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RATIONALE: Limited information is available on repeatability of inflammatory parameters in whole induced sputum samples from patients with COPD. OBJECTIVES: To study short-term and long-term repeatability in induced sputum samples in 22 patients with moderate to severe, stable COPD (mean age 64 yr, mean FEV(1) 1.91 L=65% of predicted). Samples were collected on 71 occasions twice within 1 to 7 days (mean 3.8 days) and on 12 occasions twice with an interval of 3 months while clinically stable. Cell differentials, markers of neutrophilic and eosinophilic inflammation, respiratory membrane permeability and size-selective permeation were assessed. FINDINGS: Parameters of permeability and of size-selective permeation, % eosinophils and % neutrophils showed the best short-term repeatability with intra-class correlation coefficients (Ri) of 0.61 to 0.90, followed by total cell count (TCC) with Ri of 0.52. Repeatability of soluble cell activation markers was less satisfactory (Ri 0.34 to 0.52). Mean short-term within-patient variability for TCC and permeability was approximately 2-fold and for cell activation markers 3-fold; mean between-patients variability was twice as high. Inducing sputum slightly enhanced eosinophil numbers and % neutrophils and decreased % macrophages in successive IS samples. Long-term repeatability was comparable to short-term repeatability but variability increased. CONCLUSIONS: Repeatability of parameters assessed in whole sputum is similar as reported previously for sputum plugs. In COPD an induced sputum procedure has a minor pro-inflammatory effect. The current data facilitates power calculations but also indicates that studies using inflammatory markers in sputum may easily be underpowered

Hemoptysis is a known complication in patients with bronchial artery hypertrophy due to a variety of chronic pulmonary disorders. Bronchial artery hypertrophy is observed in most patients with chronic thromboembolic pulmonary hypertension (CTEPH), but surprisingly little is known about the incidence of hemoptysis in these patients. In this paper, we report on 2 patients with CTEPH and recurrent severe hemoptysis, who were treated by bronchial artery embolization. One patient recovered and 1 patient died as a consequence of the bleeding. A systematic review revealed 21 studies on the underlying pathology in 1,844 patients with moderate to severe hemoptysis. CTEPH was reported to be the cause of bleeding in 0.1% (n = 2), pulmonary arterial hypertension without chronic thromboembolic disease in 0.2% (n = 4), and acute pulmonary embolism in 0.7% (n = 12) of the patients. In contrast to this, 5 patients (6%) in our own series of 79 CTEPH patients suffered from moderate to severe hemoptysis requiring medical intervention. Severe hemoptysis appears to be an uncommon, but possibly underreported, life-threatening complication in CTEPH patients. As most CTEPH patients require life-long anticoagulants a therapeutic dilemma may ensue. Therefore, we propose that even mild hemoptysis in CTEPH patients warrants prompt evaluation, and treatment by embolization should be offered as first choice in CTEPH patients.

Since the ability of bacteria to compete with lactoferrin for iron contributes to the pathogenesis of mucosal infections, the presence of lactoferrin receptor activity in non-encapsulated Haemophilus influenzae was investigated. The growth of 18 H. influenzae isolates from the sputum samples of chronic bronchitis patients and of six of seven H. influenzae throat isolates from healthy adults was stimulated by iron saturated human lactoferrin. Apo-lactoferrin did not stimulate the growth of H. influenzae. Human lactoferrin binding to iron limited bacteria was detected for 16 H. influenzae strains from chronic bronchitis patients and for five of seven isolates from healthy adults. We conclude that the majority of H. influenzae isolates tested bind human lactoferrin and that the iron from lactoferrin is used for growth.

Background Airway infection with influenza virus induces local expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO), which has been shown to enhance inflammatory mediator responses in vitro. Because secondary pneumococcal infections occurring shortly after recovery from influenza are associated with enhanced inflammatory responses, we hypothesized that IDO activity contributes to the enhanced response to bacterial challenges in mice previously infected with influenza virus. Methods On day 14 after influenza virus infection (with strain A/PR/8/34), C57Bl/6 mice were intranasally inoculated with 1 x 10(4) colony-forming units of S. pneumoniae (serotype 3). Matrix-driven delivery pellets that contained 70 mg of the IDO inhibitor 1-methyl-DL-tryptophan (MeTrp) released over a period of 7 days were subcutaneously implanted 48 h before pneumococcal infection. Results MeTrp treatment resulted in a 20-fold reduction in pneumococcal outgrowth 48 h after bacterial inoculation. Remarkably, pulmonary levels of interleukin-10 and tumor necrosis factor-alpha were significantly reduced in mice treated with MeTrp. Conclusions Our data suggest that IDO expression during influenza virus infection alters the inflammatory response and facilitates the outgrowth of pneumococci during secondary bacterial pneumonia.

Toll-like receptors (TLRs) are pattern-recognition receptors that have been implicated in the initiation of innate immune responses upon the first encounter with invading pathogens. The airways are frequently exposed to various types of lipopolysaccharide (LPS) from the environment or from pathogens. The current study was designed to determine the effect of LPS on TLR gene expression in human alveolar macrophages in vivo. In total, 16 healthy subjects were enrolled in a single-blinded, placebo-controlled study. Subjects inhaled 100 microg LPS or normal saline (n = 8 per group). Measurements were performed in alveolar macrophages purified from bronchoalveolar lavage fluid obtained 6 h post-challenge. Inhalation of LPS by healthy human volunteers resulted in enhanced alveolar macrophage expression of mRNAs encoding TLRs 1, 2, 7, 8 and CD14, and reduced expression of mRNAs encoding TLR4 and lymphocyte antigen 96. In conclusion, lipopolysaccharide differentially influences the toll-like receptor mRNA expression profile in human alveolar macrophages in vivo.

Airway epithelial cells are critically dependent on an intact cytoskeleton for innate defense functions. There are various pathophysiological conditions that affect the cytoskeletal architecture. We studied the effect of cytoskeletal distortion in polarized airway epithelial-like NCI-H292 cells on inflammatory gene expression, exemplified by interleukin(IL)-6 and IL-8. Disruption of microtubule structure with vinblastin and of actin with cytochalasin D did not affect TNF-alpha-induced IL-6 and IL-8 gene transcription but stabilized IL-8 and IL-6 mRNA. In line with previous studies, IL-8 mRNA stabilization was paralleled by hyperresponsive IL-8 production, but surprisingly, IL-6 production was reduced despite IL-6 mRNA stabilization. Polysome profiling revealed that, in cells with a disrupted cytoskeleton, translational efficiency of IL-6 mRNA was reduced, whereas that of IL-8 mRNA remained unaffected. Our findings indicate that distortion of the cytoskeleton in airway epithelial cells differentially affects both degradation and translation of IL-6 and IL-8 mRNA, modifying inflammatory gene expression and thus their innate defense function.

Background: There is much controversy about the ideal approach to the management of community acquired pneumonia (CAP). Recommendations differ from a pathogen directed approach to an empirical strategy with broad spectrum antibiotics. Methods: In a prospective randomised open study performed between 1998 and 2000, a pathogen directed treatment (PDT) approach was compared with an empirical broad spectrum antibiotic treatment (EAT) strategy according to the ATS guidelines of 1993 in 262 hospitalised patients with CAP. Clinical efficacy was primarily determined by the length of hospital stay (LOS). Secondary outcome parameters for clinical efficacy were assessment of therapeutic failure on antibiotics, 30 day mortality, duration of antibiotic treatment, resolution of fever, side effects, and quality of life. Results:Three hundred and three patients were enrolled in the study; 41 were excluded, leaving 262 with results available for analysis. No significant differences were found between the two treatment groups in LOS, 30 day mortality, clinical failure, or resolution of fever. Side effects, although they did not have a significant influence on the outcome parameters, occurred more frequently in patients in the EAT group than in those in the PDT group (60% v 17%, 95% CI -0.5 to -0.3; pConclusions: An EAT strategy with broad spectrum antibiotics for the management of hospitalised patients with CAP has comparable clinical efficacy to a PDT approach.

