Bronchoalveloar Lavage Fluid Research Articles

Suppression of NOD-like receptor protein 3 inflammasome activation and macrophage M1 polarization by hederagenin contributes to attenuation of sepsis-induced acute lung injury in rats

ABSTRACT Acute lung injury (ALI) is a major leading cause of death in sepsis patients. Hederagenin (HG), derived from Hedera helix Linné, has anti-inflammatory effects, while its role in sepsis-induced ALI has not been elucidated. In vivo, rats were subjected to cecal ligation and puncture to induce ALI and then treated with HG (12.5, 25, or 50 mg/kg) by gavage. Administration of HG raised survival rate, ameliorated lung injury, and decreased lung wet/dry ratio and inflammatory cell accumulation in bronchoalveloar lavage fluid (BALF) of ALI rats. HG inhibited macrophage polarization toward the M1 phenotype as evidenced by decreased CD86 expression in rat lung tissues. Moreover, HG decreased the secretion of TNF-α, IL-6 and monocyte chemoattractant protein-1 (MCP-1) in BALF and the levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung tissues. In vitro, phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 macrophages were stimulated with 100 ng/mL lipopolysaccharide. HG treatment inhibited M1 macrophage polarization and the production of M1-related pro-inflammatory mediators (IL-6, MCP-1, iNOS, and COX-2). Mechanistically, HG inhibited NLRP3 inflammasome activation and subsequent release of IL-18 and IL-1β, and suppressed NF-κB signaling pathway both in vivo and in vitro. Notably, HG treatment further emphasized the inhibitory effect of NF-κB inhibitor BAY11-7082 on NLRP3 inflammasome activation and macrophage M1 polarization. Taken together, HG exerts a protective effect against sepsis-induced ALI by reducing the inflammatory response and macrophage M1 polarization, which may involve NF-κB pathway-modulated NLRP3 inflammasome activation.

Review of Candida dubliniensis at a Pediatric Hospital

Background:Candida dubliniensis is a worldwide fungal opportunistic pathogen, closely related to C. albicans. Originally identified in patients infected with HIV in Dublin, Ireland, C. dubliniensis has emerged as a pathogen in other immunocompromised individuals, including patients receiving chemotherapy and transplant recipients. Pediatric epidemiological data for this organism are limited. Methods: We report a descriptive review of C. dubliniensis isolates recovered between January 2018 and June 2019 at a large tertiary-care pediatric institution in Columbus, Ohio. Results:C. dubliniensis was identified in 48 patients in the 18-month review period. In total, 67 positive cultures were collected in these patients with the following distribution of sources: 44 sputum (66%), 11 bronchoalveloar lavage fluid (16%), 4 blood (6%), 3 wounds (4%), 2 esophageal (3%), 2 peritoneal fluid (3%), and 1 vaginal (1%). Of the 48 patients in whom C. dubliniensis was identified, 35 (73%) were patients with cystic fibrosis. Also, 8 patients (17%) were considered to have clinical infections and received antifungal therapy: 3 patients with pneumonia, 2 patients with esophagitis, 1 patient with peritonitis, 1 patient with catheter-related bloodstream infection, and 1 patient with disseminated candidiasis. The remaining 40 patients (83%) were considered colonized. Conclusions: We report a descriptive series over 18 months of clinical isolates with C. dubliniensis recovery at a pediatric institution. Most isolates were identified as colonizing strains in patients with cystic fibrosis. C. dubliniensis was a rare cause of invasive disease in our institution, with only 8 cases identified.Funding: NoneDisclosures: None

First case of Cryptococcus gattii multilobar pneumonia in Switzerland and associated challenges

Cryptococcosis is a frequent complication in immunosuppressed patients, causing mainly central nervous system and lung infection, and leading to increased mortality risk. We present the first documented case in Switzerland of Cryptococcus gattii pneumonia in a kidney-pancreas transplant patient, with a concomitant Pneumocystis jirovecii infection mimicking an immune reconstitution syndrome. Diagnosis of cryptococcal pneumonia was based on a positive serum cryptococcal antigen and confirmed by Grocott’s methenamine silver and periodic acid-Schiff stains on bronchoalveolar lavage fliud. C. gattii was identified with mass spectrometry and antifungal susceptibility testing by microdilution was performed. After an initial successful treatment with liposomal amphotericin-B, flucytosine and tapering of immunosuppression, the patient clinically deteriorated, developing bilateral diffuse ground-glass opacities with consolidations on chest computed tomography. A diagnosis of probable P. jirovecii pneumonia versus an immune reconstitution syndrome was considered. Because of a high titre of Pneumocystis on polymerase chain-reaction testing of bronchoalveloar lavage fluid and high serum b-D-glucan, a diagnosis of probable P. jirovecii pneumonia was made. This case illustrates the potential complications of a cryptococcal infection in immunosuppressed hosts, despite timely diagnosis and appropriate antifungal therapy.

