Lung Lavage Samples Research Articles

Probiotics in milk replacer affect the microbiome of the lung in neonatal dairy calves.

Probiotics have been investigated for their many health benefits and impact on the microbiota of the gut. Recent data have also supported a gut-lung axis regarding the bacterial populations (microbiomes) of the two locations; however, little research has been performed to determine the effects of oral probiotics on the microbiome of the bovine respiratory tract. We hypothesized that probiotic treatment would result in changes in the lung microbiome as measured in lung lavage fluid. Our overall goal was to characterize bacterial populations in the lungs of calves fed probiotics in milk replacer and dry rations from birth to weaning. A group of 20 dairy calves was split into two treatment groups: probiotic (TRT; N = 10, milk replacer +5 g/d probiotics; Bovamine Dairy, Chr. Hansen, Inc., Milwaukee, WI) and control (CON; N = 10, milk replacer only). On day 0, birth weight was obtained, and calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group and then included in their dry ration. Lung lavages were performed on day 52 on five random calves selected from each treatment group. DNA was extracted from lavage fluid, and 16S ribosomal RNA (rRNA) gene hypervariable regions 1-3 were amplified by PCR and sequenced using next-generation sequencing (Illumina MiSeq) for the identification of the bacterial taxa present. Taxa were classified into both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). Overall, the evaluation of these samples revealed that the bacterial genera identified in the lung lavage samples of probiotic-fed calves as compared to the control calves were significantly different based on the OTU dataset (p < 0.05) and approached significance for the ASV dataset (p < 0.06). Additionally, when comparing the diversity of taxa in lung lavage samples to nasal and tonsil samples, taxa diversity of lung samples was significantly lower (p < 0.05). In conclusion, analysis of the respiratory microbiome in lung lavage samples after probiotic treatment provides insight into the distribution of bacterial populations in response to oral probiotics and demonstrates that oral probiotics affect more than the gut microbiome.

Effects of a water-soluble formulation of tylvalosin on disease caused by porcine reproductive and respiratory syndrome virus alone in sows or in combination with Mycoplasma hyopneumoniae in piglets

BackgroundThe effect of a water-soluble formulation of tylvalosin (Aivlosin® 625 mg/g granules) on disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (Mhyop) was investigated in two animal studies. In a PRRSV challenge model in pregnant sows (n = 18), six sows received water medicated at target dose of 5 mg tylvalosin/kg body weight/day from 3 days prior to challenge until the end of gestation. Six sows were left untreated, with a third group remaining untreated and unchallenged. Sows were challenged with PRRSV-2 at approximately 85 days of gestation. Cytokines, viremia, viral shedding, sow reproductive parameters and piglet performance to weaning were evaluated. In a dual infection study (n = 16), piglets were challenged with Mhyop on days 0, 1 and 2, and with PRRSV-1 on day 14 and euthanized on day 24. From day 10 to 20, eight piglets received water medicated at target dose of 20 mg tylvalosin/kg body weight/day and eight piglets were left untreated. Cytokines, viremia, bacteriology and lung lesions were evaluated.ResultsIn the PRRSV challenge study in pregnant sows, tylvalosin significantly reduced the levels of serum IL-8 (P < 0.001), IL-12 (P = 0.032), TNFα (P < 0.001) and GM-CSF (P = 0.001). IL-8 (P = 0.100) tended to be lower in uterus of tylvalosin sows. All piglets from tylvalosin sows surviving to weaning were PRRSV negative in faecal swabs at weaning compared to 33.3% PRRSV positive piglets from untreated sows (P = 0.08).In the dual challenge study in piglet, tylvalosin reduced serum IL1β, IL-4, IL-6, IL-8, IL-10, IL-12, IL-1α, IL-13, IL-17A, IL-18, GM-CSF, TGFβ1, TNFα, CCL3L1, MIG, PEPCAM-1 (P < 0.001) and increased serum IFNα, IL-1ra and MIP-1b (P < 0.001). In the lungs, tylvalosin reduced IL-8, IL-10 and IL-12 compared to untreated pigs (P < 0.001) and tended to reduce TNFα (P = 0.082). Lung lavage samples from all tylvalosin treated piglets were negative for Mhyop (0 cfu/mL) compared to the untreated piglets which had mean Mhyop counts of 2.68 × 104 cfu/mL (P = 0.023).ConclusionOverall, tylvalosin reduced both local and systemic proinflammatory cytokines after challenge with respiratory pathogens in sows and in piglets. Tylvalosin was effective in reducing Mhyop recovery from the lungs and may reduce virus shedding in piglets following transplacental PRRSV infection in sows.