In a prospective study to evaluate the diagnostic yield of different microbiological tests in hospitalised patients with community-acquired pneumonia, material for microbiological investigation was obtained from 262 patients. Clinical samples consisted of the following: sputum for Gram staining, culture, and detection of pneumococcal antigen; blood for culture and serological tests; urine for detection of Legionella pneumophila serogroup 1 antigen and pneumococcal antigen; and specimens obtained by fiberoptic bronchoscopy. A pathogen was identified in 158 (60%) patients, with Streptococcus pneumoniae (n=97) being the most common causative agent of community-acquired pneumonia. In 82% of the 44 patients with an adequate sputum specimen, a positive Gram stain was confirmed by positive sputum culture. S. pneumoniae infections were detected principally when adequate sputum specimens were examined by Gram stain and culture and when adequate and inadequate sputum specimens were tested for the presence of pneumococcal antigen (n=58; 60%). The urinary pneumococcal antigen test was the most valuable single test for detection of S. pneumoniae infections (n=52; 54%) when sputum pneumococcal antigen determination was not performed. Fiberoptic bronchoscopy was of additive diagnostic value in 49% of the patients who did not expectorate sputum and in 52% of those in whom treatment failed. Investigation of sputum by a combination of Gram stain, culture, and detection of pneumococcal antigen was the most useful means of establishing an aetiological diagnosis of community-acquired pneumonia, followed by testing of urine for pneumococcal antigen. Fiberoptic bronchoscopy may be of additional value when treatment failure occurs.

T helper 1 driven immune responses facilitate host defence during viral infections. Because interleukin-18 (IL-18) mediates T helper 1 driven immune responses, and since mature IL-18 is up-regulated in human macrophages after influenza virus infection in vitro, it has been suggested that IL-18 plays an important role in the immune response to influenza. To determine the role of IL-18 in respiratory tract infection with influenza, IL-18 gene-deficient (IL-18(-/-)) and normal wildtype mice were intranasally inoculated with influenza A virus. Influenza resulted in an increase in constitutively expressed IL-18 in the lungs of wildtype mice. The clearance of influenza A was inhibited by IL-18, as indicated by reduced viral loads on day 8 and day 12 after infection in IL-18(-/-) mice. This enhanced viral clearance correlated with increased CD4(+) T-cell activation in the lungs as reflected by CD69 expression on the cell surface. Surprisingly, interferon-gamma (IFN-gamma) levels were similar in the lungs of IL-18(-/-) mice and wildtype mice. Intracellular IFN-gamma staining revealed similar expression levels in lung-derived natural killer cells, CD4(+) and CD8(+) T cells, indicating that IFN-gamma production is IL-18-independent during influenza virus infection. Tumour necrosis factor-alpha production by CD4(+) T cells was significantly lower in IL-18(-/-) mice than in wildtype mice. Our data indicate that endogenous IL-18 impairs viral clearance during influenza A infection.

The lungs are frequently challenged by viruses, and resident CD8+ T cells likely contribute to the surveillance of these pathogens. To obtain insight into local T cell immunity to respiratory viruses in humans, we determined the specificity, phenotype, and function of lung-residing CD8+ T cells and peripheral blood CD8+ T cells in a paired analysis. The lung contained markedly higher frequencies of influenza (FLU)-specific and respiratory syncytial virus (RSV)-specific CD8+ T cells when compared with the circulation. This contrasted with an equal distribution of cytomegalovirus- and Epstein-Bar virus–specific CD8+ T cells. Noticeably, a substantial fraction of the lung-residing FLU- and RSV-specific CD8+ T cells had progressed to a relatively late differentiation phenotype, reflected by low expression of CD28 and CD27. Lung-derived FLU-specific CD8+ T cells had low activation requirements, as expansion of these cells could be initiated by cognate peptide in the absence of helper cell–derived signals. Thus, the human lung contains high numbers of differentiated FLU- and RSV-specific memory CD8+ T cells that can readily expand upon reexposure to virus. Resident lung T cells may provide immediate immunological protection against pulmonary virus infections.

Contains fulltext : 47718.pdf (Publisher’s version ) (Open Access) BACKGROUND: Idiopathic pulmonary fibrosis is a chronic progressive disorder with a poor prognosis. METHODS: We conducted a double-blind, randomized, placebo-controlled multicenter study that assessed the effectiveness over one year of a high oral dose of acetylcysteine (600 mg three times daily) added to standard therapy with prednisone plus azathioprine. The primary end points were changes between baseline and month 12 in vital capacity and in single-breath carbon monoxide diffusing capacity (DL(CO)). RESULTS: A total of 182 patients were randomly assigned to treatment (92 to acetylcysteine and 90 to placebo). Of these patients, 155 (80 assigned to acetylcysteine and 75 to placebo) had usual interstitial pneumonia, as confirmed by high-resolution computed tomography and histologic findings reviewed by expert committees, and did not withdraw consent before the start of treatment. Fifty-seven of the 80 patients taking acetylcysteine (71 percent) and 51 of the 75 patients taking placebo (68 percent) completed one year of treatment. Acetylcysteine slowed the deterioration of vital capacity and DL(CO): at 12 months, the absolute differences in the change from baseline between patients taking acetylcysteine and those taking placebo were 0.18 liter (95 percent confidence interval, 0.03 to 0.32), or a relative difference of 9 percent, for vital capacity (P=0.02), and 0.75 mmol per minute per kilopascal (95 percent confidence interval, 0.27 to 1.23), or 24 percent, for DL(CO) (P=0.003). Mortality during the study was 9 percent among patients taking acetylcysteine and 11 percent among those taking placebo (P=0.69). There were no significant differences in the type or severity of adverse events between patients taking acetylcysteine and those taking placebo, except for a significantly lower rate of myelotoxic effects in the group taking acetylcysteine (P=0.03). CONCLUSIONS: Therapy with acetylcysteine at a dose of 600 mg three times daily, added to prednisone and azathioprine, preserves vital capacity and DL(CO) in patients with idiopathic pulmonary fibrosis better than does standard therapy alone.

Lipopolysaccharide is ubiquitously present in the environment. To determine the effect of salmeterol, a long-acting β2-receptor agonist, on lipopolysaccharide-induced lung inflammation, mice received lipopolysaccharide (10 μg) intranasally with or without salmeterol intraperitoneally (5 mg/kg) 30 min earlier and 12 h thereafter. Salmeterol dose- and time-dependently inhibited the lipopolysaccharide-induced influx of neutrophils into bronchoalveolar lavage fluid and lung tissue, and these pulmonary neutrophils displayed a reduced expression of CD11b at their surface. To determine the contribution of the salmeterol effect on neutrophil CD11b in the attenuated neutrophil recruitment, we treated mice intranasally exposed to lipopolysaccharide with salmeterol with or without a blocking anti-CD11b antibody. Anti-CD11b profoundly reduced lipopolysaccharide-induced neutrophil influx in bronchoalveolar lavage fluid, an effect that was modestly enhanced by concurrent salmeterol treatment. These data suggest that salmeterol inhibits lipopolysaccharide-induced neutrophil recruitment to the lungs by a mechanism that possibly in part is mediated by an effect on neutrophil CD11b.

