Hoogsteden, H.C.

Incidence of viral respiratory pathogens causing exacerbations in adult cystic fibrosis patients (Article)

2013-01-01T00:00:00Z

Respiratory infections caused by respiratory viruses are common in paediatric cystic fibrosis (CF) patients and are associated with increased morbidity. There is only little data on the incidence of viral respiratory pathogens causing exacerbations in the adult CF patient population. In this observational pilot study we show, by using molecular as well as conventional techniques for viral isolation, that during 1 y a viral pathogen could be isolated in 8/24 (33%) adult CF patients who presented with a pulmonary exacerbation. This result shows that there is a considerable incidence of viral pathogens in pulmonary exacerbations in adult CF patients. Newly identified viruses such as pandemic influenza A/H1N1, human metapneumovirus, human bocavirus, and human coronavirus NL63 were not detected in our population, except for 1 human coronavirus NL63.

Expression profiling-based subtyping identifies novel non-small cell lung cancer subgroups and implicates putative resistance to pemetrexed therapy (Article)

2012-01-01T00:00:00Z

Introduction: A challenge of cancer therapy is to optimize therapeutical options to individual patients. Cancers with similar histology may show dramatically different responses to therapy, indicating that a refined approach needs to be developed to classify tumors by intrinsic characteristics that may predict response to chemotherapy. Global expression profile-based classification has the potential to identify such tumor-intrinsic subclasses. Pemetrexed effectiveness has been related to the expression of its target thymidylate synthase. The relatively frequent resistance of squamous cell carcinoma to Pemetrexed is correlated with high levels of thymidylate synthase expression. Methods: A global expression profile-based molecular classification of non-small cell lung cancer (NSCLC) was performed. Gene expression was used to predict Pemetrexed responsiveness. The distinct molecular attributes of NSCLCs predicted likely to be resistant to Pemetrexed were bioinformatically characterized. We tested if routine immunohistochemical markers can be used to distinguish putative Pemetrexed responders, predicted by gene signatures, from nonresponders. Results: Ninety NSCLCs were divided into six subclasses by gene expression signatures. The relevance of this novel phenotyping was linked to other tumor characteristics. Two of the subclasses correlated to putative Pemetrexed resistance. In addition, the identified signature genes characterizing putative Pemetrexed responsiveness predicted therapeutic benefit in a subset of squamous cell carcinoma. Conclusions: Gene expression signatures can be used to identify NSCLC subgroups and have potential to predict resistance to Pemetrexed therapy. We suggest that a combination of classical pathological markers can be used to identify molecular tumor subclasses associated with predicted Pemetrexed response. Copyright

Increased IL-17A expression in granulomas and in circulating memory T cells in sarcoidosis (Article)

2012-01-01T00:00:00Z

Sarcoidosis is a systemic inflammatory disorder characterized by granulomas. Although the aetiology is unknown, sarcoidosis is thought to be mediated by Th1 lymphocytes. Recently, IL-17A has been implicated in granuloma formation in various diseases, including tuberculosis. Therefore, we hypothesized that Th17 cells play a role in sarcoidosis, paralleling recent findings in autoimmune diseases such as RA. The aim of our study was to investigate the role of Th17 cells in sarcoidosis. T cells were investigated by intracellular flow cytometry and immunohistochemistry, in blood, bronchoalveolar lavages (BALs) and bronchial mucosal biopsies from a cohort of newly diagnosed sarcoidosis patients and healthy controls. Circulating memory CD4(+) T-cell populations of sarcoidosis patients contained significantly increased proportions of IL-17A(+) cells when compared with healthy controls. Interestingly, proportions of IL-17A/IFN-γ and IL-17A/IL-4 double-producing cells were significantly increased in blood of sarcoidosis patients and were present in substantial numbers in BAL. In granuloma-containing, but not in non-granulomatous sarcoidosis biopsies, we found significantly increased numbers of IL-17A(+) T cells, located in and around granulomas throughout the lamina propria. IL-22(+) T cells were increased in the subepithelial layer. Enhanced IL-17A expression in granulomas and the presence of IL-17A(+), IL-17A(+)IFN-γ(+) and IL-17A(+)IL-4(+)memory Th cells in the circulation and BAL indicate Th17 cell involvement in granuloma induction or maintenance in sarcoidosis. Therefore, neutralization of IL-17A activity may be a novel strategy to treat sarcoidosis.

Persistent activation of dendritic cells after resolution of allergic airway inflammation breaks tolerance to inhaled allergens in mice (Article)

2011-08-01T00:00:00Z

Rationale: Polysensitization of patients who are allergic is a common feature. The underlying immunologic mechanism is not clear. The maturation status of dendritic cells (DCs) is considered to be important for priming naive T cells in the draining lymph nodes. We hypothesized that chronic airway inflammation can induce an enhanced maturation of airway DCs and facilitate subsequent priming to neoallergens. Objectives: To investigate whether chronic airway inflammation could induce an altered activation of airway DCs in mice and whether this influences the development of allergic sensitization. Methods: Balb/c mice were repeatedly challenged withDCsto induce a chronic airway inflammation. We evaluated (1) the induction of the main characteristic features of human asthma including persistent remodeling, (2) the maturation status of airway DCs 1 month after inflammation resolved, (3) whether this influences tolerance to inhaled neoallergen, and (4) what type of T helper response would be induced by DCs. Measurements and Main Results: Airway DCs displayed a mature phenotype after complete resolution of airway eosinophilia. Inhalation of a neoallergen without any adjuvant was able to induce airway inflammation in postinflammation lungs but not in control lungs. One month after inflammation, airway DCs were able to induce Th2 polarization in naive T cells consistent with the up-regulation of the Th2 skewing molecules Ym1/2 and OX-40L compared with DCs of control airways. Conclusions: This study provides evidence that sustained maturation of DCs after resolution of Th2-mediated inflammation can contribute to polysensitization.

Highly pathogenic avian influenza virus H5N1 infects alveolar macrophages without virus production or excessive TNF-alpha induction (Article)

2011-06-01T00:00:00Z

Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 causes severe, often fatal pneumonia in humans. The pathogenesis of HPAIV H5N1 infection is not completely understood, although the alveolar macrophage (AM) is thought to play an important role. HPAIV H5N1 infection of macrophages cultured from monocytes leads to high percentages of infection accompanied by virus production and an excessive pro-inflammatory immune response. However, macrophages cultured from monocytes are different from AM, both in phenotype and in response to seasonal influenza virus infection. Consequently, it remains unclear whether the results of studies with macrophages cultured from monocytes are valid for AM. Therefore we infected AM and for comparison macrophages cultured from monocytes with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus, and determined the percentage of cells infected, virus production and induction of TNF-alpha, a pro-inflammatory cytokine. In vitro HPAIV H5N1 infection of AM compared to that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% vs up to 84%), lower virus production and lower TNF-alpha induction. In vitro infection of AM with H3N2 or H1N1 virus resulted in even lower percentages of infected cells (up to 7%) than with HPAIV H5N1, while virus production and TNF-alpha induction were comparable. In conclusion, this study reveals that macrophages cultured from monocytes are not a good model to study the interaction between AM and these influenza virus strains. Furthermore, the interaction between HPAIV H5N1 and AM could contribute to the pathogenicity of this virus in humans, due to the relative high percentage of infected cells rather than virus production or an excessive TNF-alpha induction.

Serum biomarkers in idiopathic pulmonary fibrosis (Article)

2010-12-01T00:00:00Z

Within the group of Idiopathic Interstitial Pneumonias (IIPs), above all Idiopathic Pulmonary Fibrosis (IPF) poses a considerable diagnostic and therapeutic problem. Although genetic profiling indicates that IPF, Non Specific Interstitial Pneumonia (NSIP), and chronic hypersensitivity pneumonitis (HP) are distinctly different diseases, in every day practice these diseases can be difficult to tell apart. Furthermore, treatment of these diseases is notoriously difficult. Serum biomarkers reflect our understanding of the underlying pathogenesis and potentially fulfill a role in establishing a diagnosis, prognosis and therapy. While no single biomarker is currently able to accurately predict the presence or absence of an IIP, a composite of several markers holds promise for the future. Several biomarkers, such as KL-6, surfactant proteins and circulating fibrocytes, appear to contribute to our insight into disease progression and prognosis. It is however uncertain whether these markers give us additional information to common diagnostic tests and their value has as yet to be validated for every day practice. Fortunately, the potential of biomarkers is increasingly recognized and biomarker data are prospectively gathered in current placebo-controlled therapeutic trials.

Integration of single-fiber reflectance spectroscopy into ultrasound-guided endoscopic lung cancer staging of mediastinal lymph nodes (Article)

2010-12-01T00:00:00Z

We describe the incorporation of a single-fiber reflectance spectroscopy probe into the endoscopic ultrasound fine-needle aspiration (EUS-FNA) procedure utilized for lung cancer staging. A mathematical model is developed to extract information about the physiological and morphological properties of lymph tissue from singlefiber reflectance spectra, e.g., microvascular saturation, blood volume fraction, bilirubin concentration, average vessel diameter, and Mie slope. Model analysis of data from a clinical pilot study shows that the single-fiber reflectance measurement is capable of detecting differences in the physiology between normal and metastatic lymph nodes. Moreover, the clinical data show that probe manipulation within the lymph node can perturb the in vivo environment, a concern that must be carefully considered when developing a sampling strategy. The data show the feasibility of this novel technique; however, the potential clinical utility has yet to be determined.

COX-2 inhibition improves immunotherapy and is associated with decreased numbers of myeloid-derived suppressor cells in mesothelioma. Celecoxib influences MDSC function (Article)

2010-08-30T00:00:00Z

Background: Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that accumulates in tumour-bearing hosts. These cells are induced by tumour-derived factors (e.g. prostaglandins) and have a critical role in immune suppression. MDSC suppress T and NK cell function via increased expression of arginase I and production of reactive oxygen species (ROS) and nitric oxide (NO). Immune suppression by MDSC was found to be one of the main factors for immunotherapy insufficiency. Here we investigate if the in vivo immunoregulatory function of MDSC can be reversed by inhibiting prostaglandin synthesis by specific COX-2 inhibition focussing on ROS production by MDSC subtypes. In addition, we determined if dietary celecoxib treatment leads to refinement of immunotherapeutic strategies.Methods: MDSC numbers and function were analysed during tumour progression in a murine model for mesothelioma. Mice were inoculated with mesothelioma tumour cells and treated with cyclooxygenase-2 (COX-2) inhibitor celecoxib, either as single agent or in combination with dendritic cell-based immunotherapy.Results: We found that large numbers of infiltrating MDSC co-localise with COX-2 expression in those areas where tumour growth takes place. Celecoxib reduced prostaglandin E2 levels in vitro and in vivo. Treatment of tumour-bearing mice with dietary celecoxib prevented the local and systemic expansion of all MDSC subtypes. The function of MDSC was impaired as was noticed by reduced levels of ROS and NO and reversal of T cell tolerance; resulting in refinement of immunotherapy.Conclusions: We conclude that celecoxib is a powerful tool to improve dendritic cell-based immunotherapy and is associated with a reduction in the numbers and suppressive function of MDSC. These data suggest that immunotherapy approaches benefit from simultaneously blocking cyclooxygenase-2 activity.

