Bronchoalveolar Lavage Fluid Total Cell Research Articles

Supplementation with γ-tocopherol attenuates endotoxin-induced airway neutrophil and mucous cell responses in rats

Neutrophil-mediated tissue injury is a shared pathogenesis of both chronic pulmonary diseases and acute responses to pathogens, allergens, and airborne pollutants. Interventions to minimize toxic effects of neutrophil-derived oxidants and proteases are usually limited to corticosteroids, which can have adverse side effects. We used a rodent model of endotoxin-induced lung injury to test the hypothesis that the dietary supplement γ-tocopherol (γT), a natural form of vitamin E with antioxidant and novel anti-inflammatory properties, will protect from adverse nasal and pulmonary inflammatory responses induced by endotoxin (lipopolysaccharide; LPS). Male Fisher F344 rats were intranasally (i.n.) instilled with LPS for 2 consecutive days. Beginning 2 days before i.n. LPS, the rats were gavaged daily with 30mg/kg γT. Twenty-four hours after the last i.n. LPS, bronchoalveolar lavage fluid (BALF) was collected, and pulmonary and nasal tissues were analyzed for gene expression and morphometric analyses of neutrophils and intraepithelial mucosubstances (IM). LPS caused increased BALF total cells (70% increase), neutrophils (300%), protein (35%), PGE2 (500%), and secreted mucins (75%). Robust increases in neutrophils and IM were detected in conducting airways. Pulmonary expression of MUC5AC, MIP-2, CINC-1, and MCP-1 was elevated three- to eightfold by LPS. Treatment with γT inhibited LPS-induced increases in BALF total cells, neutrophils, protein, PGE2, and secreted mucins, as well as IM and tissue neutrophil influx. Furthermore γT induced the expression of the regulatory cytokines IL-10 and IFN-γ while decreasing MUC5AC, MIP-2, CINC-1, and MCP-1. These data demonstrate novel therapeutic effects of the dietary vitamin E γT promoting anti-inflammatory pathways to protect from neutrophil-mediated lung injury.

Administration of mycobacterial Ag85A and IL-17A fusion protein attenuates airway inflammation in a murine model of asthma

Interleukin (IL)-17A contributes to the development of asthma, especially in severe asthma which has characteristic neutrophil infiltration in airways. However, IL-17A-blocking antibody could escalate T helper (Th) 2 cytokines, such as IL-13, IL-4 in murine models. We aimed at determining the effect of mycobacterial Ag85A and IL-17A fusion protein—Ag85A-IL-17A on airway inflammation in a murine model of asthma. IL-17A recombinant protein fused mycobacterial immunodominant antigen Ag85A was constructed, expressed and purified. The fusion protein was then administrated into BALB/c mice and its anti-inflammatory effects in the infiltration of inflammatory cells, Th2/Th17 cytokines in BALF, histopathological changes of lung tissues as well as chemokines in lung tissues were evaluated in the murine model of asthma. We found that administration of mycobacterial Ag85A and IL-17A fusion protein induced IL-17A specific immunoglobulin (Ig)G in sera and significantly decreased IL-17A and IL-6 levels in bronchoalveolar lavage fluid (BALF). Ag85A-IL-17A vaccinated mice also showed marked reduction in the infiltration of inflammatory cells in peribronchiolar region and significant decrease in total cells, eosinophil cells and neutrophil cells in BALF. The increased levels of IL-13 and IL-4 in BALF of ovalbumin-sensitized mice were significantly reduced by the administration of Ag85A-IL-17A. Furthermore, CD3+CD4+IL-13+ splenocytes stimulated with OVA and CXCL1 mRNA, CCL2 mRNA and GATA-3 mRNA expressed in lung tissues were decreased markedly in Ag85A-IL-17A vaccinated group. Our results demonstrate remarkable antiallergic effects of Ag85A-IL-17A in a murine model of asthma and it may have protective effects on allergic asthma.