Toll-like receptors (TLR) induce innate immune responses upon stimulation by a wide variety of pathogens. TLR4 has been implicated in innate immunity against respiratory syncytial virus (RSV) by an interaction with the viral envelope fusion (F) protein. Sendai virus (mouse parainfluenza type 1) shares many features with RSV, including a structurally and functionally similar F protein. To determine the role of TLR4 in host defense against Sendai virus respiratory tract infection, TLR4 mutant and wildtype mice were intranasally infected with Sendai virus. Sendai infection resulted in an increase in viral RNA copies in lung homogenates peaking on day 4. Pulmonary viral loads, histopathology, cytokine levels and leukocyte influx were similar in TLR4 mutant and wildtype mice. In spite of the structural similarities shared by the F proteins of Sendai virus and RSV, TLR4 is not involved in host defense against respiratory tract infection with Sendai virus. (C) 2003 Published by Elsevier B.V

Background: It remains to be established which factors contribute to the occurrence of asthma in allergic individuals. We hypothesized that differences in the late allergic inflammatory reaction to allergen between asthmatic and non-asthmatic house dust mite-allergic individuals might contribute to the difference in the clinical presentation of allergy. Aim: To compare allergen-induced changes in parameters for cellular inflammation during the phase of the late allergic reaction in the skin and nose, in house dust mite-allergic individuals with or without asthma. Material and Methods: Nasal and dermal allergen challenges with house dust mite (Dermatophagoides pteronyssinus) extract were performed in 52 house dust mite-allergic individuals, of whom 26 had mild to moderate persistent asthma and 26 had perennial rhinitis without current or past asthmatic symptoms. Serial nasal lavage samples were analyzed for the presence of inflammatory cells (eosinophils and neutrophils) and soluble markers associated with cellular inflammation [interleukin-5 (IL-5), interleukin-8 (IL-8), eosinophil cationic protein (ECP) and myeloperoxidase (MPO)]. Macroscopic late phase skin reactions were studied after intracutaneous skin tests with house dust mite extract. Results: Fixed dose nasal allergen provocation elicited a similar degree of immediate allergic reaction as judged by plasma protein exudation and histamine concentrations in asthma and non-asthmatic rhinitis. Subsequently, no differences between groups were found during the phase of the late allergic reaction (4–24 h) in inflammatory cell influx, plasma protein leakage, ECP or MPO. Likewise, there were no differences in levels of chemotactic cytokines IL-5 and IL-8. In agreement with the results of nasal challenge, the late skin reaction after dermal challenge with a fixed allergen dose and after an allergen dose 10,000 times above the skin threshold for an early skin reaction did not differ between the groups. Conclusion: House dust mite-allergic patients with or without asthma have very similar late allergic inflammatory reactions in the skin and in the nose after allergen challenge. Hence, it is unlikely that the occurrence of pulmonary symptoms in asthma is explained by a general tendency of asthmatics to have an enhanced late allergic cellular inflammatory response. Nasal and dermal allergen provocations are adequate models to study allergen-induced inflammation but probably lack the pivotal link which is essential for the development of asthma.

In vitro data suggest that salmeterol, contrary to formoterol, can partly antagonise the effect of short-acting beta(2)-agonist rescue medication. To explore whether this occurs in vivo, we compared the effects of increasing doses (200-3200 mug) of fenoterol on the recovery of methacholine induced bronchoconstriction as well as PD20 methacholine in 23 asthmatic patients, during two-week treatment periods with placebo, and standard doses of salmeterol or formoterol in a double blind, double-dummy, crossover study. Salmeterol showed a slightly higher propensity for the development of bronchodilator tolerance. The recovery of methacholine induced bronchoconstriction was more complete during regular use of formoterol relative to salmeterol. During regular use of both long-acting beta(2)-agonists the bronchoprotective efficacy of fenoterol was attenuated, but this was more pronounced during salmeterol than during formoterol. The mean maximum increase in PD20 metacholine after the highest dose of fenoterol was 3.97 DD during placebo, 2.47 DD during formoterol (p

Respiratory viral infections increase inflammatory responses to concurrent or secondary bacterial challenges, thereby worsening disease outcome. This potentiation of inflammation is explained at least in part by IFN-γ promoting increased sensitivity to TNF-α and LPS. We sought to determine whether and, if so, how IFN-γ can modulate proinflammatory responses to TNF-α and LPS by epithelial cells, which are key effector cells in the airways. Preincubation of airway epithelial-like NCI-H292 cells with IFN-γ resulted in a hyperresponsive IL-6 and IL-8 production to TNF-α and LPS. The underlying mechanism involved the induction of indoleamine 2,3-dioxygenase, which catabolized the essential amino acid, tryptophan. Depletion of tryptophan led to stabilization of IL-6 and IL-8 mRNA and increased IL-6 and IL-8 responses, whereas supplementing tryptophan largely restored these changes. This novel mechanism may be implicated in enhanced inflammatory responses to bacterial challenges following viral infection.

Non-typeable Haemophilus influenzae may infect the lower respiratory airways of chronic obstructive pulmonary disease patients. We characterized genes of non-typeable H. influenzae expressed during interaction with two human respiratory tract-derived epithelial cell lines. A library of 8000 clones was constructed in H. influenzae Rd (rec1) by cloning chromosomal fragments upstream of a promoterless cat gene. Exposure of this library to NCI-H292 epithelial cell layers in the presence of chloramphenicol (Cam) resulted in survival of bacteria expressing cat. A total of 52 clones were selected that were resistant to Cam in the presence of epithelial cells of cell line NCI-H292. These did not (n = 42) or hardly grow (n = 10) on sBHI plates containing Cam and were sensitive to Cam in cell culture medium alone. All clones, moreover, survived Cam in the presence of Hep2 epithelial cell layers. Sequence analysis showed that four clones contained sequences without homology to Rd or any other sequence, and therefore contained promoters and parts of open reading frames (ORFs) of novel genes. The other 48 clones were homologous to Rd, and characterization was based upon this genome. Six different functional classes were distinguished: (i) metabolic processes; (ii) stress response; (iii) gene expression; (iv) cell envelope biosynthesis; (v) DNA-related processes and cell division; and (vi) ORFs encoding proteins of unknown function. The contribution of identified genes to non-typeable H. influenzae adaptation to the epithelial cell environment is discussed.

In patients with allergic asthma, T-cell cytokines are implicated in the regulation of the local inflammation in the airways. The ability of sensitized mast cells to release mediators and cytokines early upon allergen stimulation makes them important candidates for local immunoregulation. We have studied the effects of human mast cells on T cells with the use of the human mast cell line HMC-1. We showed that activated human mast cells or their soluble products induced and enhanced the interferon-gamma (IFN-gamma) production by T cells up to about 60-fold. The production of interleukin (IL)-4 was hardly affected and that of IL-5 was slightly enhanced. The enhancement of IFN-gamma production was induced both in polyclonal CD4(+) and CD8(+) T cells and in CD4(+) and CD8(+) T-cell clones. Further characterization of the factors involved demonstrated a molecular mass above 30 000. Our results implicate that by this mechanism mast cells may account for a negative feedback system locally down-regulating allergen-induced T helper 2 responses via IFN-gamma production by the T cells