Zoledronic acid impairs myeloid differentiation to tumour-associated macrophages in mesothelioma (Article)

2010-08-01T00:00:00Z

Background: Suppressive immune cells present in tumour microenvironments are known to augment tumour growth and hamper efficacy of antitumour therapies. The amino-bisphosphonate Zoledronic acid (ZA) is considered as an antitumour agent, as recent studies showed that ZA prolongs disease-free survival in cancer patients. The exact mechanism is a topic of debate; it has been suggested that ZA targets tumour-associated macrophages (TAMs). Methods: We investigate the role of ZA on the myeloid differentiation to TAMs in murine mesothelioma in vivo and in vitro. Mice were intraperitoneally inoculated with a lethal dose of mesothelioma tumour cells and treated with ZA to determine the effects on myeloid differentiation and survival. Results: We show that ZA impaired myeloid differentiation. Inhibition of myeloid differentiation led to a reduction in TAMs, but the number of immature myeloid cells with myeloid-derived suppressor cell (MDSC) characteristics was increased. In addition, ZA affects the phenotype of macrophages leading to reduced level of TAM-associated cytokines in the tumour microenvironment. No improvement of survival was observed.Conclusion:We conclude that ZA leads to a reduction in macrophages and impairs polarisation towards an M2 phenotype, but this was associated with an increase in the number of immature myeloid cells, which might diminish the effects of ZA on survival.

Low-dose cyclophosphamide synergizes with dendritic cell-based immunotherapy in antitumor activitys (Article)

2010-06-18T00:00:00Z

Clinical immunotherapy trials like dendritic cell-based vaccinations are hampered by the tumor's offensive repertoire that suppresses the incoming effector cells. Regulatory T cells are instrumental in suppressing the function of cytotoxic T cells. We studied the effect of low-dose cyclophosphamide on the suppressive function of regulatory T cells and investigated if the success rate of dendritic cell immunotherapy could be improved. For this, mesothelioma tumor-bearing mice were treated with dendritic cell-based immunotherapy alone or in combination with low-dose of cyclophosphamide. Proportions of regulatory T cells and the cytotoxic T cell functions at different stages of disease were analyzed. We found that low-dose cyclophosphamide induced beneficial immunomodulatory effects by preventing the induction of Tregs, and as a consequence, cytotoxic T cell function was no longer affected. Addition of cyclophosphamide improved immunotherapy leading to an increased median and overall survival. Future studies are needed to address the usefulness of this combination treatment for mesothelioma patients. Copyright

Consolidative dendritic cell-based immunotherapy elicits cytotoxicity against malignant mesothelioma (Article)

2010-06-15T00:00:00Z

Rationale: We previously demonstrated that dendritic cell-based immunotherapy induced protective antitumor immunity with a prolonged survival rate in mice. However, the clinical relevance is still in question. To examine this, we designed a clinical trial using chemotherapy followed by antigen-pulsed dendritic cell vaccination in mesothelioma patients. Objectives: The aim of this study was to assess the safety and immunological response induced by the administration of tumor lysate-pulsed dendritic cells in patients with mesothelioma. Methods: Ten patients with malignant pleural mesothelioma received three vaccinations of clinical-grade autologous dendritic cells intradermally and intravenously at 2-week intervals after chemotherapy. Each vaccine was composed of 50 Ã 106mature dendritic cells pulsed with autologous tumor lysate and keyhole limpet hemocyanin (KLH) as surrogate marker. Delayed-type hypersensitivity activity to tumor antigens and KLH was assessed, both in vivo and in vitro. Peripheral blood mononuclear cells during the treatment were analyzed for immunological responses. Measurements and Main Results: Administration of dendritic cells pulsed with autologous tumor lysate in patients with mesothelioma was safe with moderate fever as the only side effect. There were no grade 3 or 4 toxicities associated with the vaccines or any evidence of autoimmunity. Local accumulations of infiltrating T cells were found at the site of vaccination. The vaccinations induced distinct immunological responses to KLH, both in vitro and in vivo. Importantly, after three vaccinations, cytotoxic activity against autologous tumor cells was detected in a subgroup of patients. Conclusions: This study demonstrated that autologous tumor lysate-pulsed dendritic cell-based therapy is feasible, well-tolerated, and capable of inducing immunological response to tumor cells in mesothelioma patients. Clinical trial registered with www.clinicaltrials.gov (NCT00280982).

Sequencing and quantifying igg fragments and antigen-binding regions by mass spectrometry (Article)

2010-06-01T00:00:00Z

In cancer and autoimmune diseases, immunoglobulins with a specific molecular signature that could potentially be used as diagnostic or prognostic markers are released into body fluids. An immunomics approach based on this phenomenon relies on the ability to identify the specific amino acid sequences of the complementarity-determining regions (CDR) of these immunoglobulins, which in turn depends on the level of accuracy, resolution, and sensitivity that can be achieved by advanced mass spectrometry. Reproducible isolation and sequencing of antibody fragments (e.g., Fab) by high-resolution mass spectrometry (MS) from seven healthy donors revealed 43 217 MS signals: 225 could be associated with CDR1 peptides, 513 with CDR2 peptides, and 19 with CDR3 peptides. Seventeen percent of the 43 217 MS signals did not overlap between the seven donors. The Fab isolation method used is reproducible and fast, with a high yield. It provides only one Fab sample fraction for subsequent characterization by high-resolution MS. In 17% and 4% of these seven healthy donors, qualitative (presence/absence) and quantitative (intensity) differences in Fab fragments could be demonstrated, respectively. From these results, we conclude that the identification of a CDR signature as biomarker for autoimmune diseases and cancer without prior knowledge of the antigen is feasible.

A randomized phase II study comparing two schedules of the 21-day regimen of gemcitabine and carboplatin in advanced non-small cell lung cancer (Article)

2010-06-01T00:00:00Z

Purpose: Carboplatin area under the curve (AUC) 5 ml/min on day 1 with gemcitabine 1,250 mg/m2on day 1 and day 8 is a widely used regimen in advanced non-small cell lung cancer. Grade 3-4 thrombocytopenia and neutropenia are frequent. The aim of this study is to investigate whether toxicity of gemcitabine/carboplatin could be reduced by administering carboplatin on day 8 instead of day 1 without a decrease in response rate (RR). Methods: Patients received gemcitabine 1,250 mg/m2on days 1 and 8, carboplatin AUC 5 on day 1 (arm A) or day 8 (arm B). Drugs were administered over a 21-day cycle. Toxicity and RR were evaluated weekly and every second cycle, respectively. Results: 71 patients were enrolled into the study. We found 79% (95% CI 61-91%) grade 3-4 toxicity (neutropenia and thrombocytopenia) in arm A and 50% (95% CI 32-68%) in arm B; 66% grade 3-4 thrombocytopenia in arm A and 26% in arm B. We observed 30% grade 4 hematological toxicity in arm A and 3% in arm B. In arm A an overall RR of 20% (95% CI 7.7-38.6%) was seen, and 18.2% (95% CI 7-35.5%) in arm B. Conclusions: Although the study was prematurely closed, the current data are of interest. The schedule with carboplatin on day 8 is associated with substantially lower grade 3-4 neutropenia and thrombocytopenia with comparable dose intensity and RR.

United airways: Circulating Th2 effector cells in an allergic rhinitis model are responsible for promoting lower airways inflammation (Article)

2010-03-01T00:00:00Z

Background Allergic rhinitis (AR) and asthma often coexist and are referred to as 'united airways' disease. However, the molecular and cellular pathways that are crucially involved in the interaction between upper and lower airways remain to be identified. Objective We sought to assess whether and how AR exacerbates lower airway inflammation upon allergen challenge in mice. Methods We previously developed an intranasal ovalbumin (OVA)-driven AR model, characterized by nasal eosinophilic inflammation, enhanced serum levels of OVA-specific IgE and Th2 cytokine production in cervical lymph nodes. In OVA-sensitized mice with or without AR, a lower airway challenge was given, and after 24 h, lower airway inflammation was analysed. Results We found that AR mice were more susceptible to eosinophilic inflammation following a lower airway OVA challenge than OVA-sensitized controls. AR mice manifested increased numbers of eosinophils in bronchoalveolar lavage fluid and increased inter-cellular adhesion molecule-1 (ICAM-1) expression on lung endothelium, when compared with OVA-sensitized controls. Depletion of T cells in OVA-challenged AR mice completely abrogated all hallmarks of lower airway inflammation, including enhanced IL-5 and tissue eosinophilia. Conversely, adoptive transfer of Th2 effector cells in naïve animals induced lower airway eosinophilic inflammation after challenge with OVA. Blocking T cell recirculation during AR development by the spingosine-1 analogue FTY720 also prevented lower airway inflammation including ICAM-1 expression in AR mice upon a single lower airway challenge. Conclusion Our mouse model of 'united airways' disease supports epidemiological and clinical data that AR has a significant impact on lower airway inflammation. Circulating Th2 effector cells are responsible for lung priming in AR mice, most likely through up-regulation of ICAM-1.

