and neutrophilic airway inflammation is orchestrated by the caspase-1-IL-1 cascade.