Background The models for exposure to house dust in research and clinical practice are selected with respect to their role in IgE-mediated immediate hypersensitivity. The use of isolated major allergens instead of complex allergen extracts is becoming increasingly popular as it offers some important advantages for quantitative measures in diagnosis and research. Objective To compare house dust mite extract and isolated mite major allergens with respect to their ability to induce early and late asthmatic responses and bronchial hyperreactivity. Methods Bronchial responses to house dust mite (HDM, Dermatophagoides pteronyssinus) extract and isolated major allergens from HDM (Der p 1 and Der p 2) were compared in a double-blind, randomized, cross-over study in 20 patients with mild to moderate asthma who were allergic to HDM. Allergen was titrated to a standardized early asthmatic response. Bronchial hyper-responsiveness to histamine (PC20histamine) was determined before and after allergen inhalation to assess allergen-induced bronchial hyper-responsiveness and IL-5 was measured in serum. In addition, the allergens were applied in intracutaneous skin tests and activation of basophil leucocytes and proliferation of peripheral blood mononuclear cells was tested in vitro. Results After a similar early asthmatic response (mean Δforced expiratory volume in 1 s (FEV1),max−29.4 (SD 7.2) vs. −33.1 (8.6) %; mean difference 3.6 (95% CI −0.9 to 8.2) %), the late asthmatic response (mean ΔFEV1,max−45.9 (21.9) vs. −32.7 (22.3) %; mean difference 13.2 (3.8–22.3) %), the degree of allergen-induced bronchial hyper-responsiveness (mean ΔPC20histamine, 1.8 (1.0) vs. 1.2 (0.9) doubling dose; mean difference 0.6 (0.2–1.1) doubling dose) and serum IL-5 at 6 h were found to be significantly higher after bronchial challenge with HDM extract than after challenge with an isolated HDM major allergen. Likewise, there was an increased late skin reaction with HDM compared with isolated major allergen after a similar early skin reaction. Conclusion Constituents of HDM extract, other than Der p 1 or Der p 2, with no significant influence on the IgE-mediated early asthmatic response contribute significantly to the allergen-induced late asthmatic response and bronchial hyper-reactivity.

Background Immature mast cells migrate into tissues where they differentiate into mature mast cells under the influence of local factors. In the airways of asthmatics increased numbers of chronically activated mast cells are located nearby the airway epithelium. Objective The aim of this study was to evaluate whether and, if so, which products released by epithelial cells may affect mast cell proliferation and differentiation. Methods We performed in vitro studies using the human lung mucoepidermoid carcinoma-derived H292 cell line and the immature human mast cell line, HMC-1. Proliferation was assessed by 3H-thymidine incorporation. Differentiation of HMC-1 cells was inferred from tryptase production. Results Exposure of HMC-1 cells to medium conditioned for 48 h by H292 cells resulted in a reduction of proliferation with 65 ± 4.9% (mean ± sem, n = 9) at day 5. Culturing HMC-1 cells for 8 days in the presence of H292-conditioned medium resulted in morphological changes indicative of differentiation, and in a 3.0 ± 0.4-fold increase of tryptase production (P = 0.0039, n = 9). Conditioned medium from H292 cells that were stimulated by LPS also inhibited HMC-1 proliferation. Inhibitory antibodies against two mediators from H292 cells, interleukin-6 (IL-6) and stem cell factor (SCF), abolished the increase in HMC-1 tryptase production induced by H292-conditioned medium. Recombinant human (rh) IL-6, but not rhSCF, reduced HMC-1 proliferation with 44% and 13% at day 3 and 5, respectively. Surprisingly, rhIL-6 did not increase HMC-1 tryptase production significantly whereas incubation with rhSCF did (1.5 ± 0.1-fold, P = 0.002, n = 10) although the increase was less than observed for conditioned medium. Conclusion Epithelial-derived IL-6 and SCF are implicated in differentiation of HMC-1 cells but additional factors are not excluded. As activated primary bronchial epithelial cells also express IL-6 and SCF, it should be considered that these cells are involved in mast cell differentiation within the airways, particularly in diseases where epithelial cells are activated, such as asthma.

The rising incidence of tuberculosis worldwide means an increasing burden on diagnostic facilities, so tests simpler than Ziehl-Neelsen staining are needed. Such tests should be objective, reproducible, and have at least as good a detection limit as 10 4 bacteria/ml. A capture enzyme-linked immunosorbent assay (ELISA) was developed for detection of lipoarabinomannan (LAM) in human sputum samples. As a capture antibody, we used a murine monoclonal antibody against LAM, with rabbit antiserum against Mycobacterium tuberculosis as a source of detector antibodies. The sensitivity of the capture ELISA was evaluated by using purified LAM and M. tuberculosis whole cells. We were able to detect 1 ng of purified LAM/ml and 10 4 M. tuberculosis whole cells/ml. LAM could also be detected in culture filtrate of a 3-week-old culture of M. tuberculosis . The culture filtrate contained approximately 100 μg of LAM/ml. The detection limit in sputum pretreated with N -acetyl- l -cysteine and proteinase K was 10 4 M. tuberculosis whole cells per ml. Thirty-one (91%) of 34 sputum samples from 18 Vietnamese patients with tuberculosis (32 smear positive and 2 smear negative) were positive in the LAM detection assay. In contrast, none of the 25 sputum samples from 21 nontuberculous patients was positive. This specific and sensitive assay for the detection of LAM in sputum is potentially useful for the diagnosis of tuberculosis.

Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) synergize to express polymeric immunoglobulin receptor (pIgR) but their combined effect, and that of IL-4 alone, on secretory immunoglobulin A (sIgA) release is unknown. Recently, we have developed an airway epithelial cell model that allows assessment of the integrated effect of a stimulus on pIgR gene and protein expression and sIgA release. With this model we show here that IL-4 and IFN-gamma dose-dependently increased pIgR mRNA and protein expression, and sIgA release. IFN-gamma and IL-4 induced similar maximal expression of pIgR, but IFN-gamma enhanced sIgA release more than IL-4. When added together, IL-4 and IFN-gamma synergistically increased pIgR mRNA and protein expression, but sIgA release was stimulated in an additive manner. Thus, IL-4 and IFN-gamma may be implicated in the increase of sIgA levels as found in mucosal inflammatory diseases. In addition, our results indicate that transport and release of empty pIgR is subject to regulatory mechanisms different from those of pIgR with bound dimeric IgA.

Background: IL-3, IL-5, and GM-CSF are not able to induce histamine release in purified basophils of nonallergic donors. However, we have recently found that preincubation with 2 μmol/L thapsigargin, which induces a rise in intracellular free calcium ions, renders human basophils extremely sensitive for IL-3, IL-5, or GM-CSF, leading to enhanced histamine release. Histamine release was also induced in the reverse order (first cytokine and then thapsigargin). Objective: Because these cytokines are supposed to be increased in allergic inflammation, we examined whether basophils of patients with allergic asthma showed an enhanced response to thapsigargin. Methods: We measured the histamine release induced by thapsigargin in a group of allergic asthmatic subjects (n = 24) and compared this response with those of 3 control groups. The control groups consisted of healthy control subjects (group 1, n=21); patients with a nonallergic, nonasthmatic lung disease (group 2, n=22); and patients with nonallergic asthma (group 3, n=9). Results: There was no difference in spontaneous histamine release. Also, no significant difference in histamine release was found when anti-IgE or formyl-methionyl-leucyl-phenylalanine was used as a stimulus. Histamine release induced by IL-3 alone or a combination of IL-3 and thapsigargin also did not differ. In contrast, basophils from the group with allergic asthma showed a significantly higher percentage of histamine release induced by thapsigargin (38.2% ± 13.2%) than did basophils from the 3 control groups (healthy control subjects, 22.5% ± 6.9%; subjects with lung disease, 24.9% ± 8.9%; subjects with nonallergic asthma 15.0% ± 3.0%; all mean ± SD). Conclusion: These data indicate that basophils in peripheral blood of subjects with allergic asthma have a primed phenotype and that thapsigargin-induced histamine release is a practical tool to study this phenomenon. (J Allergy Clin Immunol 1999;104:1000-7.)