Sophoraflavanone G from Sophora flavescens Ameliorates Allergic Airway Inflammation by Suppressing Th2 Response and Oxidative Stress in a Murine Asthma Model.

Sophoraflavanone G (SG), isolated from Sophora flavescens, has anti-inflammatory and anti-tumor bioactive properties. We previously showed that SG promotes apoptosis in human breast cancer cells and leukemia cells and reduces the inflammatory response in lipopolysaccharide-stimulated macrophages. We investigated whether SG attenuates airway hyper-responsiveness (AHR) and airway inflammation in asthmatic mice. We also assessed its effects on the anti-inflammatory response in human tracheal epithelial cells. Female BALB/c mice were sensitized with ovalbumin, and asthmatic mice were treated with SG by intraperitoneal injection. We also exposed human bronchial epithelial BEAS-2B cells to different concentrations of SG to evaluate its effects on inflammatory cytokine levels. SG treatment significantly reduced AHR, eosinophil infiltration, goblet cell hyperplasia, and airway inflammation in the lungs of asthmatic mice. In the lungs of ovalbumin-sensitized mice, SG significantly promoted superoxide dismutase and glutathione expression and attenuated malondialdehyde levels. SG also suppressed levels of Th2 cytokines and chemokines in lung and bronchoalveolar lavage samples. In addition, we confirmed that SG decreased pro-inflammatory cytokine, chemokine, and eotaxin expression in inflammatory BEAS-2B cells. Taken together, our data demonstrate that SG shows potential as an immunomodulator that can improve asthma symptoms by decreasing airway-inflammation-related oxidative stress.

Acute respiratory distress syndrome phenotypes with distinct clinical outcomes in PHARLAP trial cohort

Background: The Permissive Hypercapnia, Alveolar Recruitment and Low Airway Pressure (PHARLAP) randomised controlled trial compared an open lung ventilation strategy with control ventilation, and found that open lung ventilation did not reduce the number of ventilatorfree days (VFDs) or mortality in patients with moderate-to-severe acute respiratory distress syndrome (ARDS). Parsimonious models can identify distinct phenotypes of ARDS (hypo-inflammatory and hyperinflammatory) which are associated with different outcomes and treatment responses. Objective: To test the hypothesis that a parsimonious model would identify patients with distinctly different clinical outcomes in the PHARLAP study. Design, setting and participants: Blood and lung lavage samples were collected in a subset of PHARLAP patients who were recruited in Australian and New Zealand centres. A previously validated parsimonious model (interleukin-8, soluble tumour necrosis factor receptor-1 and bicarbonate) was used to classify patients with blood samples into hypo-inflammatory and hyperinflammatory groups. Generalised linear modelling was used to examine the interaction between inflammatory phenotype and treatment group (intervention or control). Main outcome measure: The primary outcome was number of VFDs at Day 28. Results: Data for the parsimonious model were available for 56 of 115 patients (49%). Within this subset, 38 patients (68%) and 18 patients (32%) were classified as having hypo-inflammatory and hyperinflammatory phenotypes, respectively. Patients with the hypo- inflammatory phenotype had more VFDs at Day 28 when compared with those with the hyperinflammatory phenotype (median [IQR], 19.5[11-24] versus 8[0-21];P= 0.03). Patients with the hyperinflammatory phenotype had numerically fewer VFDs when managed with an open lung strategy than when managed with control "protective" ventilation (median [IQR], 0 [0-19] versus 16 [8-22]). Conclusion: In the PHARLAP trial, ARDS patients classified as having a hyperinflammatory phenotype, with a parsimonious three-variable model, had fewer VFDs at Day 28 compared with patients classified as having a hypo-inflammatory phenotype. Future clinical studies of ventilatory strategies should consider incorporating distinct ARDS phenotypes into their trial design.