A novel method for isolating dendritic cells from human bronchoalveolar lavage fluid (Article)

2009-12-31T00:00:00Z

Background: Dendritic cells (DCs) play a pivotal role in linking the innate and adaptive immune response and have been implicated in a variety of pulmonary diseases. Currently, studies on the role of DCs are limited by difficulties in isolating DCs from the lung. Surgical lung specimens are not readily available and purification of DCs from digested lung tissue is likely to induce phenotypical and functional changes. DCs obtained from the alveolar spaces are thought to represent the local microenvironment and can be obtained using minimally invasive techniques. We developed a novel method of isolating DCs from bronchoalveolar lavage (BAL) fluid. Methods: After removal of macrophages, the remaining BAL cells were stained with a lineage mix (CD3-, CD14-, CD16-, CD19-, CD56-FITC), CD11c and HLA-DR and sorted with a FACS ARIA. DAPI was used as a dead-live marker. mDCs were low autofluorescent, lineage mix negative, CD11c+and HLA-DR+cells. pDCs were CD11c-but CD123+. Morphological assessment of sorted mDCs and pDCs was performed. Sorted mDCs were tested in a mixed leukocyte reaction (MLR) with naive CD4+T cells and evaluated for T cell differentiation and cytokine production. With confocal microscopy DC-T cell interaction was assessed. Results: Using our sorting strategy, mDCs and pDCs, with a high purity upon FACS analysis of the sorted fraction, were obtained. These cells showed the morphological characteristics of DCs. Most importantly, mDCs were able to induce T cell proliferation and differentiation in a MLR, and interact with T cells as assessed by confocal microscopy. These results indicate the presence of functional DCs. Freezing and thawing of the BAL cells did not affect phenotype or T cell stimulatory capacity of the isolated DCs. Conclusion: Using a novel sorting strategy, functional mDCs can be isolated from BAL fluid, enabling a detailed study in pulmonary disease.

Oral UFT, etoposide and leucovorin in recurrent non-small cell lung cancer: A non-randomized phase II study (Article)

2009-12-01T00:00:00Z

Background: Oral treatment regimens with few side effects are appealing in the 2nd or 3rd line treatment of non-small cell lung cancer (NSCLC) patients. Purpose: The aim was to investigate the efficacy and toxicity of the oral combination etoposide, Uracil-Tegafur (UFT) and leucovorin in 2nd or 3rd line in Caucasian patients with advanced NSCLC. Methods: Etoposide 50 mg/m2, UFT 250 mg/m2and leucovorin 90 mg (fixed dose) were dosed in 3 gifts approximately 8 h apart for 14 days followed by 1 week rest every 3 weeks until progressive disease (PD). Primary endpoint was response rate (RR), secondary endpoints toxicity and time to progression (TTP). Results: The median number of cycles was 3.5 (95% CI 2-5); 9 patients received ≥6 cycles, 4 > 10 cycles. The median dose intensities for etoposide and UFT were 223 mg/m2/week (95% CI 213-232) and 1092 mg/m2/week (95% CI 1032-1167), the relative dose intensities 92% and 90%, respectively. Grade 3/4 neutropenia was observed in 12% (4/32), grade 3/4 thrombocytopenia in 15% (5/32), without febrile neutropenia. Non-hematological toxicity grade 3 included hepatic toxicity (6%), lethargy (15%), diarrhea (3%) and nausea (3%). One patient developed grade 4 arterial ischemia. Fourteen percent (95% CI 4-33%) (4/28) had a confirmed partial response, 57% (95% CI 44-81%) (16/28) stable disease and 28% (95% CI 19-56%) (8/28) progressive disease. The median TTP was 3 months (95% CI 1.3-4.4), the median overall survival 6.7 months (95% CI 4.0-9.3). Conclusion: The combination of UFT, etoposide and leucovorin is active in 2nd or 3rd line therapy of Caucasian NSCLC patients and because of its favourable toxicity profile this treatment warrants further investigation.

Non-cross resistant sequential single agent chemotherapy in first-line advanced non-small cell lung cancer patients: Results of a phase II study (Article)

2009-12-01T00:00:00Z

Background. sequential chemotherapy can maintain dose intensity and preclude cumulative toxicity by increasing drug diversity. Purpose. to investigate the toxicity and efficacy of the sequential regimen of gemcitabine followed by paclitaxel in first line advanced stage non-small cell lung cancer (NSCLC) patients with good performance status (PS). Patients and methods. gemcitabine 1250mg/m2was administered on day 1 and 8 of course 1 and 2; Paclitaxel 150mg/m2on day 1 and 8 of course 3 and 4. Primary endpoint was response rate (RR), secondary endpoints toxicity and time to progression (TTP). Results. Of the 21 patients (median age 56, range 38-80 years; 62 males, 38 females) 10 (2/21) had stage IIIB, 90 (19/21) stage IV, 15% PS 0, 85 PS 1. 20 of patients had a partial response, 30 stable disease, 50 progressive disease. Median TTP was 12 weeks (range 6-52 weeks), median overall survival (OS) 8 months (range 1-27 months), 1-year survival was 33. One patient had grade 3 hematological toxicity, 2 patients a grade 3 peripheral neuropathy. Conclusions. sequential administration of gemcitabine followed by paclitaxel in first line treatment of advanced NSCLC had a favourable toxicity profile, a median TTP and OS comparable with other sequential trials and might, therefore, be a treatment option for NSCLC patients with high ERCC1 expression.

The lung vascular filter as a site of immune induction for T cell responses to large embolic antigen (Article)

2009-11-23T00:00:00Z

The bloodstream is an important route of dissemination of invading pathogens. Most of the small bloodborne pathogens, like bacteria or viruses, are filtered by the spleen or liver sinusoids and presented to the immune system by dendritic cells (DCs) that probe these filters for the presence of foreign antigen (Ag). However, larger pathogens, like helminths or infectious emboli, that exceed 20 μm are mostly trapped in the vasculature of the lung. To determine if Ag trapped here can be presented to cells of the immune system, we used a model of venous embolism of large particulate Ag (in the form of ovalbumin [OVA]-coated Sepharose beads) in the lung vascular bed. We found that large Ags were presented and cross-presented to CD4 and CD8 T cells in the mediastinal lymph nodes (LNs) but not in the spleen or liver-draining LNs. Dividing T cells returned to the lungs, and a short-lived infiltrate consisting of T cells and DCs formed around trapped Ag. This infiltrate was increased when the Toll-like receptor 4 was stimulated and full DC maturation was induced by CD40 triggering. Under these conditions, OVA-specific cytotoxic T lymphocyte responses, as well as humoral immunity, were induced. The T cell response to embolic Ag was severely reduced in mice depleted of CD11chicells or Ly6C/G+cells but restored upon doptive transfer of Ly6Chimonocytes. We conclude that the lung vascular filter represents a largely unexplored site of immune induction that traps large bloodborne Ags for presentation by monocyte-derived DCs.

Both conventional and interferon killer dendritic cells have antigen-presenting capacity during influenza virus infection (Article)

2009-09-28T00:00:00Z

Natural killer cells are innate effector cells known for their potential to produce interferon-γ and kill tumour and virus-infected cells. Recently, B220+CD11cintNK1.1+ NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220- NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.

An anti-inflammatory role for plasmacytoid dendritic cells in allergic airway inflammation (Article)

2009-07-15T00:00:00Z

It was previously shown that administration of recombinant human Fms-like tyrosine kinase receptor-3 ligand (Flt3L) before allergen challenge of sensitized mice suppresses the cardinal features of asthma through unclear mechanisms. Here, we show that Flt3L dramatically alters the balance of conventional to plasmacytoid dendritic cells (pDCs) in the lung favoring the accumulation of pDCs. Selective removal of pDCs abolished the antiinflammatory effect of Flt3L, suggesting a regulatory role for these cells in ongoing asthmatic inflammation. In support, we found that immature pDCs are recruited to the lungs of allergen-challenged mice irrespective of Flt3L treatment. Selective removal of pDCs during allergen challenge enhanced airway inflammation, whereas adoptive transfer of cultured pDCs before allergen challenge suppressed inflammation. Experiments in which TLR9 agonist CpG motifs were administered in vitro or in vivo demonstrated that pDCs were antiinflammatory irrespective of their maturation state. These effects were mediated through programmed death-1/programmed death ligand 1 interactions, but not through ICOS ligand, IDO, or IFN-α. These findings suggest a specialized immunoregulatory role for pDCs in airway inflammation. Enhancing the antiinflammatory properties of pDCs could be employed as a novel strategy in asthma treatment. Copyright

Blockade of CCR4 in a humanized model of asthma reveals a critical role for DC-derived CCL17 and CCL22 in attracting Th2 cells and inducing airway inflammation (Article)

2009-07-01T00:00:00Z

Background: As Th2 type lymphocytes orchestrate the cardinal features of allergic asthma, inhibiting their recruitment to the lungs could be of therapeutic benefit. Although human Th2 cells express the CCR4 chemokine receptor and increased production of CCR4 ligands has been found in asthmatic airways, studies in animals have reached contradictory conclusions on whether blocking this pathway would be beneficial. Objective: As a lack of efficacy might be due to differences between mouse and man, we readdressed this question using a humanized severe combined immunodeficiency model of asthma. Methods: Mice received peripheral blood mononuclear cells from house dust mite (HDM) allergic asthmatic patients and then underwent bronchial challenge with HDM. Results: This resulted in marked allergic inflammation and bronchial hyper-reactivity. Administration of CCR4 blocking antibody abolished the airway eosinophilia, goblet cell hyperplasia, IgE synthesis and bronchial hyperreactivity. In this chimeric system, human CD11c+ dendritic cells (DCs) were the predominant source of CCR4 ligands, suggesting that DC-derived chemokines attract Th2 cells. In separate experiments using human DCs, in vitro exposure to HDM of DCs from HDM allergic patients but not healthy controls caused CCL17 and CCL22 release that resulted in chemoattraction of polarized human Th2 cells in a CCR4-dependent way. Conclusions: Taken together, our data provide proof of concept that CCR4 blockade inhibits the salient features of asthma and justify further clinical development of CCR4 antagonists for this disease.

Cholera toxin B suppresses allergic inflammation through induction of secretory IgA (Article)

2009-06-30T00:00:00Z

In healthy individuals, humoral immune responses to allergens consist of serum IgA and IgG4, whereas cellular immune responses are controlled by regulatory T (Treg) cells. In search of new compounds that might prevent the onset of allergies by stimulating this type of immune response, we have focused on the mucosal adjuvant, cholera toxin B (CTB), as it induces the formation of Treg cells and production of IgA. Here, we have found that CTB suppresses the potential of dendritic cells to prime for Th2 responses to inhaled allergen. When we administered CTB to the airways of naïve and allergic mice, it strongly suppressed the salient features of asthma, such as airway eosinophilia, Th2 cytokine synthesis, and bronchial hyperreactivity. This beneficial effect was only transferable to other mice by transfer of B but not of T lymphocytes. CTB caused a transforming growth factor-β-dependent rise in antigen-specific IgA in the airway luminal secretions, which was necessary for its preventive and curative effect, as all effects of CTB were abrogated in mice lacking the luminal IgA transporting polymeric Ig receptor. Not only do these findings show a novel therapeutic avenue for allergy, they also help to explain the complex relationship between IgA levels and risk of developing allergy in humans.