This study examines the diagnostic utility of the polymerase chain reaction (PCR) in 156 patients (five human immunodeficiency virus (HIV) seropositive) suspected of extrapulmonary tuberculosis. The results of PCR in 226 samples from 11 different sites were compared with the results of microscopy and culture. Positive culture results were predicted in 86% of samples by PCR but in only 31% by microscopy. Specificity of PCR was 92%. In cases with culture-proven tuberculosis, PCR identified all 11 microscopy positive cases and 19 of 24 (79%) of the microscopy-negative cases. In four patients, PCR excluded the diagnosis of tuberculosis in microscopy-positive samples, which were later shown to contain mycobacteria other than Mycobacterium tuberculosis or laboratory contaminants. In 20 patients (microscopy, PCR and culture negative) a trial of antituberculous drugs was given, but patients showed no improvement and treatment was stopped. In 17 patients, all culture negative (in nine PCR was positive, three of whom also had positive microscopy) the diagnosis was probable tuberculosis based on clinical findings and response to treatment. This polymerase chain reaction has a much higher sensitivity than microscopy and can facilitate therapeutic decisions for those with suspected extrapulmonary tuberculosis.

Background: The use of allergen extracts will hamper studies into quantitative aspects of allergic responses because the precise amount of relevant allergen for each patient is unknown. Objective : We applied isolated IgE-binding components (major allergens) in the technique of bronchial allergen challenge to determine the role of patient characteristics in the early asthmatic response (EAR). Methods: In 30 patients with mild-to-moderate asthma, the EAR was investigated after inhalation of an isolated major allergen of Dermatophagoides pteronyssinus (i.e., Der p 1 [ n = 16] or Der p 2 [ n = 14]). The degree of early-phase bronchial responsiveness to allergen (the cumulated dose of allergen causing a 20% fall in FEV 1 [PD 20 allergen]) was related to the degree of nonspecific bronchial responsiveness (the concentration of histamine causing a 20% fall in FEV 1 [PC 20 histamine]) and the level of specific IgE or allergen thresholds as found in skin tests and basophil histamine release assays. Results: Twenty-seven patients with an immediate response during allergen and histamine challenges (ΔFEV 1 , ≥20%) were analyzed. In these patients, a strong correlation was found between PD 20 allergen and PC 20 histamine ( r = 0.81, p 20 allergen and the level of specific IgE ( r = –0.36, p = 0.07) or allergen thresholds as found in skin tests (skin prick test, r = 0.36 and p = 0.07; intracutaneous test, r = 0.49 and p = 0.01) or basophil histamine release assays ( r = 0.37, p = 0.08). Moreover, no significant contribution of these indices of IgE-mediated hypersensitivity to the prediction of PD 20 allergen by multilinear regression models with PC 20 histamine was found. Conclusion: In asthmatic patients allergic to house dust mites the degree of nonspecific bronchial hyperresponsiveness is the main determinant of early-phase bronchial responsiveness to allergen. In these patients the degree of allergic sensitivity does not contribute to the prediction of the EAR after allergen inhalation. (J Allergy Clin Immunol 1998;102:24-31.)

Restriction of eukaryotic protein synthesis affects the regulation of some transiently expressed gene transcripts resulting in their superinduction. We determined the transcriptional and post-transcriptional processes implicated in IL-6 mRNA superinduction in a human lung-derived epithelial cell line H292, and their kinetics in the absence and presence of an exogenous stimulus, tumor necrosis factor-alpha (TNF-alpha). Cycloheximide (CHI) at 10 microg/ml, which inhibited protein synthesis for 80%, caused a 80-fold induction of IL-6 mRNA level which was due predominantly to a stabilization of IL-6 mRNA (20-fold) early on. Employing transient transfection protocols we noted a small positive effect of CHI on transcription, mediated by the proximal and the distal C/EBP sites of the IL-6 promoter and paralleled by an increased C/EBP DNA-binding activity, similar to that found for exposure to TNF-alpha alone. TNF-alpha and CHI synergized on IL-6 mRNA expression (200-fold increase) which was due to an increased transcription, corresponding to a further increased C/EBP DNA-binding activity. However, the effect of CHI on IL-6 gene transcription was transient, in support of the need for ongoing protein synthesis for C/EBP activity. These findings indicate that IL-6 mRNA superinduction, at least in H292 cells, is regulated predominantly by modulating the repressive system that ensures a rapid degradation of IL-6 mRNA.

High levels of histamine can be found in the airways of asthma patients. This study describes the effects of histamine on anti-CD3-induced production of IL-4, IL-5, and IFN-gamma by T cell clones from subjects with allergic asthma and healthy subjects. T cell clones were obtained from bronchoalveolar lavage (BAL) fluid and blood. The number of clones tested, and the percentage of clones in which histamine inhibited or enhanced cytokine production by more than 25%, were as follows: IL-4, 47, 8.5%, and 4.3%; IL-5, 43, 14%, and 30%; and IFN-gamma, 52, 40%, and 15%. Inhibition of IL-5 and IFN-gamma production was reversed by IL-2. The enhancement of IFN-gamma production was associated with an enhancement of both IL-2 production and proliferation. In 21% of the clones a combined effect consisting of inhibition of IFN-gamma production and enhancement of IL-5 production was found. This response was reversed by H2-receptor antagonists and was significantly associated with a histamine-induced increase in intracellular levels of cAMP. The role of cAMP in mediating the histamine effects was supported by the observations that the beta2-agonist salbutamol had effects similar to histamine and that high concentrations of PGE2 mimicked the inhibitory effects of histamine. Clones from BAL fluid and blood showed similar responses, as did clones from patients with asthma and from control subjects. The enhancement of IFN-gamma production by histamine, however, was found only in clones from healthy subjects. The results warrant further investigations on the role of cAMP in the regulation of cytokine production.

Airway inflammation during infection is associated with increased transudation of serum proteins and increased production of protein by the airway epithelium. We therefore, assessed whether Haemophilus influenzae infections in patients with chronic bronchitis are associated with increased levels of transferrin and lactoferrin in the sputum compared to uninfected patients. Sputum sol phase and serum samples from 14 infected and 13 uninfected patients with chronic bronchitis and from 12 bronchial asthma patients were included in the study. The median Q-values (the concentration in sputum sol phase/the concentration in serum) x 10(3) of transferrin appeared increased in chronic bronchitis patients with an H. influenzae infection (26.0, n=13) compared to uninfected controls (9.5, n=11) and bronchial asthma patients (4.5, n=6). The ratio of the Q(transferrin)/Q(albumin) was >1 in infected chronic bronchitis patients, indicating local production of transferrin. Growth of H. influenzae was stimulated more in sputum from infected and uninfected patients with chronic bronchitis than in sputum from patients with bronchial asthma. The concentrations of lactoferrin were not significantly different in infected (n=14) and uninfected (n=13) chronic bronchitis patients and bronchial asthma patients (n=12) (median 137.4, 84.6, 87.1 mg x L(-1), respectively). We conclude that in patients with chronic bronchitis with Haemophilus influenzae infections, the levels of transferrin are increased and the levels of lactoferrin are not associated with infections.

Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.