PSI-4 Changes in serum cortisol concentrations and cytokine profiles of growing pigs subjected to low-intensity heat stress

Abstract This study was to determine the impact of low-intensity heat stress on inflammatory biomarkers in serum, ileum, jejunum and lung lavage samples. Pigs (n = 64; 24.6 ± 3.7 kg BW) were housed four per pen in thermoneutral conditions (CON; 23.9°C ± 2.5°C) or constant high ambient temperatures (HS; 28.7°C ± 1.3°C) for 16 d, followed by thermoneutral conditions (24.4°C ± 1.8°C) for both groups through d 21, and subsequent heat stress (28.0°C ± 3.0°C) for both groups through d 30. Respiration rate (P &amp;lt; 0.01) was greater in HS than CON pigs from d 2 through d 15 (mean: 82.54 breaths/min vs. 59.26 breaths/min). HS pigs had greater cortisol concentration (40.1 ng/mL vs. 27.3 ng/mL) than CON on d 2, but lower concentrations on d 22 (32.9 ng/mL vs. 48.9 ng/mL) and d 25 (25.4 ng/mL vs. 42.0 ng/mL; treatment x day: P &amp;lt; 0.001). HS pigs tended to have greater serum IL1β (190.1 vs. 143.4 pg/mL; P = 0.06) and greater serum IL12 concentrations (914.9 vs. 785.4 pg/mL; P &amp;lt; 0.01) than CON pigs. HS pigs tended to have greater ileal mucosa IL6 concentrations than CON pigs (50.4 vs. 30.9 pg/mL; P = 0.08). Jejunal mucosa IL1α concentrations tended (P = 0.10) to be greater for CON than HS pigs (13.5 vs. 9.0 pg/mL) and CON pigs had greater jejunal IL12 concentrations (18.1 vs. 11.8 pg/mL) than HS pigs (P = 0.02). Bronchioalveolar lavage from HS pigs tended (P = 0.10) to have a greater granulocyte-macrophage colony-stimulating factor concentration (6.9 vs. 5.3 pg/mL) than CON pigs and had greater IL4 (7.7 vs. 4.2 pg/mL) and IL12 concentrations (34.8 vs. 24.5 pg/mL) than CON pigs (P = 0.01). Increased inflammatory activity and endocrine stress response occurred during low-intensity heat stress, indicating it may be more detrimental than previously accepted.

Active immunoprophylaxis with a synthetic DNA-encoded monoclonal anti-respiratory syncytial virus scFv-Fc fusion protein confers protection against infection and durable activity

ABSTRACT Respiratory Syncytial virus (RSV) is a major threat to many vulnerable populations. There are currently no approved vaccines, and RSV remains a high unmet global medical need. Here we describe the employment of a novel synthetic DNA-encoded antibody technology platform to develop and deliver an engineered human DNA-encoded monoclonal antibody (dMAbTM) targeting the fusion protein (F) of RSV as a new approach to prevention or therapy of at risk populations. In in vivo models, a single administration of synthetic DNA-encoding the single-chain fragment variable-constant fragment (scFv-Fc) RSV-F dMAb resulted in robust and durable circulating levels of a functional antibody systemically and in mucosal tissue. In cotton rats, which are the gold-standard animals to model RSV infection, we observed sustained scFv-Fc RSV-F dMAb in the sera and lung-lavage samples, demonstrating the potential for both long-lasting immunity to RSV and effective biodistribution. The scFv-Fc RSV-F dMAb harbored in the sera exhibited RSV antigen-specific binding and potent viral neutralizing activity. Importantly, in vivo delivery of synthetic DNA-encoding, the scFv-Fc RSV-F dMAb protected animals against viral challenge. Our findings support the significance of dMAbs as a potential platform technology for durable protection against RSV disease.