Intervention effect and dose-dependent response of Tanreqing injection on airway inflammation in lipopolysaccharide-induced rats

ObjectiveTo assess the effect of Tanreqing injection on airway inflammation in rats. MethodsA rat model of airway inflammation was generated with lipopolysaccharide (LPS). Tanreqing injection was given by intratracheal instillation, and bronchoalveolar lavage fluid (BALF) from the right lung was collected. BALF total cell and neutrophil counts were then determined. In addition, BALF levels of inflammatory cytokines interleukin-1β, cytokine-induced neutrophil chemoattractant-1 (CINC-1), and tumor necrosis factor-α were measured using enzyme linked immunosorbent assay (ELISA). The middle lobe of the right lung was stained with hematoxylin-eosin and histological changes examined. ResultsLPS increased airway inflammation, decreased BALF inflammatory cell count, inflammatory cytokine levels, and suppressed leukocyte influx of the lung. The LPS-induced airway inflammation peaked at 24 h, decreased beginning at 48 h, and had decreased markedly by 96 h. ConclusionTanreqing injection contains anti-inflammatory properties, and inhibits airway inflammation in a dose-dependent manner.

Transcriptional Events during the Recovery from MRSA Lung Infection: A Mouse Pneumonia Model

Community associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging threat to human health throughout the world. Rodent MRSA pneumonia models mainly focus on the early innate immune responses to MRSA lung infection. However, the molecular pattern and mechanisms of recovery from MRSA lung infection are largely unknown. In this study, a sublethal mouse MRSA pneumonia model was employed to investigate late events during the recovery from MRSA lung infection. We compared lung bacterial clearance, bronchoalveolar lavage fluid (BALF) characterization, lung histology, lung cell proliferation, lung vascular permeability and lung gene expression profiling between days 1 and 3 post MRSA lung infection. Compared to day 1 post infection, bacterial colony counts, BALF total cell number and BALF protein concentration significantly decreased at day 3 post infection. Lung cDNA microarray analysis identified 47 significantly up-regulated and 35 down-regulated genes (p<0.01, 1.5 fold change [up and down]). The pattern of gene expression suggests that lung recovery is characterized by enhanced cell division, vascularization, wound healing and adjustment of host adaptive immune responses. Proliferation assay by PCNA staining further confirmed that at day 3 lungs have significantly higher cell proliferation than at day 1. Furthermore, at day 3 lungs displayed significantly lower levels of vascular permeability to albumin, compared to day 1. Collectively, this data helps us elucidate the molecular mechanisms of the recovery after MRSA lung infection.

NF-κB activation in myeloid cells mediates ventilator-induced lung injury

BackgroundAlthough use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated.MethodsTo study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKβ△mye), IL-6-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI.ResultsOur results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβ△mye mice as well as in IL6-/- to WT chimeric mice.ConclusionGiven that IKKβ△mye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB–IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.

EFFECT OF THERMONEUTRAL HOUSING ON FUNGAL‐INDUCED RESPIRATORY ALLERGIC DISEASE IN MICE.

Climate change is projected to increase the number of fungal agents and may become a significant health impact. It is important to consider how respiratory allergic responses could be altered with global warming. Core temperature (Tc) was monitored by telemetry in female, BALB/c mice while acclimated for one week to ambient temperatures (Ta) of 22 or 30 °C. Exposure to 30 °C led to small increase in Tc that recovered in ~3 d. Mice were exposed to house dust mite (HDM) allergen that was administered by intratracheal aspiration 4X over a 4 week period at doses ranging from 1.25 to 20 ug. Airway responses to methacholine challenge were assessed 1 day following the last exposure to HDM and suggest a trend toward more elevated responses in higher dose mice housed at 30 °C. Bronchoalveolar lavage fluid (BALF) and blood were collected 2 days after the final exposure. BALF total cell counts increased at 10 and 20 ug in mice acclimated to 22 C. Allergic responses were manifested at lower doses of HDM in mice acclimated to 30 °C. Tc appeared to be unaffected by HDM exposure at 22 and 30 °C. The data suggest that housing in a thermoneutral environment of 30 °C may exacerbate the respiratory response to allergic agents compared to housing at a Ta of 22 °C that is a mild cold stress in mice. Future studies will address how higher Ta that elevate Tc will affect respiratory allergic responses. (Abstract does not represent USEPA policy).