During the course of persistent infections by nonencapsulated Haemophilus influenzae in patients with chronic bronchitis, the major outer membrane protein (MOMP) P5 varies in molecular weight. The nature of this variability was determined by DNA sequence analysis of the P5 gene from five different H. influenzae strains and their seven MOMP P5 variants which were isolated from patients with chronic infections of the lower respiratory tract. Analysis of the P5 sequence data from the different strains revealed four well-defined, heterogeneous regions. These regions of variable sequence appeared to correspond to the regions of the gene encoding the putative surface-exposed loops of MOMP P5. The MOMP P5 variants with alterations in MOMP P5 were shown to result from DNA point mutations and codon deletions. In addition, in three variants derived sequentially from one H. influenzae strain, a frameshift mutation resulted in the formation of a stop codon in the region encoding the signal sequence of the MOMP P5 gene. Strikingly, all nucleotide substitutions in the MOMP P5 loop regions of variants were nonsynonymous, suggesting that variants with alterated amino acid compositions of the surface-exposed parts of MOMP P5 obtained a selective advantage during persistence of the infection by nonencapsulated H. influenzae in chronic bronchitis patients.

Background: In our outpatient population, approximately one third of patients sensitized to grass pollen were found to have significant serum levels of anti-peanut IgE in the RAST, without positive peanut skin prick test (SPT) response and without peanut-related allergic symptoms. It was suggested earlier that poor biologic activity of IgE antibodies directed to cross-reactive carbohydrate determinants (CCD) of glycoproteins might explain these discrepancies. Objective: In this study we investigated the biologic activity of IgE directed to CCD. Methods: Sera of 32 patients allergic to grass pollen with significant levels of anti-peanut IgE, a negative response on peanut SPT, and no symptoms of peanut allergy were tested for the presence of anti-CCD IgE. Eleven of these patients with greater than 3.0 IU/ml anti-peanut IgE (patients 1 to 11) were selected together with four control patients allergic to peanut, on the basis of a positive response on peanut SPT and a history of peanut allergy (patients 12 to 15). Inhibition of the peanut RAST was performed by using proteinase K–treated grass pollen extract as a CCD source. Basophil histamine release assays (BHRAs) were performed with peanut extract and the isolated peanut major allergens Ara h 1 and Ara h 2. In addition, intracutaneous tests with peanut extract were performed. Results: In 29 (91%) of 32 patients with discrepant peanut RAST and SPT responses, anti-CCD IgE (≥0.1 IU/ml) was detected. In patients 1 to 11 almost complete inhibition of the peanut RAST with CCD was found (94.3% ± 5.5%; mean ± SD). In contrast, in the patients allergic to peanut only partial inhibition (59%) was found in one subject ( p = 0.002, Mann-Whitney test). In the BHRAs and the intracutaneous tests of patients with discrepant peanut RAST and SPT results, reactivity was found only at high concentrations of peanut allergens. When related to specific IgE levels, reactivity to peanut allergens in the BHRAs of these patients was found to be at least a factor of 1000 less when compared with reactivity to control inhalant allergens. Conclusion: We conclude that cross-reactive IgE directed to carbohydrate determinants of glycoproteins, as found in grass pollen–sensitized patients, has poor biologic activity. It can therefore cause positive RAST results without apparent clinical significance. (J Allergy Clin Immunol 1997;100:327-34.)

Nonencapsulated Haemophilus influenzae strains isolated from patients with chronic bronchitis can be divided into those that persist in the lower respiratory tract and those that do not. We tested the hypothesis that persisting and nonpersisting strains differ in the extent to which they activate epithelial cells to produce two potent inflammatory mediators, interleukin (IL)-6 and IL-8. A suspension of 10(7) and 10(8) colony forming units (cfu) x mL(-1) of H. influenzae, persisting and nonpersisting, induced a dose- and time-dependent production of IL-6 and IL-8 by the human pulmonary mucoepidermoid carcinoma-derived cell line H292, but levels of IL-6 were lower after exposure to persisting H. influenzae (p

Pulmonary hypertension is frequently observed in advanced idiopathic pulmonary fibrosis (IPF) and is associated with poor prognosis. Cardiopulmonary exercise testing (CPET) can be used to detect less advanced pulmonary vascular impairment, and therefore may be of prognostic use. We studied the predictive value of non-invasive exercise parameters that were associated with elevated systolic pulmonary artery pressure (sPAP) for survival in IPF patients.From our interstitial lung disease database, we reviewed records of consecutive patients with IPF in whom CPET and echocardiography were performed within 2 weeks (n = 38).Eleven patients (29%) had increased sPAP (≥40 mm Hg). From all non-invasive CPET parameters, only the ventilatory equivalent for CO2 (V'E /V'CO2 ) at anaerobic threshold differed significantly between patients with and without sPAP ≥ 40 mm Hg. The receiver-operator characteristic curve for V'E /V'CO2 resulted in areas under the curve of 0.77 (95% CI: 0.569-0.970; P = 0.026), with a cut-off value for predicting sPAP ≥ 40 mm Hg of45.0. Patients with V'E /V'CO2 45.0 had significantly worse survival compared with patients with V'E /V'CO2 ≤ 45.0 (P = 0.001). In contrast, sPAP did not predict survival.V'E /V'CO2 , the only CPET parameter associated with elevated sPAP, appears a potentially useful non-invasive marker for early detection of pulmonary vascular impairment, and therefore may be of use for a more accurate prognostic assessment in IPF patients.

Supernatants of cultured human mononuclear cells contain factors that induce histamine release from basophils. Some of the histamine-releasing factors present are IgE-independent, but an IgE-dependent form has also been described. The IgE that does respond to the IgE-dependent histamine-releasing factor was defined as IgE+. This study was conducted to analyze the contribution of chemokines, such as monocyte chemotactic protein-1 (IgE-independent), to improve the detection of IgE-dependent histamine-releasing factor. Supernatants were prepared from mononuclear cells of three subjects. Monocyte chemotactic protein-1 content and histamine-releasing activity (HRA) were measured. Depletion of chemokine activity was carried out with heparin-Sepharose (Pharmacia, Uppasala, Sweden). Replacing IgE- on basophils with IgE+ serum decreased the correlation between monocyte chemotactic protein-1 and histamine release (rho = 0.80, n = 280 vs p = 0.12, n = 18; p for difference between p values; < 0.05). After depletion of chemokines in three supernatants derived from mononuclear cells, IgE-dependent HRA was still present. We conclude that in supernatants derived from mononuclear cells, the IgE-independent HRA masks the IgE-dependent HRA. The latter can be more clearly detected after depletion of chemokine activity with heparin-Sepharose

Atopic allergy is associated with allergen-specific CD4+ T cells showing a bias to production of the type-2 cytokines interleukin (IL)-4 and IL-5. There are indications that this bias is also evident in atopic T-helper (Th) cells with other antigen specificities. The balance between the production of type-1 and type-2 cytokines is influenced by various factors present in the microenvironment of the Th cells during their activation. Factors of special interest are antigen-presenting cell-derived IL-12 and prostaglandin E2, skewing to type-1 and type-2 cytokine production, respectively. The production of IL-12 and prostaglandin E2 is induced by the interaction between CD40 and CD40 ligand expressed by Th cells, and by biologically active agents such as micro-organisms or their products. The IL-12/prostaglandin E2 production ratio depends on the antigen-presenting cell type, the type of stimulus and the presence of certain cytokines. Other Th-cytokine-skewing factors are autocrine or paracrine IFN-gamma and IL-4. In fact, the type-1/type-2 cytokine production balance in Th cells is under the control of various soluble products that act in a complex network of type-1 (e.g. IL-12, IFN-gamma) or type-2 (e.g. IL-4, IL-10 and prostaglandin E2) factors and are produced by Th cells and their accessory antigen-presenting or bystander cells. The levels of these factors may further be determined by gene polymorphisms or aberrant hormone levels. Despite the growing knowledge of the regulation of Th-cell cytokine production, the etiology of biased cytokine production in atopic allergic individuals is still enigmatic.