From Hendra to Wuhan: what has been learned in responding to emerging zoonotic viruses

From Hendra to Wuhan: what has been learned in responding to emerging zoonotic viruses

Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice.

Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for understanding how innate immune cells respond to infection but is technically challenging due to the small number of bacteria that initially infect the lung. Previous studies either evaluated Mtb-infected cells at later stages of infection when the number of bacteria in the lung is much higher or used in vitro model systems to assess the response of myeloid cells to Mtb. Here, we describe a method that uses fluorescent bacteria, a high-dose aerosol infection model, and flow cytometry to track Mtb-infected cells in the lung immediately following aerosol infection and fluorescence-activated cell sorting (FACS) to isolate naïve, bystander, and Mtb-infected cells for downstream applications, including RNA-sequencing. This protocol provides the ability to monitor Mtb-infection and cell-specific responses within the context of the lung environment, which is known to modulate the function of both resident and recruited populations. Using this protocol, we discovered that alveolar macrophages respond to Mtb infection in vivo by up-regulating a cell protective transcriptional response that is regulated by the transcription factor Nrf2 and is detrimental to early control of the bacteria.

Mass spectrometry profiling of oxylipins, endocannabinoids, and N-acylethanolamines in human lung lavage fluids reveals responsiveness of prostaglandin E2 and associated lipid metabolites to biodiesel exhaust exposure

The adverse effects of petrodiesel exhaust exposure on the cardiovascular and respiratory systems are well recognized. While biofuels such as rapeseed methyl ester (RME) biodiesel may have ecological advantages, the exhaust generated may cause adverse health effects. In the current study, we investigated the responses of bioactive lipid mediators in human airways after biodiesel exhaust exposure using lipidomic profiling methods. Lipid mediator levels in lung lavage were assessed following 1-h biodiesel exhaust (average particulate matter concentration, 159 μg/m3) or filtered air exposure in 15 healthy individuals in a double-blinded, randomized, controlled, crossover study design. Bronchoscopy was performed 6 h post exposure and lung lavage fluids, i.e., bronchial wash (BW) and bronchoalveolar lavage (BAL), were sequentially collected. Mass spectrometry methods were used to detect a wide array of oxylipins (including eicosanoids), endocannabinoids, N-acylethanolamines, and related lipid metabolites in the collected BW and BAL samples. Six lipids in the human lung lavage samples were altered following biodiesel exhaust exposure, three from BAL samples and three from BW samples. Of these, elevated levels of PGE2, 12,13-DiHOME, and 13-HODE, all of which were found in BAL samples, reached Bonferroni-corrected significance. This is the first study in humans reporting responses of bioactive lipids following biodiesel exhaust exposure and the most pronounced responses were seen in the more peripheral and alveolar lung compartments, reflected by BAL collection. Since the responsiveness and diagnostic value of a subset of the studied lipid metabolites were established in lavage fluids, we conclude that our mass spectrometry profiling method is useful to assess effects of human exposure to vehicle exhaust.

Angiostrongylus chabaudi in felids: New findings and a review of the literature

Cardiopulmonary infections by Angiostrongylus chabaudi affect domestic and wild felids but, due to limited information on the biology of this nematode, its pathogenicity remains unclear. This article describes the histopathological alterations associated with Angiostrongylus infection in a wildcat from Bulgaria, and reviews current literature on this feline angiostrongylid. Nematodes were isolated from lung lavage and faecal samples of a road killed wildcat in Southern Bulgaria. The morphological identification of parasite larvae as A. chabaudi was confirmed by molecular analysis of part of the 18S ribosomal RNA gene. Upon histopathological examination, severe granulomatous pneumonia, ranging from multifocal to coalescing, and pulmonary vascular lesions were observed. Extensive alveolar collapse, alveolar emphysematous changes, parenchymal haemorrhages and small artery wall hyperplasia were observed in the parenchyma adjacent to the granulomas. Histopathological examination revealed the presence of cross-sections of adult female parasites within the lumen of the pulmonary artery branches, the intima altered markedly by subendothelial proliferation and oedematous changes. This study compliments current knowledge of the pathogenesis of feline angiostrongylosis by A. chabaudi in wildcats, as well as of the distribution of this little-known parasite.