Safety of and Cellular Response to Segmental Bronchoprovocation in Allergic Asthma

RationaleDespite its incorporation into research studies, the safety aspects of segmental allergen bronchoprovocation and differences in cellular response among different allergens have received limited consideration.MethodsWe performed 87 segmental challenges in 77 allergic asthma subjects. Allergen dose was based on each subject’s response to whole lung allergen challenge. Bronchoalveolar lavage was performed at 0 and 48 hours. Safety indicators included spirometry, oxygen saturation, heart rate, and symptoms.ResultsAmong subjects challenged with ragweed, cat dander, or house dust mite, there were no differences in safety indicators. Subjects demonstrated a modest oxygen desaturation and tachycardia during the procedure that returned to normal prior to discharge. We observed a modest reduction in forced vital capacity and forced expiratory volume in one second following bronchoscopy. The most common symptoms following the procedure were cough, sore throat and fatigue. Total bronchoalveolar lavage fluid cell numbers increased from 13±4 to 106±108×104 per milliliter and eosinophils increased from 1±2 to 44±20 percent, with no significant differences among the three allergens.ConclusionsIn mild allergic asthma, segmental allergen bronchoprovocation, using individualized doses of aeroallergens, was safe and yielded similar cellular responses.

The Effect of Oral Tolerance on the Allergic Airway Response in Younger and Aged Mice

Background. The effect of increased age on the induction of oral tolerance by low-dose antigen feeding and its effect on the response to antigen airway challenge in aged mice have not been well characterized. Objective. To determine whether oral tolerance can be induced in aged mice and its impact on the development of allergic airway inflammation. Methods. Younger (6 weeks old) and aged (18 months old) mice were fed ovalbumin (OVA) prior to sensitization to induce antigen tolerance. Serum antigen-specific immunoglobulins (Igs), bronchoalveolar lavage fluid (BALF), lung histology, enumeration of CD4 + Foxp3+ Treg cells, and airway hyperresponsiveness (AHR) were determined after the final antigen challenge. Results. Feeding antigen to aged mice prior to sensitization induced oral tolerance as determined by a decrease in antigen-specific IgE and IgG1; however, the effect was greater in younger mice. Induction of oral tolerance was associated with a greater increase in airway Treg cells in the younger mice. Despite these differences, oral tolerance significantly suppressed features of asthma in aged mice, including BALF total cell and eosinophil numbers, cytokine production, and AHR. Conclusions. Aged mice developed oral tolerance to antigen, which suppressed several features of allergic airway inflammation.

The Effect of Intravenous Lidocaine Infusion on Bronchoalveolar Lavage Cytology in Equine Recurrent Airway Obstruction