Therapeutic Treatment with Abdominal Adipose Mesenchymal Cells Does Not Prevent Elastase-Induced Emphysema in Rats.

Emphysema and chronic bronchitis have different pathophysiologies but both are significant components of chronic obstructive lung disease (COPD). The levels of Matrix metalloproteinase (MMP)-9 in the bronchoalveloar lavage fluid (BALF) and in serum indicate the presence of emphysema. Intratracheal administration of elastase has been used to create a rat model of emphysema. Adipose tissue-derived mesenchymal stem cells (MSC) have been postulated to prevent or reverse emphysema, however, this has not been examined in the rat model of elastase-induced emphysema. In this study, 31 Wistar albino rats aged 6-8 weeks and weighing 250-300 g were assessed. On day 1, the animals were treated intratracheally with 0.5 mL saline (control group, n=10), i.e., 0.5 mL saline solution containing 0.1 IU porcine pancreatic elastase (PPE) (Elastase group, n=12) or PPE plus MSC (Elastase-MSC group, n=9) was adminstered per animal. MSCs suspended in serum were injected via the caudal vein on day 21. At least 106 cells were injected. All animals were sacrificed on day 42 and the emphysema index (EI) was calculated, along with measuring the BALF and serum MMP-9 concentrations. Porcine pancreatic elastase induced a significant degree of emphysema in the PPE groups as compared to the control group, which was determined by the EI index (p=0.008). This was not reversed by MSC treatment. The EI remained significantly low in comprison with the controls (p=0.001) and measured no different from the Elastase-treated animals. There was no statistically significant difference between the BALF and serum MMP-9 levels between the control and treatment groups. Our findings suggest that therapeutic treatment with adipose tissue-derived MSC in rats has no effect on emphysema or on MMP9 expression, which is a known marker of emphysema.

Pulmonary toxicity following inhalation exposure to VX in anesthetized rats: Possible roles for compromised immunity and oxidative stress-induced lung injury

The nerve agent VX is one of the most deadly threat agents available in weapons stockpiles for intentional release. While mostly considered a percutaneous toxicant, it can be fatal when aerosolized. The objective of this study was to investigate toxic responses in the lung up to two weeks following a single 10-minute exposure to inhaled VX. Anesthetized rats were exposed singly and only once to VX. The nebulization rate in this system was 0.2–0.3 ml per minute with the delivery of a consistent particle size of 2.1 µm. Following exposure, all rats were removed from the ventilator and allowed to recover in the glovebox for 10–15 minutes. Results showed that inhaled VX altered several respiratory parameters and caused increased lung resistance up to 6 h post-exposure (PE). There was a trending increase in SOD and xanthine oxidoreductase (XOR) activities, both of which are indicative of oxidative stress. Based on increased lung tissue p38 signaling, MAP kinase expression was activated after VX exposure. IL-6 expression was also increased at 6 h post-inhalation for the 31.6 mg/m3 exposed group. Innate survival response mechanisms in rats may be present due to increased lung tissue mRNA AChE expression 6 h after exposure. Immunohistochemistry showed reduced staining for surfactant D and increased expression of iNOS, indicating that the activation of •NO precursor pathways. Bronchoalveloar lavage fluid (BALF) results from 1 h to 2 weeks PE show that inflammatory cells are highly active as evidenced by the increased production of cytokines and chemokines. This is the first study linking VX-induced lung injury to a possible innate survival amplification of AChE and possibly compromised immune function. These results could supplement medical treatment strategies with regard to therapeutic approaches against VX inhalational challenge.

Effectiveness of biomarker-based exclusion of ventilator-acquired pneumonia to reduce antibiotic use (VAPrapid-2): study protocol for a randomised controlled trial.