Humoral immunity in phenotypes of chronic lung allograft dysfunction: A broncho-alveolar lavage fluid analysis

BackgroundRecently, antibody mediated rejection (AMR) has been associated with a higher incidence of chronic lung allograft dysfunction (CLAD) and mortality after lung transplantation (LTx). We investigated markers related to AMR and matrix remodeling in CLAD, with special attention for its two phenotypes being bronchiolitis obliterans syndrome (BOS) and restrictive CLAD (rCLAD). MethodsImmunoglobulins (IgA, IgE, IgG1–IgG4, total IgG and IgM) and complement (C4d and C1q) were quantified in lung lavage samples at the moment of BOS (n=15) or RAS (n=16) diagnosis; and were compared to stable transplant patients who served as control (n=14). Also, airway remodeling and metalloproteinases (MMPs) were investigated via zymography and gelatin degradation. The presence of DSA was additionally assessed in blood. ResultsTotal IgG, IgG1-IgG4 and IgM were increased in rCLAD versus control (p<0.001) and BOS patients (p<0.01). IgA and IgE were increased in rCLAD compared to control (respectively p<0.05 and p<0.01), but not to BOS. Total IgG and IgE were increased in BOS versus control (respectively p<0.01 and p<0.05). Complement proteins were exclusively present in rCLAD and correlated positively with immunoglobulins. Additionally, in blood, DSA were more present in rCLAD (p=0.041). MMP-9 levels increased in RAS and BOS versus control (p<0.001) and MMP-9 induced gelatin degradation was only increased in BOS compared to control (p<0.01). ConclusionWe demonstrated increased levels of immunoglobulins and complement proteins dominantly present in rCLAD. This leads to the belief that antibodies and AMR might play a more important role in rCLAD compared to BOS. Therefore, anti B-cell therapy could offer beneficial therapeutic effects in patients diagnosed with rCLAD, which needs further research.

Androstenetriol enhances lung macrophage host defense against bacterial pneumonia in mice

The active metabolite of dihydroepiandrosterone, 17beta‐androstenetriol (AET), has potent immunomodulatory effects but its potential to improve host defense against the common problem of bacterial pneumonia has not been studied. Mice (C57BL/6, male) were pre‐treated with AET (0.5 mg, s.c.) or vehicle before infection with Strep. pneumoniae (serotype 3, 100K CFU, i.n.). Evaluation showed 8‐fold improved bacterial clearance at 24 h (p &lt;.001, n&gt;20/group) accompanied by decreased inflammation (5‐fold fewer neutrophils in lung lavage samples at 24 h, p &lt; .001). Secondary bacterial pneumonia after influenza remains a major clinical problem. AET treatment of mice on day 7 after non‐lethal influenza infection produced resulted in 15‐fold improved bacterial clearance (p &lt;.001, n &gt;5/group). Survival analysis showed 37% survival in AET‐treated vs. 0% survival in vehicle‐treated mice in this model of secondary pneumococcal pneumonia. In vitro, AET improved macrophage killing of bacteria in vitro (e.g. % bacteria alive after ingestion, vehicle vs AET‐treated: 54 ± 8 vs. 36 ± 7, n&gt;5). To explore mechanisms, we analyzed the role of MARCO, a macrophage scavenger receptor with important functions in host defense against bacteria. Western blot analysis showed that in vitro AET increased MARCO expression in both murine and human macrophages. Comparison of bacterial clearance after vehicle or AET pre‐treatment showed marked improvement in wild‐type mice, but no benefit in MARCO −/− mice. We conclude that AET enhances innate resistance to primary and secondary pneumococcal pneumonia, in part through increased expression of the scavenger receptor MARCO, and is a potential immunomodulator for improving lung host defense against bacterial pneumonia.Support or Funding InformationNIH ES00002 ES11008

Oxidation of the alarmin IL-33 regulates ST2-dependent inflammation.