Lidocaine decreases neutrophilic inflammation in models of acute lung injury and decreases inflammation in asthmatic patients. Neutrophilic bronchiolitis develops in recurrent airway obstruction (RAO), but it remains unknown if lidocaine infusion decreases neutrophil migration into the airways. Lidocaine decreases neutrophilic inflammation as measured in BALF in RAO-affected horses. Six RAO-susceptible horses in remission. In a randomized cross-over design, horses received lactated Ringer's solution (LRS) IV or lidocaine hydrochloride IV with a minimum of 4 weeks at pasture between treatments. Treatments were delivered as continuous infusions beginning 4 hours before and for 68 hours during exposure to hay and straw challenge. Clinical score (CS, grade 0-8), maximal change in pleural pressure (∆Ppl(max) ), and bronchoalveolar lavage fluid (BALF) cytology were measured at baseline and the end of challenge (day 4). Plasma lidocaine concentrations were monitored daily. At baseline, there were no significant differences in variables between treatments. Plasma lidocaine concentration was consistently > 1100 ng/mL. After challenge, CS increased significantly [baseline: 2/8 (2-3), [median (interquartile range)]; day 4: 4/8 (4-5) P = .0006] as did ∆Ppl(max) [baseline: 3.6 (2.63-4.95) cmH(2) 0; day 4: 9.62 (6.5-16) P = .0036], but there was no difference between treatments. Percentage of neutrophils was not different between treatments, but lidocaine infusion significantly increased BALF total cells [baseline: LRS 2.18 ± 0.82 × 10(5) cells/mL (mean ± SD), lidocaine 1.6 ± 0.3 × 10(5) , day 4: LRS 2.0 ± 0.88 × 10(5) , lidocaine 4.4 ± 2 × 10(5) (P = .0045)]. Lidocaine does not decrease neutrophilic inflammation in RAO.

Glutathione Peroxidase-1 Reduces Influenza A Virus–Induced Lung Inflammation

Oxidative stress caused by excessive reactive oxygen species production is implicated in influenza A virus-induced lung disease. Glutathione peroxidase (GPx)-1 is an antioxidant enzyme that may protect lungs from such damage. The objective of this study was to determine if GPx-1 protects the lung against influenza A virus-induced lung inflammation in vivo. Male wild-type (WT) or GPx-1(-/-) mice were inoculated with HKx31 (H3N2, 1 × 10(4) plaque-forming units), and bronchoalveolar lavage fluid (BALF)/lung compartments were analyzed on Days 3 and 7 after infection for inflammatory marker expression, histology, and viral titer. WT mice infected with HKx31 had significantly more BALF total cells, macrophages, neutrophils, and lymphocytes at Days 3 and 7 compared with naive WT animals (n = 5-8; P < 0.05). However, infected GPx-1(-/-) mice had significantly more BALF inflammation, which included more total cells, macrophages, and neutrophils, compared with WT mice, and this was abolished by treatment with the GPx mimetic ebselen. BALF inflammation persisted in GPx-1(-/-) mice on Day 10 after infection, and GPx-1(-/-) mice had significantly more influenza-specific CD8(+) T cells in spleen compared with WT mice (n = 3-4; P < 0.05). Infected GPx-1(-/-) mice had greater peribronchial and parenchymal inflammation than WT mice, and viral titer was significantly reduced in GPx-1(-/-) mice at Day 3 (n = 5; P < 0.05). Gene expression analysis revealed that infected GPx-1(-/-) mice had higher whole lung TNF-α, macrophage inflammatory protein (MIP)-1α, MIP-2, KC, and matrix metalloproteinase (MMP)-12 mRNA compared with infected WT mice. GPx-1(-/-) mice had more MIP-2 protein in BALF at Day 3 and more active MMP-9 protease in BALF at Days 3 and 7 than WT mice. These data indicate that GPx-1 reduces influenza A virus-induced lung inflammation.

Accumulation of Myeloid-Derived Suppressor Cells in the Lungs during Pneumocystis Pneumonia