BackgroundVentilator-acquired pneumonia (VAP) is a common reason for antimicrobial therapy in the intensive care unit (ICU). Biomarker-based diagnostics could improve antimicrobial stewardship through rapid exclusion of VAP. Bronchoalveloar lavage (BAL) fluid biomarkers have previously been shown to allow the exclusion of VAP with high confidence.Methods/DesignThis is a prospective, multi-centre, randomised, controlled trial to determine whether a rapid biomarker-based exclusion of VAP results in fewer antibiotics and improved antimicrobial management. Patients with clinically suspected VAP undergo BAL, and VAP is confirmed by growth of a potential pathogen at > 104 colony-forming units per millilitre (CFU/ml). Patients are randomised 1:1, to either a ‘biomarker-guided recommendation on antibiotics’ in which BAL fluid is tested for IL-1β and IL-8 in addition to routine microbiology testing, or to ‘routine use of antibiotics’ in which BAL undergoes routine microbiology testing only. Clinical teams are blinded to intervention until 6 hours after randomisation, when biomarker results are reported to the clinician. The primary outcome is a change in the frequency distribution of antibiotic-free days (AFD) in the 7 days following BAL. Secondary outcome measures include antibiotic use at 14 and 28 days; ventilator-free days; 28-day mortality and ICU mortality; sequential organ failure assessment (SOFA) at days 3, 7 and 14; duration of stay in critical care and the hospital; antibiotic-associated infections; and antibiotic-resistant pathogen cultures up to hospital discharge, death or 56 days. A healthcare-resource-utilisation analysis will be calculated from the duration of critical care and hospital stay. In addition, safety data will be collected with respect to performing BAL. A sample size of 210 will be required to detect a clinically significant shift in the distribution of AFD towards more patients having fewer antibiotics and therefore more AFD.DiscussionThis trial will test whether a rapid biomarker-based exclusion of VAP results in rapid discontinuation of antibiotics and therefore improves antibiotic management in patients with suspected VAP.Trial registrationISRCTN65937227. Registered on 22 August 2013. ClinicalTrials.gov, NCT01972425. Registered on 24 October 2013.Electronic supplementary materialThe online version of this article (doi:10.1186/s13063-016-1442-x) contains supplementary material, which is available to authorized users.

Vitamin D deficiency and bacterial load in a murine model of sepsis-induced lung injury

BackgroundWe have previously shown that patients with acute lung injury are severely vitamin D deficient. Several studies have reported a high prevalence of vitamin D deficiency in critically ill patients with sepsis, associated with increased morbidity and mortality, but whether this association is cause or effect is unknown. Bacteraemic sepsis is more common in the winter months when serum vitamin D concentrations are lowest. The purpose of this study was to investigate the local and systemic effects of vitamin D deficiency in a murine model of sepsis-induced lung injury where we can predictably time the initiating insult. MethodsWe fed eight wild-type C57BL/6 mice a diet completely devoid of vitamin D for 6 weeks to induce severe vitamin D deficiency (serum concentration 9 nmol/L) and compared them with seven mice fed a vitamin D sufficient diet (42 nmol/L). We used caecal ligation and puncture (CLP) to establish sepsis. Animals were culled 16 h after CLP, and blood, peritoneal lavage fluid (PLF), and bronchoalveloar lavage fluid (BALF) were collected. Cell infiltrates were assessed by flow cytometry. Fluid protein concentrations were measured, and tissue protein permeability index was calculated as the ratio between fluid and serum protein. Bacterial load was evaluated as colony-forming units (CFU) per ml after 24 h incubation on appropriate media. Statistical analysis was done with Minitab 16 statistical software. FindingsVitamin D deficient mice had significantly increased bacterial load compared with dietary sufficient mice in BALF, blood, and PLF (BALF median 2·51 × 103 CFU per mL [IQR 0·65 × 103–1097 ×103] vs 0·51 ×103 [0·0–1·15 × 103], p=0·038; blood 66·1 × 103 [1·93 × 103–175 × 103] vs 0·56 × 103 [0·0–5·56 × 103], p=0·042; and PLF 336 × 103 [52 × 103–115 × 105] vs 8·22 × 103 [1·54 ×103–20·6 × 103], p=0·005). BALF protein permeability index was higher in deficient mice than in sufficient mice (median 3·3 [IQR 2·61–4·64] vs 1·8 [1·22–2·09], p=0·0003) but there was no difference in cell numbers recruited to the lung. PLF protein permeability index was also higher in the deficient group than in sufficient mice (46·8 [28·1–59·3] vs 27·2 [14·2–37·3], p=0·05), with an associated increase in neutrophils recruited to the peritoneum (p=0·04). InterpretationVitamin D deficiency significantly increased the bacterial load systemically, locally, and within the lung in a murine model of peritonitis. This increase was associated with a rise in tissue permeability locally and within the lung. These data support pre-existing vitamin D deficiency as a determinant of the severity of bacteraemic sepsis and might account for some of the seasonal variations observed in the incidence of sepsis. FundingUK Medical Research Council.