In response to infections and irritants, the respiratory epithelium releases the alarmin interleukin (IL)-33 to elicit a rapid immune response. However, little is known about the regulation of IL-33 following its release. Here we report that the biological activity of IL-33 at its receptor ST2 is rapidly terminated in the extracellular environment by the formation of two disulphide bridges, resulting in an extensive conformational change that disrupts the ST2 binding site. Both reduced (active) and disulphide bonded (inactive) forms of IL-33 can be detected in lung lavage samples from mice challenged with Alternaria extract and in sputum from patients with moderate–severe asthma. We propose that this mechanism for the rapid inactivation of secreted IL-33 constitutes a ‘molecular clock' that limits the range and duration of ST2-dependent immunological responses to airway stimuli. Other IL-1 family members are also susceptible to cysteine oxidation changes that could regulate their activity and systemic exposure through a similar mechanism.

Synthesis, characterization, and bioactivity of carboxylic acid-functionalized titanium dioxide nanobelts.

BackgroundSurface modification strategies to reduce engineered nanomaterial (ENM) bioactivity have been used successfully in carbon nanotubes. This study examined the toxicity and inflammatory potential for two surface modifications (humic acid and carboxylation) on titanium nanobelts (TNB).MethodsThe in vitro exposure models include C57BL/6 alveolar macrophages (AM) and transformed human THP-1 cells exposed to TNB for 24 hrs in culture. Cell death and NLRP3 inflammasome activation (IL-1β release) were monitored. Short term (4 and 24 hr) in vivo studies in C57BL/6, BALB/c and IL-1R null mice evaluated inflammation and cytokine release, and cytokine release from ex vivo cultured AM.ResultsBoth in vitro cell models suggest that the humic acid modification does not significantly affect TNB bioactivity, while carboxylation reduced both toxicity and NLRP3 inflammasome activation. In addition, short term in vivo exposures in both C57BL/6 and IL-1R null mouse strains demonstrated decreased markers of inflammation, supporting the in vitro finding that carboxylation is effective in reducing bioactivity. TNB instillations in IL-1R null mice demonstrated the critical role of IL-1β in initiation of TNB-induced lung inflammation. Neutrophils were completely absent in the lungs of IL-1R null mice instilled with TNB for 24 hrs. However, the cytokine content of the IL-1R null mice lung lavage samples indicated that other inflammatory agents, IL-6 and TNF-α were constitutively elevated indicating a potential compensatory inflammatory mechanism in the absence of IL-1 receptors.ConclusionsTaken together, the data suggests that carboxylation, but not humic acid modification of TNB reduces, but does not totally eliminate bioactivity of TNB, which is consistent with previous studies of other long aspect ratio nanomaterials such as carbon nanotubes.

5-Aminosalicylic Acid attenuates allergen-induced airway inflammation and oxidative stress in asthma

Pro-inflammatory cytokines regulate the magnitude of allergic reactions during asthma. Tumor necrosis factor – alpha (TNF-α), interleukin-6 (IL-6) and interleukin-13 (IL-13) play a crucial role in aggravating the inflammatory conditions during allergic asthma. In addition, oxidative stress contributes to the pathogenesis of asthma by altering the physiological condition resulting in the development of status asthmaticus. Anti-inflammatory corticosteroids are being widely used for treating allergic asthma. In the present study 5-aminosalicylic acid (5-ASA), a salicylic acid derivative, was evaluated, in vivo for its potential to suppress TNF-α, IL-6 and IL-13 using ovalbumin (OVA) induced allergic asthma in Balb/C mice. Oral administration of 65, 130 and 195 mg/kg 5-ASA significantly reduced the OVA induced total and differential leucocyte count, TNF-α, IL-6, IL-13, nitrite, nitrate, MDA, MPO and TPL levels in the lung lavage samples. Collectively, these findings suggest that 5-ASA is a potent immunomodulator and suppresses key Th2 cytokines production and oxidative stress in OVA-induced asthma.