Pre-operative pulmonary evaluation of lung cancer patients: A review of the literature (Article)

2009-05-01T00:00:00Z

Complete anatomical resection of the primary tumour is still the standard of care in patients with early stage lung cancer. Because these patients are usually smokers who also suffer from chronic obstructive pulmonary disease, regional differences in pulmonary function due to lung tissue destruction exist. he purpose of the present article is to evaluate the currently available guidelines and to discuss novel methods for the pre-operative functional and anatomical pulmonary evaluation in lung cancer patients. espite the fact that knowledge on the pre-operative evaluation of the pulmonary function has substantially increased during the past decade, the majority of the studies are small, underpowered and, with exception of a proposed algorithm, not prospectively validated in independent cohorts.The future harmonisation of guidelines is required and novel imaging techniques should be incorporated in the pre-operative evaluation in chronic obstructive pulmonary disease patients with borderline pulmonary function. Copyright

Stem cells and the natural history of lung cancer: Implications for lung cancer screening (Article)

2009-04-01T00:00:00Z

Lung cancer is not simply a single disease, but a collection of several phenotypically very diverse and regionally distinct neoplasias. Its natural history is complex and not yet fully understood. Stem cells and the complex interaction with the microenvironment of the tumor and the immune system play an important role in tumor progression and metastasizing capacity. This finding explains why lung cancer does not always follow the multistep carcinogenetic and exponential growth model and why small lesions do not always equate to early-stage disease. Despite the fact that volume doubling times are increasingly used as surrogate markers for the natural history of lung cancer and as estimates for the proportion of overdiagnosed cases, it is only a momentary impression. At baseline screening especially, screen-detected lung cancer cases are preferably detected when they are in the indolent phase of their growth curve (length-biased sampling), from which it can by no means be concluded that they may not progress or metastasize at a later stage. Because the natural history of lung cancer is only partly elucidated, conclusions on the impact of overdiagnosis in lung cancer screening are premature.

The effects of guideline implementation for proton pump inhibitor prescription on two pulmonary medicine wards (Article)

2009-01-01T00:00:00Z

Background: It has been demonstrated that 40% of patients admitted to pulmonary medicine wards use proton pump inhibitors (PPIs) without a registered indication. Aim: To assess whether implementation of a guideline for proton pump inhibitor (PPI) prescription on pulmonary medicine wards could lead to a decrease in use and improved appropriateness of prescription. Methods: This prospective study comprised two periods, i.e. the situation before and after guideline implementation. In each period, 300 consecutive patients were included. We registered patient characteristics, medications and occurrence of upper gastrointestinal-related disorders. Results: After implementation, fewer patients were started on PPIs [21% vs. 13%; odds ratio (OR): 0.56; 95% confidence interval (CI): 0.33-0.97] and more users discontinued their use; however, the latter was not significant (3% vs. 6%; OR for continuation: 0.56; 95% CI: 0.14-2.23). Multivariable logistic regression analysis confirmed that PPI use during hospitalization decreased after implementation (adjusted pooled OR: 0.54; 95% CI: 0.32-0.90). Implementation did not result in a change in reported reasons for PPI prescription. There was no significant difference in the occurrence of upper GI-related disorders in the first 3 months after discharge. Conclusions: Guideline implementation for PPI prescription on two pulmonary medicine wards resulted in a reduction in the number of patients starting PPIs during hospitalization, but appropriateness of prescribing PPIs was not affected. Further studies are needed to determine how appropriateness of PPI prescription on pulmonary medicine wards can be further improved.

Sensitization by intratracheally injected dendritic cells is independent of antigen presentation by host antigen-presenting cells (Article)

2009-01-01T00:00:00Z

Adoptive transfer of antigen-pulsed dendritic cells (DC) in the airways of mice has been used as a model system for eosinophilic airway inflammation, which allows studying the DC-specific contribution of genes of interest or reagents to induced inflammation by genetically modifying DC or exposure of DC to compounds prior to injection in the airways. Antigen transfer and CD4+T cell priming by endogenous antigen-presenting cells (APCs) may interfere with the correct interpretation of the data obtained in this model, however. We therefore examined antigen transfer and indirect CD4+T cell priming by host APCs in this model system. Transfer of antigen between injected DC and host cells appeared to be minimal but could not be totally excluded. However, only direct antigen presentation by injected DC resulted in robust CD4+T cell priming and eosinophilic airway inflammation. Thus, this adoptive transfer model is well suited to study the role of DC in eosinophilic airway inflammation.

Inducible costimulator blockade prolongs airway luminal patency in a mouse model of obliterative bronchiolitis (Article)

2008-11-27T00:00:00Z

BACKGROUND.: In human lung transplantation, chronic rejection is accompanied by obliterative bronchiolitis (OB), a fibrosing inflammatory condition that leads to occlusion of the bronchial lumen and graft failure. The pathogenesis of this disorder is poorly understood, but likely involves antigen presentation by dendritic cells (DC). We studied the presence and activation status of DCs in transplanted tracheas in a mouse model of OB and studied the effect on graft luminal patency of blocking the costimulatory B7RP-1/inducible costimulator (ICOS) pathway. METHODS.: Tracheas from Balb/C or from C57Bl/6 mice were transplanted heterotopically under the dorsal skin of C57Bl/6 mice. Histologic, fluorescence-activated cell sorter, and quantitative-polymerase chain reaction analyses were performed after 1, 2, or 4 weeks. In some groups, treatment with blocking rat anti-mICOS antibodies or irrelevant rat immunoglobulin G was administered during the entire observation period. RESULTS.: After heterotopic transplantation, both CD103CD11b and CD103CD11bMHC IIDCs accumulated in the airway epithelium as early as 1 week after allogeneic (mismatched) but not syngeneic (matched) transplantation. Four weeks after Tx, infiltration with CD11c MHCIIDCs and CD8 lymphocytes, luminal fibrosis and epithelial damage were more pronounced in the allogeneic than in the syngeneic setting. There was a 10-fold up-regulation of ICOS mRNA and of chemokines involved in T-cell influx in the mismatched setting compared with the matched setting. Strikingly, anti-ICOS treatment without other immunosuppression prevented luminal fibrosis in mismatched transplants. CONCLUSIONS.: Our results suggest that early infiltration by DC occurs in posttransplant OB. Blocking critical costimulatory molecules expressed on DCs, as in the B7RP1-ICOS pathway, prevents epithelial damage and luminal fibrosis.

Clearance of influenza virus from the lung depends on migratory langerin+CD11b- but not plasmacytoid dendritic cells (Article)

2008-07-07T00:00:00Z

Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b-and CD11b+conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b-CD11chiDCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chiDCs in the trachea and lung interstitium. In the MLNs, the CD11b+DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b-CD8α+DCs presented to CD8 cells, and migratory CD11b-CD8α-DCs presented to CD4 and CD8 T cells. When lung CD11chiDCs and macrophages or langerin+CD11b-CD11chiDCs were depleted using either CD11c - diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specif c CD8+T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+CD11cintpDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.

N-terminal pro-brain natriuretic peptide testing in the emergency department: Beneficial effects on hospitalization, costs, and outcome (Article)

2008-07-01T00:00:00Z

Background: N-terminal pro-brain natriuretic peptide (NT-proBNP) is an established biomarker for heart failure. Assessment of this biomarker in patients with acute dyspnea presenting to the emergency department (ED) may aid diagnostic decision-making, resulting in improved patient care and reduced costs. Methods: In a prospective clinical trial, patients presenting with acute dyspnea to the ED of the Erasmus Medical College, Rotterdam, the Netherlands, were randomized for either rapid measurement or no measurement of NT-proBNP. For ruling out heart failure, cutoff values of 93 pg/mL in male and 144 pg/mL in female patients were used, and for ruling in heart failure, a cutoff value of 1,017 pg/mL was used. Time to discharge from the hospital and costs related to hospital admission were primary end points. Bootstrap analysis was used for comparison of costs and 30-day mortality between the NT-proBNP and control group. Results: A total of 477 patients (54% male) was enrolled. The mean age was 59 years, with 44% of patients having a history of cardiac disease. Median time to discharge from the hospital was 1.9 days (interquartile range [IQR], 0.12-8.4 days) in the NT-proBNP group (n = 241) compared with 3.9 days (IQR, 0.16-11.0 days) in the control group (n = 236) (P = .04). Introduction of NT-proBNP testing resulted in a trend toward reduction in costs related to hospital admission and diagnostic investigations of $1,364 per patient (95% CI $-246 to $3,215), whereas 30-day mortality was similar (15 patients in the NT-proBNP and 18 patients in the control group). Conclusions: Introduction of NT-proBNP testing for heart failure in the ED setting reduces the time to discharge and is associated with a trend toward cost reduction.

Inappropriate prescription of proton pump inhibitors on two pulmonary medicine wards (Article)

2008-07-01T00:00:00Z

Objective: We recently noticed that proton pump inhibitor (PPI) use was high on a pulmonary medicine ward of a university clinic and reasons for this high usage were not clear. Our aim was to determine the indications for PPI use on two pulmonary medicine wards and to assess whether this use was appropriate. Methods: We assessed prospectively the number of patients on PPIs and the indications for PPI use on two pulmonary medicine wards, one from a university and one from a regional clinic in The Netherlands. Results: On admission, 88 of 300 (29%) patients already used PPIs. The use of PPIs was discontinued in three (1%) patients, whereas PPIs were initiated in 45 (15%) patients, resulting in 130 (43%) patients on PPIs during hospitalization. The most common indication for PPI use was the prevention of medication-associated complications. In 78 of 130 (60%) patients on PPIs, this medication was used for a registered indication, whereas in 52 (40%) patients a registered indication was not present (overuse). In contrast, 19 of 300 (6%) patients were not prescribed PPIs despite the presence of a registered indication for its use (underuse). No differences were found in prescription rate and indications for PPI use between the university and the regional clinic. Conclusion: PPI use was very common on two pulmonary medicine wards in the Netherlands. Forty percent of the patients used their PPIs for a nonregistered indication. As use of PPIs is costly and may be associated with side effects, hospital physicians should to be better educated on guidelines for its use.