S98 Vitamin D deficiency increases bacterial load in a murine model of sepsis-induced lung injury

IntroductionWe have previously shown that patients with Acute Lung Injury (ALI) are severely vitamin D deficient. Several studies have reported a high prevalence of vitamin D deficiency in critically ill...

Mepacrine alleviates airway hyperresponsiveness and airway inflammation in a mouse model of asthma

Asthma is a multifactorial respiratory disease. Though its incidence is increasing rapidly all over the world, the available therapeutic strategies are neither sufficient nor safe for long term use. Mepacrine, a known antimalarial drug, has been shown to possess antioxidant, anti-inflammatory, platelet anti-aggregant, and PLA 2 inhibitory activities. However, its possible use in asthma has not been studied yet. The objective of this study was to investigate the anti-asthmatic property of mepacrine using a mouse model of asthma. To accomplish this, male BALB/c mice were sensitized and challenged with ovalbumin and treated with increasing concentrations of mepacrine. Airway hyperresponsiveness (AHR) to methacholine was assessed using unrestrained whole body plethysmography. Mepacrine (1 mg/kg) has shown marked attenuation of AHR. Cytokines such as IL-4, IL-5, IL-13 and IFN-γ and OVA-specific IgE levels were measured in BAL (bronchoalveloar lavage) fluid and sera, respectively. Mepacrine effectively reduced the rise in IL-4, IL-5, IL-13, and OVA-specific IgE and restored IFN-γ levels. Mepacrine also significantly prevented the increase of sPLA 2 (secretory phospholipase A 2) activity in BAL fluid supernatant and Cys-LT (cysteinyl leukotrienes) in lung tissue homogenates of asthmatic mice. In addition, mepacrine treatment reduced BAL fluid eosinophilia and signs of allergic airway inflammation such as perivascular and peribronchial distribution of inflammatory cells. These findings indicate that mepacrine reduces the asthmatic features in ovalbumin induced asthma by acting on PLA 2-Cys-LT axis. Thus, it could be useful for the development of better asthma therapy.

RU486 blocks the anti-inflammatory effects of exercise in a murine model of allergen-induced pulmonary inflammation

In an ovalbumin (OVA)-driven murine model of allergic pulmonary inflammation, we have shown previously that moderate-intensity aerobic exercise training attenuates inflammatory responses, disease progression, and NF-κB activation within the sensitized lung. Glucocorticoids (GCs), potent anti-inflammatory agents, have been shown to alter transcriptional events that are important in asthmatic pathogenesis, such as NF-κB activation. Notably, exercise training can alter the production and signaling capacity of endogenous GCs. Because GCs exert their anti-inflammatory effects through binding to intracellular glucocorticoid receptors (GRs), we examined the role of the GR in facilitating the anti-inflammatory effects of exercise. Results show that, in exercised OVA-sensitized mice, treatment with the GR antagonist RU486 blocked the exercise-induced reductions in cellular infiltration of the airways ( p < .05), KC and soluble VCAM-1 protein levels in the bronchoalveloar lavage fluid ( p < .05), and NF-κB translocation and DNA binding within the lung to levels similar to those observed in sedentary OVA-sensitized mice. Importantly, RU486 treatment also blocked exercise-induced increases in GR nuclear translocation to the levels seen in sensitized control mice. Together, these results suggest that GR nuclear translocation and NF-κB activation play roles in mediating the anti-inflammatory effects of exercise in allergen-mediated lung pathology.