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of hematopoietic precursors with the ability to adversely affect host immunity. They have been shown to accumulate in pathological conditions, such as cancer and some microbial diseases. In the mouse and rat models of Pneumocystis pneumonia (PcP), we found a distinct population of cells with MDSC-like morphology in the bronchoalveolar lavage (BAL) fluid, constituting up to 50% of the total cells in BAL fluid. These cells were not seen in the BAL fluid from normal animals or from Pneumocystis-infected animals that had been successfully treated for PcP with a combination of trimethoprim and sulfamethoxazole. With flow cytometry, these cells were found to express the characteristic MDSC surface markers Gr-1 and CD11b in mice or CD11bc and His48 in rats. Using reverse transcription-PCR, we demonstrated that these cells produced high levels of arginase-1 and inducible nitric oxide synthase (iNOS) mRNA. These cells were shown to suppress CD4(+) T-cell proliferation in response to stimulation by anti-CD3 and anti-CD28 antibodies. Adoptive transfer of these cells to normal mice caused lung damage, as indicated by elevated levels of albumin and lactate dehydrogenase in the BAL fluid. These experiments provide evidence of the presence of MDSCs in the lungs during PcP. Further studies on the roles of MDSCs in PcP are warranted in order to develop treatment strategies which can reduce the number of MDSCs and the damage caused by these cells.

Oral administration of Enterococcus faecalis FK-23 suppresses Th17 cell development and attenuates allergic airway responses in mice

Evidence is increasing that oral administration of probiotics can attenuate asthmatic responses both in murine models and clinical trials. T-helper 17 (Th17) cells, a subset of CD4+ T cells have been implicated as having an important role in the development of several allergic disorders, but the relationship between oral administration of probiotics and Th17 development has not been well studied. BALB/c mice were given lysed Enterococcus faecalis FK-23 (LFK) orally for 28 days. After sensitization by subcutaneous injection of ovalbumin (OVA) on Days 14 and 21 and 1% OVA inhalation on Days 25, 26 and 27, they were challenged with a 5% OVA aerosol on Day 28. Twenty-four hours later, airway resistance and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung tissues were determined. Ιnterleukin (IL)-17-expressing CD4+ lymphocytes isolated from lung, spleen and lamina propria of the intestine were detected by flow cytometry. The expression of IL-6 and TGF-β mRNA was assessed by real-time PCR. Increases in airway hyperresponsiveness, and numbers of total leukocytes and mast cells in BALF induced by OVA challenge were significantly suppressed by oral administration of LFK. The increased percentage of IL-17-expressing CD4+ cells from lung, spleen and intestine in OVA-challenged mice was reduced following LFK treatment. We conclude that the oral administration of LFK suppresses the asthmatic response and that this is associated with attenuation of Th17 cell development.

Fructose-1,6-bisphosphate Protects against Zymosan-induced Acute Lung Injury in Mice

It has been previously showed that fructose-1,6-bisphosphate (FBP) has anti-inflammatory and immunomodulatory effects on several experimental inflammation models. However, the effects and mechanism of FBP on Zymosan-induced acute lung injury (ALI) in mice had not been tested. In this study, our aim was to assess the anti-inflammatory activities of FBP on Zymosan-induced ALI. We found that in vivo treatment with FBP (500mg/kgi.p.) markedly decreased the nitric oxide (NO) levels in the lungs and significantly reduced bronchoalveolar lavage fluid total cell and neutrophil counts and protein exudation after Zymosan challenge. Furthermore, FBP inhibited inducible nitric oxide synthase (iNOS) activities in RAW macrophages. Meanwhile, FBP did not inhibit the cyclooxigenase 2, interleukin-6, and nuclear factor kappa B transcription. Taken together, these results suggest that FBP shows anti-inflammatory effects through inhibiting lung edema, NO, and iNOS activities.

Metformin attenuates ventilator-induced lung injury.