In view of increasing evidence suggesting an active immunoregulatory role of the skin keratinocytes and the observation that the differentiation of allergen specific T lymphocytes is critical in the development of allergy, we evaluated epidermal expression of HLA-DR antigen in skin reactions induced with an atopen (house dust mite) and with an non-atopic antigen (Hemocyanin). Two groups of patients with house dust mite (Dermatophagoides pteronyssinus [Der p]) allergy were compared, one group was skin tested with Der p, the other group was immunized and subsequently skin tested with Helix pomatia Hemocyanin (HPH). Biopsy specimens taken at 48 h after the HPH (n = 11) and Der p (n = 11) tests were analysed immunohistologically. Reactions in both groups were comparable in size. Immunohistological analysis showed domination by CD4+ lymphocytes. Expression of HLA-DR antigen by epidermal keratinocytes was observed in six out of 11 of the HPH induced reactions, but in none of the Der p induced reactions. Eosinophils were spotted only throughout the Der p induced reactions, showing a good correlation with the number of CD4 positive lymphocytes. The lack of HLA-DR expression by keratinocytes during the allergen-induced reaction, compared with the Hemocyanin induced reaction can be the result of a difference in cytokine profile of the lymphocytes dominating the dermal infiltrate. On the other hand evidence exists that defective HLA-DR expression by keratinocytes enhances antigen induced lymphocyte activation, and may thus contribute to the development of allergen-specific T-lymphocytes.

In normal airways, invasion of small numbers of pathogenic bacteria will not give rise to injury or local colonization. In chronic obstructive pulmonary disease, however, local conditions are changed, which may impair local defense systems and facilitate infection. These include: (1) factors promoting bacterial adherence and growth like the observed damaged airway epithelium in chronic bronchitis. The consequent changes in the composition of the epithelial lining fluid and impaired mucociliary clearance may be the most important pathophysiologic airway sequele in this respect; (2) aggravating generalized airway obstruction including mucus secretion, increase in mucus viscosity, and proliferation of submucosal smooth muscles, resulting in significant changes in airway geometry. This may change local conditions dramatically and so sustain factors promoting bacterial infections; (3) subversion of normally protective defense mechanisms into damaging host tissue at the mucosal level. Activated epithelial cell layers induce increased submucosal vascular leakage, edema, and inflammatory cell infiltration with subsequent tissue injury by locally produced cell products like free elastase. Specific bacterial products but also generally produced bacterial endotoxins may induce a local immune response resulting in the local production of high concentrations of antibodies and an invasion of specific effector cells. The role of these reactive cells or proteins may even be a primary one, in that they attack on (cellular) proteins, which increase their susceptibility for dysfunction in the defense line. For the clinicians, the new insights in the role of these aggravating factors in the development of recurrent bacterial airway infections in chronic bronchitis may be of major importance.(ABSTRACT TRUNCATED AT 250 WORDS)

Since nonencapsulated Haemophilus influenzae persists in the lower respiratory tracts of patients with chronic bronchitis despite the presence of specific antibodies, complement, and polymorphonuclear leukocytes (PMNs), opsonophagocytosis of H. influenzae was analyzed. Nonencapsulated H. influenzae isolated from the sputa of chronic bronchitis patients was labeled with fluorescein isothiocyanate and incubated with human PMNs in the presence of complement and antibodies for 30 min at 37 degrees C. Candida albicans was added to each sample as an internal standard, and the reduction of the number of bacteria was determined by flow cytometry. Fluorescence quenching with ethidium bromide was used to discriminate between intracellular and extracellular bacteria. Opsonophagocytosis of viable H. influenzae d1 was 17% +/- 29% in the presence of complement and human pooled sera containing high titers of strain-specific antibodies. Opsonophagocytosis of six other H. influenzae strains was also poor. Under the same conditions, opsonophagocytosis of Staphylococcus aureus was 90% +/- 5%, and opsonophagocytosis of C. albicans was 55% +/- 23%. About half of the number of H. influenzae bacteria associated with PMNs was internalized. Opsonophagocytosis of heat-killed H. influenzae d1 (41% +/- 20%) was higher than that of viable bacteria of the same strain (P < 0.05). This result suggests that the accessibility of epitopes on H. influenzae for opsonizing antibodies is better on killed than on viable bacteria. We conclude that viable nonencapsulated H. influenzae is poorly opsonophagocytized in the presence of strain-specific antibodies and complement.

The ability of monoclonal antibodies (MAbs) specific for variable and conserved epitopes of outer membrane protein (OMP) P2 (b,c) of nonencapsulated Haemophilus influenza to promote opsonophagocytosis of this bacterium by human polymorphonuclear leucocytes (PMNs) was determined by flow cytometry. MAbs rendering PMNs fluorescent because of association with fluorescein isothiocyanate-labelled bacteria were defined as stimulating opsonophagocytosis. Opsonophagocytosis was dependent on the presence of both antibodies and complement. Of the 14 MAbs directed to the variable parts of OMP P2 (L. van Alphen, P. Eijk, L. Geelen-van den Broek, and J. Dankert, Infect. Immun. 59:247-252, 1991), 9 stimulated opsonophagocytosis. Four of the five nonopsonophagocytic MAbs that were immunoglobulin G1 were unable to cause complement activation. The MAbs promoting opsonophagocytosis included MAbs specific for one or more OMP P2 antigenic variants of H. influenzae strains isolated from patients with chronic bronchitis during persistent infection. MAbs cross-reacting in enzyme-linked immunosorbent assays with nonrelated H. influenzae did not promote opsonophagocytosis of strains from other patients. Opsonophagocytosis was not observed in the presence of three MAbs reacting with OMP P2 epitopes common in H. influenzae. These results indicate that OMP P2-dependent opsonophagocytosis of nonencapsulated H. influenzae is strictly strain specific.

The molecular basis for the diversity of the major outer membrane protein (MOMP) P2 of non-encapsulated Haemophilus influenzae was analyzed by direct sequencing of fragments of the P2 genes, obtained by the polymerase chain reaction. Genetically divergent H. influenzae strains were isolated from patients with otitis media and from infected patients with chronic obstructive pulmonary disease (COPD). The nucleotide sequences of these P2 genes were determined and compared with P2 gene sequences of three non-encapsulated strains and with the P2 gene of encapsulated H. influenzae type b. Variation in the nucleotide sequence of the P2 genes of non-encapsulated H. influenzae was localized in eight distinct regions which were interchanged with relatively conserved regions. Regions 2, 4, 5 and 8 were found to be hypervariable, since they were characterized by multiple deletions or substitutions of nucleotides. According to the proposed structure of MOMP P2 the variable regions were localized in eight loop structures exposed at the outside of the outer membrane. Loops 2, 4, 5 and 8 were hypervariable in all isolates whereas loop 6 varied only in isolates from COPD patients with persistent infections.