Efficacy of infliximab in extrapulmonary sarcoidosis: Results from a randomised trial (Article)

2008-06-01T00:00:00Z

The aim of the present study was to investigate the efficacy of infliximab for the treatment of extrapulmonary sarcoidosis. A prospective, randomised, double-blind, placebo-controlled trial was conducted, with infliximab at 3 and 5 mg·kg-1body weight administered over 24 weeks. Extrapulmonary organ severity was determined by a novel severity tool (extrapulmonary physician organ severity tool; ePOST) with an adjustment for the number of organs involved (ePOSTadj). In total, 138 patients enrolled in the trial of infliximab versus placebo for the treatment of chronic corticosteroid-dependent pulmonary sarcoidosis. The baseline severity of extrapulmonary organ involvement, as measured by ePOST, was similar across treatment groups. After 24 weeks of drug-therapy study, the change from baseline to week 24 in ePOST was greater for the combined infliximab group compared with the placebo group. After adjustment for the number of extrapulmonary organs involved, the improvement in ePOSTadj observed in the combined infliximab group was also greater than that observed in placebo-treated patients, after 24 weeks of therapy. The improvements in ePOST and ePOSTadj were not maintained during a subsequent 24-week washout period. Infliximab may be beneficial compared with placebo in the treatment of extrapulmonary sarcoidosis in patients already receiving corticosteroids, as assessed by the severity tool described in the present study. Copyright

How to deal with incidentally detected pulmonary nodules less than 10 mm in size on CT in a healthy person (Article)

2008-05-01T00:00:00Z

The high frequency of non-calcified pulmonary nodules (NCN) <10 mm incidentally detected on a multi-detector CT (MDCT) of the chest raises the question of how clinicians and radiologists should deal with these nodules. Management algorithms for solitary pulmonary nodules >10 mm do not carry across to sub-centimeter lesions. Purpose of this review is to provide a 10-step approach for routinely detected sub-centimeter NCN on a MDCT in healthy persons in order to be able to make an optimal discrimination between benign and malignant NCNs. Recommendations are primarily based on individual cancer risk, the presence or absence of calcifications and nodule size. In nodules >4-5 mm nodule consistency, margin and shape should be taken into account. Next steps in the nodule evaluation are the assessment of localization, nodule number, presence or absence of growth and volume doubling time. Growth is defined as a volume doubling time of 400 days or less, based on volumetry. For nodules <4 mm, a follow-up CT at 12 months is recommended in high risk persons, whilst for low-risk persons no follow-up is needed. If no growth is observed at 12 months, no further follow-up is required. For solid, smooth or attached indeterminate NCN between 5 and 10 mm we recommend an annual repeat scan, whilst for purely intra-parenchymal nodules a 3-month repeat scan should be made to assess growth. Growing lesions with a volume doubling time <400 days require further work-up and diagnosis, otherwise an annual repeat scan to assess growth is recommended.

Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells (Article)

2008-04-14T00:00:00Z

Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)-resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c+monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting "nature's adjuvant," the inflammatory DC through induction of the endogenous danger signal uric acid. JEM

Modulation of neurotrophin and neurotrophin receptor expression in nasal mucosa after nasal allergen provocation in allergic rhinitis (Article)

2008-04-01T00:00:00Z

Background: Patients with allergic rhinitis (AR) feature both allergic airway inflammation and a hyperresponsiveness to nonspecific stimuli which is partly neuronally controlled. Still, it is unclear whether or not neurotrophins are involved in airway pathophysiology of AR and in nasobronchial interaction. Methods: Nine AR patients with mono-allergy to grass pollen and nine healthy controls underwent nasal allergen provocation (NP). Serum samples, nasal and bronchial biopsies were taken before (T0) and 24 h after (T24) NP. Pan-neurotrophin receptor p75NTR, tyrosine kinase A (trkA), trkB, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) were assessed with immunohistochemistry, and NGF and BDNF levels with ELISA. Results: At T24, BDNF and NGF were upregulated in nasal mucosa (P < 0.05) and increased in the peripheral blood of AR compared with T0. The increase in nasal BDNF expression correlated positively with the maximum increase in total nasal symptom score in AR (P = 0.02). p75NTRwas expressed on peripheral nerves and epithelial layer, trkA on endothelial cells, and trkB on mast cells. trkB + mast cells significantly decreased after NP in AR (P < 0.01). NP did not modulate p75NTRand trkA expression in nasal mucosa and had no effect on the expression of neurotrophins and receptors in bronchial mucosa. Conclusion: This study shows that neurotrophins and their receptors are expressed in human airways. Allergic rhinitis was characterized by a modulation of BDNF, NGF, and trkB in nasal mucosa after NP and a correlation of nasal BDNF with the maximal increase of total nasal symptom score. Therefore, our data suggest that neurotrophins participate in upper-airway pathophysiology in AR, whereas their role in nasobronchial interaction remains unclear.

HIF1a expression in bronchial biopsies correlates with tumor microvascular saturation determined using optical spectroscopy (Article)

2007-09-01T00:00:00Z

Tumor hypoxia is generally considered to be related to aggressive behaviour of a tumor. As in lung cancer direct determination of oxygenation is difficult, hypoxia-related proteins have been studied. A number of studies on these proteins show different results and the usefulness of these protein expressions remains questionable. In this article, we relate one of these hypoxia-related proteins (hypoxia-inducible factor, HIF1a) to a direct in vivo spectroscopic measurement of tumor blood saturation performed during bronchoscopy. Seventeen samples from malignancies and non-malignant tissues were studied. Microvascular saturation levels in the no malignancy group equalled 87 ± 11.5% (range 71-100%) and in the malignant group 43 ± 21% (range 6-63%). This difference was statistically significant (p < 0.0002). There was a significant difference in the spectroscopically determined saturations between the biopsies with negative expression of HIF1a and the biopsies with positive expression of HIF1a (p < 0.005). From these data, it can be concluded that HIF1a expression is related to a low microvascular blood saturation as determined in vivo by optical spectroscopy. This study may lead to a better acceptance of the usage of different techniques to establish hypoxia in order to study the effect of hypoxia on therapeutic interventions and prognosis of lung cancer.

Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells (Article)

2007-08-01T00:00:00Z

Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X or P2Y receptors. Here we show that allergen challenge causes acute accumulation of ATP in the airways of asthmatic subjects and mice with experimentally induced asthma. All the cardinal features of asthma, including eosinophilic airway inflammation, Th2 cytokine production and bronchial hyper-reactivity, were abrogated when lung ATP levels were locally neutralized using apyrase or when mice were treated with broad-spectrum P2-receptor antagonists. In addition to these effects of ATP in established inflammation, Th2 sensitization to inhaled antigen was enhanced by endogenous or exogenous ATP. The adjuvant effects of ATP were due to the recruitment and activation of lung myeloid dendritic cells that induced Th2 responses in the mediastinal nodes. Together these data show that purinergic signaling has a key role in allergen-driven lung inflammation that is likely to be amenable to therapeutic intervention.

Activation of the D prostanoid 1 receptor suppresses asthma by modulation of lung dendritic cell function and induction of regulatory T cells (Article)

2007-02-01T00:00:00Z

Prostaglandins (PGs) can enhance or suppress inflammation by acting on different receptors expressed by hematopoietic and nonhematopoietic cells. Prostaglandin D2binds to the D prostanoid (DP)1 and DP2 receptor and is seen as a critical mediator of asthma causing vasodilation, bronchoconstriction, and inflammatory cell influx. Here we show that inhalation of a selective DP1 agonist suppresses the cardinal features of asthma by targeting the function of lung dendritic cells (DCs). In mice treated with DP1 agonist or receiving DP1 agonist-treated DCs, there was an increase in Foxp3+CD4+regulatory T cells that suppressed inflammation in an interleukin 10-dependent way. These effects of DP1 agonist on DCs were mediated by cyclic AMP-dependent protein kinase A. We furthermore show that activation of DP1 by an endogenous ligand inhibits airway inflammation as chimeric mice with selective hematopoietic loss of DP1 had strongly enhanced airway inflammation and antigen-pulsed DCs lacking DP1 were better at inducing airway T helper 2 responses in the lung. Triggering DP1 on DCs is an important mechanism to induce regulatory T cells and to control the extent of airway inflammation. This pathway could be exploited to design novel treatments for asthma. JEM

Comparison of total-breath and single-breath diffusing capacity in healthy volunteers and COPD patients (Article)

2007-01-01T00:00:00Z

Background: The measurement of single-breath diffusing capacity (DLCOSB) assumes that diffusing capacity per liter of alveolar volume (DLCO/VA) determined in a 750-mL gas sample represents the diffusing capacity (DLCO) of the entire lung. Fast-responding gas analyzers provide the opportunity to verify this assumption because of the possibility to measure CO and CH4fractions continuously throughout the entire expiration. Continuous gas sampling provides more information per measurement, but this information cannot be expressed in the traditional parameters. Our goals were to find new parameters to express the extra information of the continuous gas sampling, and to compare these new parameters with the traditional parameters. Methods: We compared a new method to determine DLCO with the traditional method in 62 healthy volunteers and 26 COPD patients. Traditionally, DLCOSBis determined by multiplying DLCO/VA with alveolar volume, both calculated from gas concentrations in a 750-mL gas sample. The new method calculates total-breath DLCO (DLCOTB) by integration of DLCO/VA against exhaled volume. Results: In healthy volunteers, DLCO/VA shows a slight upward slope during exhalation, while in COPD patients DLCO/VA shows a horizontal line. Total-breath total lung capacity (TLC) is larger than single-breath TLC both in healthy volunteers and in COPD patients, leading to a DLCOTBthat is significantly larger than DLCOSBin both groups (p < 0.001). Conclusion: The assumption that a 750-mL gas sample represents the entire lung seems to be correct for DLCO/VA but not for the CH4fraction in case of ventilation inhomogeneity.