IntroductionDiabetic patients may develop acute lung injury less often than non-diabetics; a fact that could be partially ascribed to the usage of antidiabetic drugs, including metformin. Metformin exhibits pleiotropic properties which make it potentially beneficial against lung injury. We hypothesized that pretreatment with metformin preserves alveolar capillary permeability and, thus, prevents ventilator-induced lung injury.MethodsTwenty-four rabbits were randomly assigned to pretreatment with metformin (250 mg/Kg body weight/day per os) or no medication for two days. Explanted lungs were perfused at constant flow rate (300 mL/min) and ventilated with injurious (peak airway pressure 23 cmH2O, tidal volume ≈17 mL/Kg) or protective (peak airway pressure 11 cmH2O, tidal volume ≈7 mL/Kg) settings for 1 hour. Alveolar capillary permeability was assessed by ultrafiltration coefficient, total protein concentration in bronchoalveolar lavage fluid (BALF) and angiotensin-converting enzyme (ACE) activity in BALF.ResultsHigh-pressure ventilation of the ex-vivo lung preparation resulted in increased microvascular permeability, edema formation and microhemorrhage compared to protective ventilation. Compared to no medication, pretreatment with metformin was associated with a 2.9-fold reduction in ultrafiltration coefficient, a 2.5-fold reduction in pulmonary edema formation, lower protein concentration in BALF, lower ACE activity in BALF, and fewer histological lesions upon challenge of the lung preparation with injurious ventilation. In contrast, no differences regarding pulmonary artery pressure and BALF total cell number were noted. Administration of metformin did not impact on outcomes of lungs subjected to protective ventilation.ConclusionsPretreatment with metformin preserves alveolar capillary permeability and, thus, decreases the severity of ventilator-induced lung injury in this model.

S100A8/A9: A Mediator of Neutrophilic Airway Inflammation, Airway Hyperresponsiveness and Biomarker of Severe Asthma.

Background: Most severe asthmatics exhibit steroid refractory disease that limits effectiveness of therapy and typically associ ated with chronic neutrophilia, and release of a unique repertoire of “steroid refractory” mediators. Thus, a better understanding of severe disease, including triggers of these steroid refractory mediators is critical to address unmet clinical needs. Our aim was to identify whether S100A9 knock-out mice (the subunit that stabilizes the functional S100 protein heterodimer) elicits attenuated hallmarks of asthma including, airway inflammation (neutrophilia and eosinophilia), airway hyperresponsiveness and airway remodelling in a murine model of allergic asthma. Methods: Eight week old Balb/c mice were subjected to intranasal (i.n) challenges with house dust mite (HDM) (Dermatophagoides pteronnyssinus) extract (0.7mg/mL in saline) - acute (5 consecutive daily i.n challenge for 2weeks) and chronic (3 daily mid-week HDM i.n challenge for additional 5 weeks). Animals were assessed for S100A8/A9 at: ~8hrs and 48hrs post acute protocol (peak neutrophilia and eosinophilia respectively); and 48 hrs post chronic protocol (established airway remodeling). We also assessed baseline respiratory mechanics in S100A9 knockout (KO) mice via flexiVent. Using Hu man endobronchial biopsies (severe asthma) and patient-matched bronchoalveolar lavage fluid (BALF), we also performed immunohistochemistry and Enzyme Linked ImmunoabSorbent Assay (ELISA) to assay S100A8/A9 and Receptor for Advanced Glycation Endproducts (RAGE) expression. Results: Expression of S100A8/A9 and its primary receptor, RAGE, are increased in asthmatic biopsies compared to healthy biopsies. S100A8/A9 release in BALF fluid was elevated by 6 fold in human asthmatic [605 ng/ml (24-2420)] compared to healthy subject BALF [107 ng/ml (38-400)]. In mice, acute HDM challenge induces and sustains S100A8/A9 release in BALF, and this tracks with neutrophil cell numbers in BALF. S100A8/A9 and RAGE from lung tissue is increased in acute and chronic HDM challenged mice compared to naive controls. Using the acute HDM protocol, HDM S100A9 KO mice elicit reduced airway resistance (AR), tissue resistance (TR) and tissue elastance (TE) to methacholine challenge (6-50 mg/ml) compared to HDM wild-type mice. Similarly basal AR and TE are both reduced in the HDM S100A9 KO mice compared to HDM wild-type mice. Furthermore, total BALF cells including eosinophils and neutrophils appear reduced in S100A9 KO mice for both naive and HDM groups. Conclusion: In severe asthma and murine models of allergic asthma there is an elevated release and expression of S100A9/ A9 and RAGE. Improved lung mechanics and attenuated lung inflammation in S100A9 KO mice suggest that sustained levels of S100A8/A9 may contribute to disease pathogenesis.