Sulforaphane improves MARCO expression, bacterial clearance and survival in post‐influenza bacterial pneumonia (145.1)

Mechanisms that increase susceptibility for post‐influenza bacterial pneumonia include down‐regulation of the alveolar macrophage (AM) scavenger receptor MARCO by interferon‐γ (IFN‐γ), leading to diminished bacterial clearance. Since MARCO expression can be increased by NRF2 signaling, we tested whether the NRF2‐inducer sulforaphane (SFN) can up‐regulate MARCO expression and improve bacterial clearance after influenza. Human AM‐like monocyte‐derived macrophages (HMDMs) were treated with IFN‐γ (10‐50 IU/ml, 24h) to simulate influenza infection, followed by sulforaphane (SFN, 10µM) for 24h before quantitation of MARCO expression or challenge with green fluorescence protein (GFP)‐expressing S. aureus. Scanning cytometry was used to quantitate MARCO surface labeling and uptake of bacteria. C57/BL6 mice were inoculated i.n. on day 0 with 3500PFU of PR8 influenza virus, and on day 7 with 500 CFU of S. pneumoniae serotype 3. Mice were injected with SFN 25 or 50 mg/kg, or PBS daily from day 6 to 9 after influenza infection. Bacterial clearance was measured on day 1 after S. pneumoniae infection using lung lavage samples. IFN‐γ down‐regulated MARCO expression (p&lt;0.001) of HMDMs and impaired phagocytosis of S. aureus (p&lt;0.001, n=4). SFN added after IFN‐γ significantly improved MARCO expression (p=0.003) and S. aureus phagocytosis (p=0.05), an effect that was inhibited by scavenger receptor blockers poly(I) (p=0.001) or MARCO blocking peptide (P&lt;0.001). In vivo, sulforaphane‐treated mice showed improved bacterial clearance (e.g. 84%, 95% reduction in SFN‐treated mice in 2 trials) and a significantly higher survival rate compared to PBS‐treated mice (75.0% vs. 12.5%, n = 8/grp, p=0.022). Therapeutic targeting of the NRF2 pathway may be beneficial to reduce risk or severity of secondary bacterial pneumonia after influenza.Grant Funding Source: Supported by ES00002

Detection of Toxoplasma gondii from Clinical Specimens of Patients Receiving Renal Transplant Using ELISA and PCR

BackgroundToxoplasma gondii is an opportunistic parasitic organism causing infection in many mammals, including immunosuppressed patients. Toxoplasmosis as an opportunistic infection is highly prevalent among patients receiving a kidney transplant.ObjectivesThe purpose of this study was to identify and determine the prevalence of Toxoplasma gondii in clinical samples collected from patients receiving renal transplants.Patients and MethodsA total of 50 blood samples and 40 lung lavage samples from transplanted patients admitted to the infectious wards and the patients undergoing bronchoscopy were collected. The B1 Gene of Toxoplasma gondii was amplified using PCR of the blood and bronoalveolar lavage BAL samples, and IgG and IgM antibodies against Toxoplasma were detected in serum samples using ELISA.ResultsOur results indicated that anti-toxoplasma specific IgG and IgM antibodies were prevalent among transplant recipients with values of 54% and 4% respectively. PCR was performed to detect Toxoplasma gondii in 3 blood and lavage samples (3.3%) with 100% sensitivity and 97.9% specificity.ConclusionsToxoplasma gondii pulmonary infection is measured along with brain toxoplasmosis in patients receiving a kidney transplant. After serological methods, PCR is the second useful method for Toxoplasma gondii screening. Proper prophylaxis before and after receiving a kidney transplant together with Toxoplasma gondii screening of donor and transplant is recommended.