Formoterol added to low-dose budesonide has no additional antiinflammatory effect in asthmatic patients. (Article)

2005-09-01T00:00:00Z

STUDY OBJECTIVE: Adding inhaled long-acting beta2-agonists to a low dose of inhaled corticosteroids (ICSs) results in better asthma control than increasing the dose of ICSs. An important, but as yet unresolved, question is whether this is due to an additional reduction of airway inflammation. DESIGN: Double-blind, parallel-group trial. PATIENTS: Forty asthma patients (FEV1, 50 to 90% predicted; provocative concentration of a substance [methacholine] causing a 20% fall in FEV1 of < 8 mg/mL; no ICSs in the last 4 weeks). INTERVENTIONS: Randomization to 8 weeks of treatment with 100 microg of budesonide bid plus placebo (BUD200) or 100 microg of budesonide bid plus 12 microg of formoterol (BUD200 + F). Then the dose of budesonide (BUD) was increased to 400 microg bid in both groups for another 8 weeks. Bronchial biopsy specimens were collected before, and after 8 and 16 weeks of treatment. Eosinophils (major basic protein [MBP]) and mast cells (tryptase) were analyzed by immunohistochemistry. RESULTS: BUD200 reduced the MBP staining (p = 0.008) and tryptase staining (p = 0.048) in the epithelium compared to baseline levels. There were no significant differences between the BUD200 and BUD200 + F groups. In both groups, increasing the dosage of BUD to 800 microg had no significant additional antiinflammatory effect. CONCLUSIONS: Our results demonstrate that BUD administered at a low dose has significant antiinflammatory effects in patients with mild asthma. No significant additional antiinflammatory effects could be demonstrated either by adding formoterol or by increasing the dose of BUD.

Measurement of hypoxia-related parameters in bronchial mucosa by use of optical spectroscopy. (Article)

2005-05-15T00:00:00Z

RATIONALE: Tumor hypoxia has both prognostic and therapeutic consequences for solid tumors. We developed a novel noninvasive technique, differential path-length spectroscopy (DPS), which allows the measurement of hypoxia-related parameters in the superficial microvasculature of tissue. OBJECTIVES: The aim of this study was to measure the microvascular oxygenation of histologically normal endobronchial mucosa and of neoplastic lesions during bronchoscopy using DPS. METHODS: Sixty-four patients with known or suspected malignancies of the lung were studied. One hundred and five endobronchial lesions (38 histologically normal, 37 metaplastic/mild dysplastic lesions, and 30 invasive carcinomas) were detected by white and/or autofluorescence bronchoscopy and measured using DPS. RESULTS: We observed that bronchial tumors are characterized by a lower blood oxygen saturation and a higher blood content than normal mucosa. No differences were observed between normal and metaplastic/mild dysplastic mucosa. CONCLUSION: DPS is a new optical technique allowing the noninvasive study of endobronchial tumor hypoxia.

Immunotherapy of murine malignant mesothelioma using tumor lysate-pulsed dendritic cells. (Article)

2005-05-15T00:00:00Z

RATIONALE: Exploiting the immunostimulatory capacities of dendritic cells holds great promise for cancer immunotherapy. Currently, dendritic cell-based immunotherapy is evaluated clinically in a number of malignancies, including melanoma and urogenital and lung cancer, showing variable but promising results. OBJECTIVE: To evaluate if pulsed dendritic cells induce protective immunity against malignant mesothelioma in a mouse model. METHODS: Malignant mesothelioma was induced in mice by intraperitoneal injection of the AB1 mesothelioma cell line, leading to death within 28 days. For immunotherapy, dendritic cells were pulsed overnight either with AB1 tumor cell line lysate, AB1-derived exosomes, or ex vivo AB1 tumor lysate, and injected either before (Days -14 and -7) at the day of (Day 0) or after (Days +1 and +8) tumor implantation. MAIN RESULTS: Mice receiving tumor lysate-pulsed dendritic cells before tumor implantation demonstrated protective antitumor immunity with prolonged survival (> 3 months) and even resisted secondary tumor challenge. Tumor protection was associated with strong tumor-specific cytotoxic T-lymphocyte responses. Adoptive transfer of splenocytes or purified CD8+ T lymphocytes transferred tumor protection to unimmunized mice in vivo. When given after tumor implantation in a therapeutic setting, pulsed dendritic cells prevented mesothelioma outgrowth. With higher tumor load and delayed administration after tumor implantation, dendritic cells were no longer effective. CONCLUSIONS: We demonstrate in this murine model that immunotherapy using pulsed dendritic cells may emerge as a powerful tool to control mesothelioma outgrowth. In the future, immunotherapy using dendritic cells could be used as adjuvant to control local recurrence after multimodality treatment for malignant mesothelioma.

In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma (Article)

2005-01-01T00:00:00Z

Although dendritic cells (DCs) play an important role in sensitization to inhaled allergens, their function in ongoing T helper (Th)2 cell-mediated eosinophilic airway inflammation underlying bronchial asthma is currently unknown. Here, we show in an ovalbumin (OVA)-driven murine asthma model that airway DCs acquire a mature phenotype and interact with CD4(+) T cells within sites of peribronchial and perivascular inflammation. To study whether DCs contributed to inflammation, we depleted DCs from the airways of CD11c-diphtheria toxin (DT) receptor transgenic mice during the OVA aerosol challenge. Airway administration of DT depleted CD11c(+) DCs and alveolar macrophages and abolished the characteristic features of asthma, including eosinophilic inflammation, goblet cell hyperplasia, and bronchial hyperreactivity. In the absence of CD11c(+) cells, endogenous or adoptively transferred CD4(+) Th2 cells did not produce interleukin (IL)-4, IL-5, and IL-13 in response to OVA aerosol. In CD11c-depleted mice, eosinophilic inflammation and Th2 cytokine secretion were restored by adoptive transfer of CD11c(+) DCs, but not alveolar macrophages. These findings identify lung DCs as key proinflammatory cells that are necessary and sufficient for Th2 cell stimulation during ongoing airway inflammation.

Proteomic analysis of exosomes isolated from human malignant pleural effusions. (Article)

2004-07-01T00:00:00Z

Exosomes are membrane vesicles from endosomal origin secreted by various cells such as hematopoietic, epithelial, and tumor cells. Exosomes secreted by tumor cells contain specific antigens potentially useful for immunotherapeutic purposes. Our aim was to determine if exosomes are present in human cancerous pleural effusions and to identify their proteomic content. Exosomes were purified by sucrose gradient ultracentrifugation, and electron microscopy was used to check both concentration and purity of exosomes. Proteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and protein bands were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blotting. Exosomes were present in pleural fluid obtained from patients suffering from mesothelioma (n = 4), lung cancer (n = 2), breast cancer (n = 2), and ovarian cancer (n = 1). As previously reported by others, antigen-presenting molecules, cytoskeletal proteins, and signal transduction-involved proteins were present. Proteins not previously reported were identified (SNX25, BTG1, PEDF, thrombospondin 2). Different types of immunoglobulins and complement factors were abundantly present in the sucrose fractions containing exosomes. Exosome-directed specificity of these immunoglobulins was not observed. In conclusion, sucrose gradient ultracentrifugation allows isolation of exosomes from malignant pleural effusions. However, pleural fluid proteins and especially immunoglobulins are coisolated and may hamper the use of exosomes isolated from malignant effusion for immunotherapy programs.

Activation of peroxisome proliferator-activated receptor-gamma in dendritic cells inhibits the development of eosinophilic airway inflammation in a mouse model of asthma (Article)

2004-01-01T00:00:00Z

Peroxisome proliferator-activated receptors (PPARs) are activated by an array of polyunsaturated fatty acid derivatives, oxidized fatty acids, and phospholipids and are proposed to be important modulators of immune and inflammatory responses. Recently, we showed that activation of PPAR-gamma alters the maturation process of dendritic cells (DCs), the most potent antigen-presenting cells. In the present report, we investigated the possibility that, by targeting DCs, PPAR-gamma activation may be involved in the regulation of the pulmonary immune response to allergens. Using a model of sensitization, based on the intratracheal transfer of ovalbumin (OVA)-pulsed DCs, we show that rosiglitazone, a selective PPAR-gamma agonist, reduces the proliferation of Ag-specific T cells in the draining mediastinal lymph nodes but, surprisingly enough, dramatically increases the production of the immunoregulatory cytokine interleukin (IL)-10 by T cells, as compared to control mice sensitized with OVA-pulsed DCs. After aerosol challenge, the recruitment of eosinophils in the bronchoalveolar lavage fluids was strongly reduced compared to control mice. Finally, T cells from the mediastinal lymph nodes produced higher amounts of IL-10 and interferon-gamma. Inhibition of IL-10 activity with anti-IL-10R antibodies partly restored the inflammation. The specificity of the phenomenon was confirmed by treating OVA-pulsed DCs with ciglitazone, another PPAR-gamma agonist, and by using GW9662, a PPAR-gamma antagonist. Our data suggest that PPAR-gamma activation prevents induction of Th2-dependent eosinophilic airway inflammation and might contribute to immune homeostasis in the lung.

Proteomic analysis of exosomes secreted by human mesothelioma cells (Article)

2004-01-01T00:00:00Z

Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins present in exosomes secreted by tumor cells have been poorly defined. This study describes the protein composition of mesothelioma cell-derived exosomes in more detail. After electrophoresis of exosome preparations, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to characterize the protein spots. MHC class I was found to be present together with the heat shock proteins HSC70 and HSP90. In addition, we found annexins and PV-1, proteins involved in membrane transport and function. Cytoskeleton proteins and their associated proteins ezrin, moesin, actinin-4, desmoplakin, and fascin were also detected. Besides the molecular motor kinesin-like protein, many enzymes were detected revealing the cytoplasmic orientation of exosomes. Most interesting was the detection of developmental endothelial locus-1 (DEL-1), which can act as a strong angiogenic factor and can increase the vascular development in the neighborhood of the tumor. In conclusion, mesothelioma cells release exosomes that express a discrete set of proteins involved in antigen presentation, signal transduction, migration, and adhesion. Exosomes may play an important role in the interaction between tumor cells and their environment.

Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen (Article)

2004-01-01T00:00:00Z

Tolerance is the usual outcome of inhalation of harmless antigen, yet T helper (Th) type 2 cell sensitization to inhaled allergens induced by dendritic cells (DCs) is common in atopic asthma. Here, we show that both myeloid (m) and plasmacytoid (p) DCs take up inhaled antigen in the lung and present it in an immunogenic or tolerogenic form to draining node T cells. Strikingly, depletion of pDCs during inhalation of normally inert antigen led to immunoglobulin E sensitization, airway eosinophilia, goblet cell hyperplasia, and Th2 cell cytokine production, cardinal features of asthma. Furthermore, adoptive transfer of pDCs before sensitization prevented disease in a mouse asthma model. On a functional level, pDCs did not induce T cell division but suppressed the generation of effector T cells induced by mDCs. These studies show that pDCs provide intrinsic protection against inflammatory responses to harmless antigen. Therapies exploiting pDC function might be clinically effective in preventing the development of asthma.