A comparison of the allergic responses induced by Penicillium chrysogenum and house dust mite extracts in a mouse model

A report by the Institute of Medicine suggested that more research is needed to better understand mold effects on allergic disease, particularly asthma development. We compared the ability of the fungal Penicillium chrysogenum (PCE) and house dust mite (HDM) extracts to induce allergic responses in BALB/c mice. The extracts were administered by intratracheal aspiration (IA) at several doses (0, 2.5, 5, 10, 20, 40, and 80 μg) four times over a 4-week period. Three days after the last IA exposure, serum and bronchoalveolar lavage fluid (BALF) were collected. The relative allergenicity of the extracts was evaluated based on the lowest dose able to induce a significant response compared to control (0 μg) and the robustness of the response. PCE induced the most robust response at the lowest dose for most endpoints examined: BALF total, macrophage, neutrophil, and eosinophil cell counts, and antigen-specific IgE. Taken together, our data suggest that PCE may induce a more robust allergic and inflammatory response at lower doses than HDM. Our data suggest that Penicillium chrysogenum is a robust allergen and may be a more potent allergen source than house dust mite (HDM) in this mouse model. Two critical factors in the development of human allergic disease, exposure levels and sensitization thresholds, are unknown for most allergens including molds/fungi. Human exposure levels are not within the scope of this article. However, the data presented suggest a threshold dose for the induction of allergic responsiveness to P. chrysogenum. Additionally, P. chrysogenum as well as other molds may play an important role in asthma development in our society.

Cbl-b regulates airway mucosal tolerance to aeroallergen

As an E3 ubiquitin ligase and a molecular adaptor, Cbl-b controls the activation threshold of the antigen receptor and negatively regulates CD28 costimulation, functioning as an intrinsic mediator of T cell anergy that maintains tolerance. However, the role of Cbl-b in the airway immune response to aeroallergens is unclear. To determine the contribution of Cbl-b in tolerance to aeroallergens, we examined ovalbumin (OVA)-induced lung inflammation in Cbl-b-deficient mice. Cbl-b(-/-) mice and wild-type (WT) C57BL/6 mice were sensitized and challenged with OVA intranasally, a procedure normally tolerated by WT mice. We analysed lung histology, bronchoalveolar lavage fluid total cell counts and differential, cytokines and chemokines in the airway, and cytokine response by lymphocytes after re-stimulation by OVA antigen. Compared with WT mice, OVA-challenged Cbl-b(-/-) mice showed significantly increased neutrophilic and eosinophilic infiltration in the lung and mucus hyperplasia. The serum levels of IgG2a and IgG1, but not IgE, were increased. The levels of inflammatory mediators IFN-γ, IL-10, IL-12, IL-13, IP-10, MCP-1, MIP-1α, eotaxin, and RANTES, but not IL-17A or IL-6, were elevated in the airway of Cbl-b(-/-) mice. Lymphocytes from Cbl-b(-/-) mice released increased amount of IFN-γ, IL-10, IL-13, and IP-10 in response to OVA re-stimulation. However, no significant changes were noted in the CD4(+) CD25(+) T regulatory cell populations in the lung tissues after OVA stimulation and there was no difference between WT and Cbl-b(-/-) mice. These results demonstrate that Cbl-b deficiency leads to a breakdown of tolerance to OVA allergen in the murine airways, probably through increased activation of T effector cells, indicating that Cbl-b is a critical factor in maintaining lung homeostasis upon environmental exposure to aeroallergens.