A serologic test for the diagnosis of tuberculosis was evaluated in 91 newly diagnosed tuberculosis (TB) patients, of whom 15 were HIV positive, in 17 TB patients during treatment, and in 220 control subjects (including individuals from endemic areas and patients with sarcoidosis or Crohn's disease). Purified proteins of M. tuberculosis with molecular weights of 10,000, 16,000, 24,000, 30,000, 38,000, and 70,000 were tested by ELISA. In addition, monoclonal antibody TB72 was tested by competition ELISA. The cutoff values were set at the mean plus three standard deviations of the values obtained in 100 healthy Dutch army recruits. Only the ELISA with the 10,000, 16,000, and 24,000 antigens and the TB72 assay discriminated between patients with TB who were not HIV positive and control subjects. Specificity varied from 95 to 98% and sensitivity from 29 to 51% with the different antigens. Combination of the test results of the ELISA with the 16,000 antigen and the TB72 assay had a sensitivity of 65% (95% confidence interval, 53 to 75%) and a specificity of 96% (95% confidence interval, 92 to 98%). The assay was useful for the diagnosis of both pulmonary and extrapulmonary TB. Optimal use of the serologic assay could be obtained when likelihood ratios for each test value are calculated instead of using the test dichotomized (positive or negative). High posttest probabilities indicate the presence of TB; low posttest probabilities do not exclude the disease and should lead to additional investigations.

Rationale: The role of antibiotics in acute exacerbations is controversial and their efficacy when added to systemic corticosteroids is unknown. Objectives: We conducted a randomized, placebo-controlled trial to determine the effects of doxycycline in addition to corticosteroids on clinical outcome, microbiological outcome, lung function, and systemic inflammation in patients hospitalized with an acute exacerbation of chronic obstructive pulmonary disease. Methods: Of 223 patients, we enrolled 265 exacerbations defined on the basis of increased dyspnea and increased sputum volume with or without increased sputum purulence. Patients received 200 mg of oral doxycycline or matching placebo for 7 days in addition to systemic corticosteroids. Clinical and microbiological response, time to treatment failure, lung function, symptom scores, and serum C-reactive protein were assessed. Measurements and Main Results: On Day 30, clinical success was similar in intention-to-treat patients (odds ratio, 1.3; 95% confidence interval, 0.8 to 2.0) and per-protocol patients. Doxycycline showed superiority over placebo in terms of clinical success on Day 10 in intention-to-treat patients (odds ratio, 1.9; 95% confidence interval, 1.1 to 3.2), but not in per-protocol patients. Doxycycline was also superior in terms of clinical cure on Day 10, microbiological outcome, use of open label antibiotics, and symptoms. There was no interaction between the treatment effect and any of the subgroup variables (lung function, type of exacerbation, serum C-reactive protein, and bacterial presence). Conclusions: Although equivalent to placebo in terms of clinical success on Day 30, doxycycline showed superiority in terms of clinical success and clinical cure on Day 10, microbiological success, the use of open label antibiotics, and symptoms

Background: Inadequate technique reduces the effects of inhalation medication. Errors in inhalation technique have been reported to range up to 85%. Not only various patients' characteristics but also the device has an effect on correct inhalation technique. The purpose of this study was to determine the effect of patients' characteristics and type of inhaler device on inhalation technique in patient with asthma or chronic obstructive pulmonary disease (COPD). Methods: A validated scoring method was used that consisted of triple viewing of video-recorded inhalations, using device-specific checklists. The following patient characteristics were investigated: gender, age, education level, diagnosis, treatment by a pulmonary physician, previously received inhalation instruction, exacerbation frequency, knowledge, self-management competence, pulmonary function, and use of multiple inhaler devices. Chi-square statistics were used for univariate associations between potential determinants and correctness of inhalation technique. Relevant determinants were entered into a multivariate logistic regression model. Moreover, inhalation technique errors were examined for six inhaler devices: three prefilled dry powder inhalers, one single-dose dry powder inhaler, a pressurized metered-dose inhaler (pMDI) and a pMDI with a spacer. Results: Overall, 40% of the patients made at least one essential mistake in their inhalation technique. Patients who never received inhalation instruction and patients who used more than one inhaler device made significantly more errors (odds ratio both 2.2). Comparison between devices showed that a correct inhalation technique most likely occurred with the use of prefilled dry powder devices. Conclusion: Incorrect inhalation technique is common among asthma and COPD patients in a pulmonary outpatient clinic. Our study suggests that the use of prefilled dry powder inhalers as well as inhalation instruction increases correct inhalation technique. Simultaneous use of different types of inhalation devices has to be discouraged

Various characteristics of atopic allergic disorders seem to be causally related with the activation of allergen-specific T helper lymphocytes with a type 2 cytokine secretion profile, including high levels of interleukin-4 and interleukin-5. These cytokines are responsible for the occurrence of elevated levels of serum allergen-specific IgE and eosinophilia and play an important role in local inflammatory reactions.

Mice from four different inbred strains were infected with live Mycobacterium tuberculosis and the immune response to M. tuberculosis was followed for 24 weeks, using Western blotting. Nearly all mice, irrespective of H-2 type, reacted with the 3S-kDa protein band. Antibodies against this secreted 38-kDa protein were ihe first to appear, 4 weeks after infection. Thereafter the secreted 19-kDa protein and non-secreted antigens, such as the 65-kDa and 33-kDa proteins, were recognized. The immune response against the non-secreted antigens was influenced by the mouse strain. However, the 33-kDa protein band was recognized by all mouse strains after a second injection wilh live M. tuberculosis. The specificity of the antibodies was analysed in Western blot using sonicates of M. tuberculosis, M. kansasii, M avium. M. terrae, M. gordonae and Escherichia coli. Antibodies against the 38-kDa and 33-kDa protein bands seemed to be specific for M. tuberculosis, while antibodies against the 19-kDa protein band showed limited cross-reactivity. Antibodies against the 65-kDa prolein were strongly cross-reactive. These results suggest that the 38-kDa protein is secreted in vivo and, therefore, may be available to the humoral immune system at an early stage of infection. The non-secreted 33-kDa protein is only recognized by all mouse strains afler prolonged contact with M, tuberculosis.

BackgroundPreviously we showed that reduced availability of the essential amino acid tryptophan per se attenuates post-transcriptional control of interleukin (IL)-6 and IL-8 leading to hyperresponsive production of these inflammatory mediators by airway epithelial cells. Availability of the non-essential amino acid arginine in the inflamed airway mucosa of patients with asthma is reduced markedly, but it is not known whether this can also lead to an exaggerated production of IL-6 and IL-8.MethodsIL-6 and IL-8 were determined by ELISA in culture supernatants of NCI-H292 airway epithelial-like cells and normal bronchial epithelial (NHBE) cells that were exposed to TNF-α, LPS or no stimulus, in medium with or without arginine. Arginine deficiency may also result from exposure to poly-L-arginine or major basic protein (MBP), which can block arginine uptake. Epithelial cells were exposed to these polycationic proteins and L-14C-arginine uptake was assessed as well as IL-6 and IL-8 production. To determine the mode of action, IL-6 and IL-8 mRNA profiles over time were assessed as were gene transcription and post-transcriptional mRNA degradation.ResultsFor both NCI-H292 and NHBE cells, low arginine concentrations enhanced basal epithelial IL-6 and IL-8 production and synergized with TNF-α-induced IL-6 and IL-8 production. Poly-L-arginine enhanced the stimulus-induced IL-6 and IL-8 production, however, blocking arginine uptake and the enhanced IL-6 and IL-8 production appeared unrelated. The exaggerated IL-6 and IL-8 production due to arginine deficiency and to poly-L-arginine depend on a post-transcriptional and a transcriptional process, respectively.ConclusionWe conclude that both reduced arginine availability per se and the presence of polycationic proteins may promote airway inflammation by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.