Airway eosinophils accumulate in the mediastinal lymph nodes but lack antigen-presenting potential for naive T cells (Article)

2003-01-01T00:00:00Z

Asthma is characterized by infiltration of the airway wall with eosinophils. Although eosinophils are considered to be effector cells, recent studies have reported their ability to activate primed Th2 cells. In this study, we investigated whether eosinophils are capable of presenting Ag to unprimed T cells in draining lymph nodes (DLN) of the lung and compared this capacity with professional dendritic cells (DC). During development of eosinophilic airway inflammation in OVA-sensitized and challenged mice, CCR3(+) eosinophils accumulated in the DLN. To study their function, eosinophils were isolated from the bronchoalveolar lavage fluid of mice by sorting on CCR3(+)B220(-)CD3(-)CD11c(dim) low autofluorescent cells, avoiding contamination with other APCs, and were intratracheally injected into mice that previously received CFSE-labeled OVA TCR-transgenic T cells. Eosinophils did not induce divisions of T cells in the DLN, whereas DC induced on average 3.7 divisions in 45.7% of T cells. To circumvent the need for Ag processing or migration in vivo, eosinophils were pulsed with OVA peptide and were still not able to induce T cell priming in vitro, whereas DC induced vigorous proliferation. This lack of Ag-presenting ability was explained by the very weak expression of MHC class II on fresh eosinophils, despite expression of the costimulatory molecules CD80 and ICAM-1. This investigation does not support any role for airway eosinophils as APCs to naive T cells, despite their migration to the DLN at times of allergen exposure. DC are clearly superior in activating T cells in the DLN of the lung.

Lipopolysaccharide-induced suppression of airway Th2 responses does not require IL-12 production by dendritic cells (Article)

2003-01-01T00:00:00Z

The prevalence of atopic asthma, a Th2-dependent disease, is reaching epidemic proportions partly due to improved hygiene in industrialized countries. There is an inverse correlation between the level of environmental endotoxin exposure and the prevalence of atopic sensitization. As dendritic cells (DC) have been implicated in causing sensitization to inhaled Ag, we studied the effect of endotoxin on Th2 development induced by bone marrow DC in vitro and by intratracheal injection in vivo, with particular emphasis on the role played by the polarizing cytokine IL-12. Bone marrow-derived DC stimulated with Escherichia coli O26:B6 LPS produced IL-12p70 for a limited period of time, after which production became refractory to further stimulation with CD40 ligand, a phenomenon previously called "exhaustion." The level of IL-12 production of DC did not correlate with Th1 development, as exhausted OVA-pulsed DC were still capable of shifting the cytokine pattern of responding OVA-specific Th cells toward Th1 in vitro and in vivo. When mice were first immunized by intratracheal injection of OVA-DC and subsequently challenged with OVA aerosol, prior in vitro stimulation of DC with LPS reduced the development of airway eosinophilia and Th2 cytokine production. Most surprisingly, the capacity of LPS to reduce Th2-dependent eosinophilic airway inflammation was IL-12-independent altogether, as IL-12p40 knockout DC had a similar reduced capacity to prime for Th2 responses. These results suggest that LPS reduces sensitization to inhaled Ag by reducing DC-driven Th2 development, but that IL-12 is not necessary for this effect.

Prostaglandin D2 inhibits airway dendritic cell migration and function in steady state conditions by selective activation of the D prostanoid receptor 1 (Article)

2003-01-01T00:00:00Z

PGD(2) is the major mediator released by mast cells during allergic responses, and it acts through two different receptors, the D prostanoid receptor 1 (DP1) and DP2, also known as CRTH2. Recently, it has been shown that PGD(2) inhibits the migration of epidermal Langerhans cells to the skin draining lymph nodes (LNs) and affects the subsequent cutaneous inflammatory reaction. However, the role of PGD(2) in the pulmonary immune response remains unclear. Here, we show that the intratracheal instillation of FITC-OVA together with PGD(2) inhibits the migration of FITC(+) lung DC to draining LNs. This process is mimicked by the DP1 agonist BW245C, but not by the DP2 agonist DK-PGD(2). The ligation of DP1 inhibits the migration of FITC-OVA(+) DCs only temporarily, but still inhibits the proliferation of adoptively transferred, OVA-specific, CFSE-labeled, naive T cells in draining LNs. These T cells produced lower amounts of the T cell cytokines IL-4, IL-10, and IFN-gamma compared with T cells from mice that received FITC-OVA alone. Taken together, our data suggest that the activation of DP receptor by PGD(2) may represent a pathway to control airway DC migration and to limit the activation of T cells in the LNs under steady state conditions, possibly contributing to homeostasis in the lung.

Allergen-induced accumulation of airway dendritic cells is supported by an increase in CD31(hi)Ly-6C(neg) bone marrow precursors in a mouse model of asthma (Article)

2002-01-01T00:00:00Z

Airway dendritic cells (DCs) are held responsible for inducing sensitization to inhaled antigen, leading to eosinophilic airway inflammation, typical of asthma. However, less information is available about the role of these cells in ongoing inflammation. In a mouse model of asthma, sensitization to ovalbumin (OVA) was induced by intratracheal injection of myeloid OVA-pulsed DCs. Upon OVA aerosol challenge and induction of eosinophilic airway inflammation in sensitized mice, there was a time-dependent and almost 100-fold increase in the number of MHCII(+) CD11b(+) CD11c(+) endogenous airway DCs as well as CD11b(+) blood DCs. The mechanism of this increase was studied. Adoptive transfer experiments demonstrated that accumulation of airway DCs was not due to reduced migration to the mediastinal lymph nodes. Rather, the massive increase in airway and lymph node DCs was supported by an almost 3-fold expansion of myeloid CD31(hi)Ly-6C(neg) hematopoietic precursor cells in the bone marrow (BM). There was no change in any of the other 5 populations revealed by CD31/Ly-6C staining. When these CD31(hi)Ly-6C(neg) BM precursors were sorted and grown in granulocyte macrophage-colony-stimulating factor, they differentiated into MHCII(+) CD11c(+) DCs. The same CD31(hi)Ly-6C(neg) precursors also expressed the eotaxin receptor CCR3 and differentiated into eosinophils when grown in interleukin 5. Serum levels of eotaxin were doubled in mice with inflammation. These findings in an animal model of asthma suggest that the BM increases its output of myeloid precursors to meet the enhanced demand for DCs and eosinophils in inflamed airways.

Effects of fluticasone propionate in COPD patients with bronchial hyperresponsiveness (Article)

2002-01-01T00:00:00Z

BACKGROUND: Treatment of chronic obstructive pulmonary disease (COPD) with inhaled corticosteroids does not appear to be as effective as similar treatment of asthma. It seems that only certain subgroups of patients with COPD benefit from steroid treatment. A study was undertaken to examine whether inhaled fluticasone propionate (FP) had an effect on lung function and on indices of inflammation in a subgroup of COPD patients with bronchial hyperresponsiveness (BHR). METHODS: Twenty three patients with COPD were studied. Patients had to be persistent current smokers between 40 and 70 years of age. Non-specific BHR was defined as a PC(20) for histamine of

Dyspnoea perception during clinical remission of atopic asthma (Article)

2002-01-01T00:00:00Z

Symptoms of atopic asthma often disappear around puberty. The authors recently demonstrated that this clinical remission is accompanied with ongoing airways inflammation in most subjects. The discrepancy between lack of symptoms and persistent airway inflammation suggests that perception of the symptoms is unclear. In the present study, young adults in clinical remission of atopic asthma assigned themselves a modified Borg score during methacholine and adenosine-5'-monophosphate induced bronchoconstriction. Borg scores of subjects in clinical remission were compared with those of symptomatic asthmatic subjects. A marked variation in the Borg scores at a 20% fall in the forced expiratory volume in one second was found. Significant differences in Borg scores between remission patients and asthmatics could not be detected. It was concluded that perception of dyspnoea, induced with methacholine and adenosine challenge, is similar in young adults in clinical remission of atopic asthma compared to that of patients with symptomatic asthma. Hence, an unclear perception seems to be an unlikely explanation for the discrepancy between lack of symptoms and ongoing inflammation. Other factors, including both physical and psychological ones, may play a role in the apparent absence of symptoms, thereby potentially leading to undertreatment.

Segmental bronchoprovocation in allergic rhinitis patients affects mast cell and basophil numbers in nasal and bronchial mucosa (Article)

2001-01-01T00:00:00Z

Mast cells and basophils are cells that play an important role in the initiation and control of allergic inflammation in asthma and rhinitis. This study was undertaken to determine the presence and dynamics of mast cells and basophils in the nasal and bronchial mucosa of allergic rhinitis patients after segmental bronchial provocation (SBP). Eight nonasthmatic, grass pollen-allergic rhinitis patients and eight healthy controls were included. Bronchial and nasal biopsies, as well as blood samples, were taken before (T(0)) and 24 h (T(24)) after SBP. Immunohistochemical staining was performed for mast cells (tryptase and chymase; phenotypes MC(T), MC(TC), MC(C)) and basophils (BB1). In the bronchial mucosa, the number of BB1(+) cells increased significantly (p < 0.05) in allergic rhinitis patients after SBP. In the nasal mucosa, the numbers of MC(C) and MC(TC) cells decreased significantly, whereas the numbers of [BB1(+)] cells increased significantly in allergic rhinitis patients after SBP (p < 0.05). In blood, the number of basophils decreased (p < 0.05) and the level of interleukin (IL)-5 increased (p < 0.05) in atopic patients after SBP. No significant changes could be observed in healthy controls. This study shows that SBP in nonasthmatic allergic rhinitis patients reduces numbers of mast cells in the nose as a result of enhanced degranulation. At the same time, there is evidence for an influx of basophils from the blood into the nasal and bronchial mucosae.

Lung dendritic cells and host immunity to infection (Article)

2001-01-01T00:00:00Z

The lung is a portal of entry for numerous microbial pathogens, against which evolution has created an adequate innate and adaptive immune response. Dendritic cells (DCs) are central to the integration of innate and specific immunity. These cells are located within the epithelium and interstitium of the lung where they are influenced by the innate immune system. Upon recognition and internalization of microbial antigens, DCs migrate to the draining lymph nodes of the lung to initiate the specific cellular and humoral immune response. By their capacity to integrate stimuli derived from the pathogen, the host and the environment, they are specialized to induce a protective immune response while at the same time avoiding damage to the host. It is becoming increasingly clear that dendritic cells are involved in the induction of immunity to viruses, bacteria, mycobacteria and fungi. Some pathogens subvert the function of dendritic cells to escape immune recognition. Not surprisingly, if dendritic cell function fails, the consequence for the host is immunodeficiency.