Profibrotic Activities for Matrix Metalloproteinase-8 during Bleomycin-Mediated Lung Injury

Matrix metalloproteinase-8 (MMP-8) is a potent interstitial collagenase thought to be expressed mainly by polymorphonuclear neutrophils. To determine whether MMP-8 regulates lung inflammatory or fibrotic responses to bleomycin, we delivered bleomycin by the intratracheal route to wild-type (WT) versus Mmp-8(-/-) mice and quantified MMP-8 expression, and inflammation and fibrosis in the lung samples. Mmp-8 steady state mRNA and protein levels increase in whole lung and bronchoalveolar lavage samples when WT mice are treated with bleomycin. Activated murine lung fibroblasts express Mmp-8 in vitro. MMP-8 expression is increased in leukocytes in the lungs of patients with idiopathic pulmonary fibrosis compared with control lung samples. Compared with bleomycin-treated WT mice, bleomycin-treated Mmp-8(-/-) mice have greater lung inflammation, but reduced lung fibrosis. Whereas bleomycin-treated Mmp-8(-/-) and WT mice have similar lung levels of several pro- and antifibrotic mediators (TGF-β, IL-13, JE, and IFN-γ), Mmp-8(-/-) mice have higher lung levels of IFN-γ-inducible protein-10 (IP-10) and MIP-1α. Genetically deleting either Ip-10 or Mip-1α in Mmp-8(-/-) mice abrogates their lung inflammatory response to bleomycin, but reconstitutes their lung fibrotic response to bleomycin. Studies of bleomycin-treated Mmp-8 bone marrow chimeric mice show that both leukocytes and lung parenchymal cells are sources of profibrotic MMP-8 during bleomycin-mediated lung fibrosis. Thus, during bleomycin-mediated lung injury, MMP-8 dampens the lung acute inflammatory response, but promotes lung fibrosis by reducing lung levels of IP-10 and MIP-1α. These data indicate therapeutic strategies to reduce lung levels of MMP-8 may limit fibroproliferative responses to injury in the human lung.

The harmful effect of prolonged high-dose methylprednisolone in acute lung injury

Although many literatures have shown that prolonged high-dose administration of corticosteroids is hazardous and not indicated to therapy acute lung injury (ALI), there is little information on the harmful effect of prolonged high-dose corticosteroids in acute lung injury. In this study, we aimed to investigate the effect of prolonged high-dose methylprednisolone (MPL) on ALI and improve knowledge regarding the appropriate use of corticosteroids in ALI. The different doses of MPL (3, 30, 180mg·kg−1) were given via tail vein injection 1h after the first time LPS administration and were daily administrated for 14days. Lung tissues and lavage samples were isolated for biochemical determinations and histological measurements at 12h, 7days and 14days after LPS administration. Single administration of 180mg·kg−1 MPL decreased the lung injury score, wet-to-dry ratio, the total cell numbers and level of procollagen type III in BALF at 12h after LPS challenge. However, prolonged therapy with 180mg·kg −1 MPL for 7days and 14days decreased the number of AMs in BALF and increased the above-mentioned indexes. These results suggested that the prolonged high-dose MPL has harmful effects to treat LPS-induced ALI in rats.

Fas/FasL pathway-mediated alveolar macrophage apoptosis involved in human silicosis

In vitro and in vivo studies have demonstrated that lung cell apoptosis is associated with lung fibrosis; however the relationship between apoptosis of alveolar macrophages (AMs) and human silicosis has not been addressed. In the present study, AM apoptosis was determined in whole-lung lavage fluid from 48 male silicosis patients, 13 male observers, and 13 male healthy volunteers. The relationships between apoptosis index (AI) and silica exposure history, soluble Fas (sFas)/membrane-bound Fas (mFas), and caspase-3/caspase-8 were analyzed. AI, mFas, and caspase-3 were significantly higher in lung lavage fluids from silicosis patients than those of observers or healthy volunteers, but the level of sFas demonstrated a decreasing trend. AI was related to silica exposure, upregulation of mFas, and activation of caspase-3 and -8, as well as influenced by smoking status after adjusting for confounding factors. These results indicate that AM apoptosis could be used as a potential biomarker for human silicosis, and the Fas/FasL pathway may regulate this process. The present data from human lung lavage samples may help to understand the mechanism of silicosis and in turn lead to strategies for preventing or treating this disease.