Deactivation of alveolar macrophages in neutropenic acute respiratory distress syndrome (ARDS) in children with hematologic malignancy

Objective To study the alveolar macrophages (AM) activation in neutropenic ARDS in children with hematologic malignancy. Methods Thirteen patients of ARDS were prospectively enrolled into the study and divided into two groups,neutropenic (5 cases) and nonneutropenic (8 cases). Bronchoalveolar lavage fluid (BALF) samples were analyzed for differential and total cell counts, and alveolar activation marker expression (HLA-DR) was determined. Results In neutropenic patients,the BALF total cell count and the macrophage absolute count were significantly lower compared to those in nonneutropenic group ((62. 6 ±9. 6)/μl vs ( 124. 0 ± 6. 7)/μl,P ＜ 0. 01 and (40. 8 ±4. 3)/μl vs (67. 6 ± 10. 7)/μl,P ＜ 0. 01,respectively). As compared with the nonneutropenic group, the percentage of AM in neutropenic patients was significantly higher (65.9% ±9. 0% vs 54. 6% ± 8. 7% ,P ＜0. 05) ,the mean percentage of AM HLA-DR expression was significantly lower (35. 3% ± 5.8% vs 62. 2% ± 5. 8%, P ＜ 0. 01 ). Conclusion These results suggest an alveolar hypocellularity and a deactivation of AM in neutropenic ARDS in children with hematologic malignancy. Key words: Alveolar macrophage; Acute respiratory distress syndrome; Neutropenia; Human leukocyte antigen-DR

Functions of Type II Pneumocyte-Derived Vascular Endothelial Growth Factor in Alveolar Structure, Acute Inflammation, and Vascular Permeability

Vascular endothelial growth factor-A (VEGF) is a potent regulator of vascular permeability, inflammatory response, and cell survival in the lung. To explore the functions of VEGF produced locally in type II pneumocytes, we generated mice with a conditional deletion of VEGF-A using Cre recombinase driven by the human surfactant protein C (SPC) promoter. In 7- to 10-week-old VEGF-knockout (SPC-VEGF-KO) mice, lung histology and physiology were essentially normal, except for higher dynamic lung compliance and lower pulmonary vascular permeability. Emphysema was seen in 28- to 32-week-old animals. To investigate the role of type II pneumocyte-derived VEGF in acute lung injury, we challenged 7- to 10-week-old SPC-VEGF-KO mice and their wild-type littermates with intestinal ischemia-reperfusion. Bronchoalveolar lavage fluid total cell count, pulmonary permeability, and lung injury score were significantly attenuated, and total lung VEGF levels were significantly lower in SPC-VEGF-KO mice compared with wild-type controls. In SPC-VEGF-KO mice, activated caspase 3-positive type II epithelial cells were increased after intestinal ischemia-reperfusion, even though there was no significant difference in the total number of cells positive for terminal deoxynucleotidyl transferase dUTP nick-end labeling. We conclude that VEGF in type II cells helps protect alveolar epithelial cells from caspase-dependent apoptosis. However, VEGF produced from type II cells may contribute to increased vascular permeability during acute lung injury.

Influence of age on bronchoscopic findings in healthy beagle dogs

The aims of this study were to evaluate the effect of age on bronchoscopic features and bronchoalveolar lavage fluid (BALF) cellularity in dogs. Thirty healthy beagle dogs from three age groups were included: young dogs (10 months to 4.5 years of age; n=8), middle-aged dogs (5-8 years old; n=13) and older dogs (>8 years; n=9). Haematology, thoracic radiography, bronchoscopy and bronchoalveolar lavage were performed; bronchoscopic findings were scored and BALF total and differential cell counts were determined. The total bronchoscopic score was higher in older dogs; these dogs had more irregular bronchial mucosa, more prominent mucosal vessels and bronchiectasis. Younger dogs had a higher percentage of neutrophils in BALF compared with middle-aged and old dogs and a higher percentage of lymphocytes in BALF compared with middle-aged dogs. The results show that age has an effect on bronchoscopic features of airways and the composition of BALF in the dog.