Airway inflammation is present during clinical remission of atopic asthma (Article)

2001-01-01T00:00:00Z

Symptoms of atopic asthma often disappear at puberty. However, asthmatic subjects in clinical remission will frequently have a relapse later in life. The aim of this study was to investigate whether subjects in clinical remission of atopic asthma have persistent airway inflammation and/or airway remodeling. Bronchial biopsies were obtained from subjects in clinical remission, asthmatic subjects, and healthy control subjects. The presence and/or activation state of eosinophils, mast cells, macrophages, T lymphocytes, interleukin (IL)-5, eotaxin, and inducible nitric oxide synthase (iNOS) were analyzed. Results were compared with less invasive indicators of airway inflammation. Also aspects of airway remodeling were determined. Eosinophils, T cells, mast cells, and IL-5 were significantly elevated in the airway mucosa of subjects in remission compared with control subjects. Also, blood eosinophil cell counts were significantly higher in subjects in clinical remission. Blood eosinophil cell counts, exhaled nitric oxide (eNO) levels, and bronchial response to adenosine-5'-monophosphate correlated significantly with the quantity of tissue eosinophils. Significant airway remodeling was found in subjects in clinical remission. Our study has shown ongoing airway inflammation and airway remodeling in adolescents in clinical remission of atopic asthma. Subclinical airway inflammation may well determine the risk of an asthma relapse later in life.

Influence of lung parenchymal destruction on the different indexes of the methacholine dose-response curve in COPD patients (Article)

2000-01-01T00:00:00Z

STUDY OBJECTIVES: The interpretation of nonspecific bronchial provocation dose-response curves in COPD is still a matter of debate. Bronchial hyperresponsiveness (BHR) in patients with COPD could be influenced by the destruction of the parenchyma and the augmented mechanical behavior of the lung. Therefore, we studied the interrelationships between indexes of BHR, on the one hand, and markers of lung parenchymal destruction, on the other. PATIENTS AND METHODS: COPD patients were selected by clinical symptoms, evidence of chronic, nonreversible airways obstruction, and BHR, which was defined as a provocative dose of a substance (histamine) causing a 20% fall in FEV(1) (PC(20)) of

Segmental bronchial provocation induces nasal inflammation in allergic rhinitis patients (Article)

2000-01-01T00:00:00Z

Allergic rhinitis and asthma often coexist and share a genetic background. Pathophysiologic connections between the nose and lungs are still not entirely understood. This study was undertaken to compare allergic inflammation and clinical findings in the upper and lower airways after segmental bronchial provocation (SBP) in nonasthmatic allergic rhinitis patients. Eight nonasthmatic, grass pollen-sensitive patients with allergic rhinitis and eight healthy controls were included. Bronchial biopsies and blood samples were taken before (T(0)) and 24 h (T(24)) after SBP. Nasal biopsies were obtained at T(0), 1 h after SBP (T(1)), and T(24). Immunohistochemical staining was performed for eosinophils (BMK13), interleukin (IL)-5, and eotaxin. The number of eosinophils increased in the challenged and unchallenged bronchial mucosa (p < 0.05) and in the blood (p = 0.03) of atopic subjects at T(24). We detected an increase of BMK13-positive and eotaxin-positive cells in the nasal lamina propria and enhanced expression of IL-5 in the nasal epithelium of atopic subjects only at T(24) (p < 0.05). SBP induced nasal and bronchial symptoms as well as reductions in pulmonary and nasal function in the allergic group. No significant changes could be observed in healthy controls. The study shows that SBP in nonasthmatic allergic rhinitis patients results in peripheral blood eosinophilia, and that SBP can induce allergic inflammation in the nose.

Tumor necrosis factor-alpha enhances mRNA expression and secretion of interleukin-6 in cultured human airway smooth muscle cells (Article)

2000-01-01T00:00:00Z

Airway smooth muscle (ASM) is considered to be an end-target cell for the effects of mediators released during airway wall inflammation. Several reports suggest that activated ASM may be capable of generating various proinflammatory cytokines. We investigated the effects of tumor necrosis factor (TNF)-alpha, a potent proinflammatory cytokine, on cultured human ASM cells by examining the expression and release of the cytokine interleukin (IL)-6, cell proliferation, and the expression pattern of c-fos and c-jun, two nuclear proto-oncogenes constituting the activator protein-1 transcription factor. Growth-arrested cell monolayers were stimulated with human recombinant TNF-alpha in a concentration- and time-dependent manner. TNF-alpha stimulated the expression of IL-6 messenger RNA (mRNA), which was detected after 15 min, reaching a maximum at 1 h. IL-6 protein was readily detected in ASM cell-conditioned medium after 2 h of TNF-alpha stimulation. Protein levels increased in a time- and concentration-dependent manner. Release of IL-6 elicited by TNF-alpha was significantly inhibited by dexamethasone, cycloheximide, and nordihydroguaiaretic acid (NDGA). TNF-alpha did not alter DNA biosynthesis up to 48 h or cell numbers up to 120 h. Northern blot analysis of proto-oncogene expression revealed that c-fos and c-jun mRNA levels were elevated after 30 min of TNF-alpha incubation with maximum levels at 1 h and 45 min, respectively. Expression of c-fos mRNA was downregulated by NDGA. Four hours of TNF-alpha treatment resulted in translocation of c-jun immunofluorescence from the cytoplasm to the nucleus in human ASM cells. Our results suggest that despite the lack of a mitogenic response to TNF-alpha, upregulation of primary response genes in human ASM cells may account for the induction of proinflammatory cytokines, such as IL-6, in human airways.

Adolescents in clinical remission of atopic asthma have elevated exhaled nitric oxide levels and bronchial hyperresponsiveness (Article)

2000-01-01T00:00:00Z

Symptoms of atopic asthma often decrease or even seem to disappear around puberty. The aim of this study was to investigate whether this so-called clinical remission is accompanied by remission of airway inflammation, since symptoms relapse in a substantial proportion of subjects later in life. To assess indicators of inflammation and/or structural damage of the airways, exhaled nitric oxide (eNO) and bronchial responsiveness to adenosine-5'-monophosphate (AMP) and methacholine (MCh) were determined in 21 subjects in clinical remission of atopic asthma. Clinical remission was defined as complete absence of symptoms of asthma without the use of any medication in the year preceding the study. Results were compared with those of 21 patients with current asthma and 18 healthy control subjects. We found significantly higher eNO values in the remission group than in healthy controls (geometric mean, 18.9 and 1.0 ppb, respectively; p < 0.001) whereas eNO values of the remission group and those of the subjects with current asthma (geometric mean, 21.9 ppb) were similar (p = 0.09). The responsiveness to both AMP and MCh of subjects in clinical remission was significantly higher as compared with responsiveness of healthy controls, and lower than responsiveness of subjects with current asthma. A significant correlation could be established between eNO and responsiveness to AMP, but not between eNO and responsiveness to MCh. The results of this study are suggestive of persistent airway inflammation during clinical remission of atopic asthma. We speculate that subclinical inflammation is a risk factor for asthma relapse later in life, and that eNO and responsiveness to both AMP and MCh can be used as different, noninvasive indices of the inflammatory process of the airways.

Eosinophils in the bronchial mucosa in relation to methacholine dose-response curves in atopic asthma (Article)

1999-01-01T00:00:00Z

Asthma is characterized by both local infiltration of eosinophils in the bronchial mucosa and bronchial hyperreactivity (BHR). A detailed characterization of BHR implies analysis of a histamine or methacholine dose-response curve yielding not only the dose at 20% fall of baseline forced expiratory volume in 1 s (FEV1), but also a plateau (P) representing the maximal narrowing response in terms of percent change in FEV1 and reactivity as the steepest slope at 50% of P (%FEV1/doubling dose). In the baseline condition, the specific airway conductance (sGaw) may be considered closely related to airway lumen diameter. In 20 nonsmoking asthmatic patients, methacholine dose-response curves were obtained, and a sigmoid model fit yielded the BHR indexes. Immunohistochemistry with the monoclonal antibodies (EG1 and EG2) was used to recognize the total number of eosinophils and activated eosinophils, respectively. The number of activated eosinophils was significantly correlated to both P (r = 0.62; P < 0.05) and sGaw (r = -0.52; P < 0.05), whereas weaker and nonsignificant correlations were found for dose at 20% fall of baseline FEV1 and the total number of eosinophils. We conclude that the number of activated eosinophils can be considered a marker of the inflammation-induced decrease of airway lumen diameter as represented by the plateau index and sGaw.

Effects of fluticasone propionate on methacholine dose-response curves in nonsmoking atopic asthmatics (Article)

1996-01-01T00:00:00Z

Methacholine is frequently used to determine bronchial hyperresponsiveness (BHR) and to generate dose-response curves. These curves are characterized by a threshold (provocative concentration of methacholine producing a 20% fall in forced expiratory volume in one second (PC20) = sensitivity), slope (reactivity) and maximal response (plateau). We investigated the efficacy of 12 weeks of treatment with 1,000 microg fluticasone propionate in a double-blind, placebo-controlled study in 33 atopic asthmatics. The outcome measures used were the influence on BHR and the different indices of the methacholine dose-response (MDR) curve. After 2 weeks run-in, baseline lung function data were obtained and a MDR curve was measured with doubling concentrations of the methacholine from 0.03 to 256 mg x mL(-1). MDR curves were repeated after 6 and 12 weeks. A recently developed, sigmoid cumulative Gaussian distribution function was fitted to the data. Although sensitivity was obtained by linear interpolation of two successive log2 concentrations, reactivity, plateau and the effective concentration at 50% of the plateau value (EC50) were obtained as best fit parameters. In the fluticasone group, significant changes occurred after 6 weeks with respect to means of PC20 (an increase of 3.4 doubling doses), plateau value fall in forced expiratory volume in one second (FEV1) (from 58% at randomization to 41% at 6 weeks) and baseline FEV1 (from 3.46 to 3.75 L) in contrast to the placebo group. Stabilization occurred after 12 weeks. Changes for reactivity were less marked, whereas changes in log, EC50 were not significantly different between the groups. We conclude that fluticasone is very effective in decreasing the maximal airway narrowing response and in increasing PC20. However, it is likely that part of this increase is related to the decrease of the plateau of maximal response.