Joachim Bargon

Epithelial Defensins Impair Adenoviral Infection: Implication for Adenovirus-Mediated Gene Therapy

Epithelial cells have been to participate actively in host defense by producing small cationic peptides called defensins. To investigate the biological activity of epithelial defensins in more detail, we expressed two defensins, hBD-1 and HD-5, in eukaryotic cell lines. Defensins were localized in the cytoplasm and in cell culture medium and exhibited strong microbicidal activity toward Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Moreover, our data indicate that the presence of defensins protected the cells from adenoviral infection. The presence of HD-5 or hBD-1 reduced the infectivity of Av1CF2 three- to fivefold. These results imply that defensins must be considered a serious obstacle whenever adenovirus is used to deliver genes to epithelial cells.

Expression of human β-defensin-1 promotes differentiation of keratinocytes

Abstract Epithelial cells have been shown to express the antibiotic peptides human β-defensins-1 and 2. While β-defensin-2 is known to be up-regulated by bacterial factors and proinflammatory mediators, the expression of β-defensin-1 does not appear to be affected by these mediators. To determine the regulation and function of β-defensin-1 we analyzed its expression upon stimulation of inflammatory mediators in vitro and ex vivo. In immortalized human cell lines (HaCaT) and nasal polyps β-defensin-1 was not induced upon incubation with bacteria or proinflammatory mediators, suggesting that the inertness of β-defensin-1 expression levels is not the result of the shortcoming of HaCaT cells. As proliferation and regeneration play an important role at sites of inflammation, we examined the expression level of β-defensin-1 in relation to the differentiation and proliferation of HaCaT cells. β-defensin-1 mRNA levels remained low during proliferation but were highly induced upon differentiation. In contrast, β-defensin-2 expression was unaffected under these conditions. To examine the function of β-defensin-1 in cellular proliferation and differentiation processes β-defensin-1 was overexpressed in keratinocytes. Protein expression analysis of the differentiation marker keratin 10 revealed that its expression is highly induced in the presence of increased concentrations of β-defensin-1. Hence our data indicate that high expression of β-defensin-1 promotes cell differentiation processes of keratinocytes.

Sonographic findings of the hepatobiliary-pancreatic system in adult patients with cystic fibrosis

Sonography of the liver, biliary system, and pancreas in adult patients with cystic fibrosis is by far less systematically documented than in pediatric patients with cystic fibrosis. In this prospective study, duplex sonographic findings of the liver, biliary system, and pancreas in adult patients with cystic fibrosis were compared with those of healthy control subjects.

Peroxisome Proliferator-Activated Receptor α (PPARα) down-regulation in cystic fibrosis lymphocytes

BackgroundPPARs exhibit anti-inflammatory capacities and are potential modulators of the inflammatory response. We hypothesized that their expression and/or function may be altered in cystic fibrosis (CF), a disorder characterized by an excessive host inflammatory response.MethodsPPARα, β and γ mRNA levels were measured in peripheral blood cells of CF patients and healthy subjects via RT-PCR. PPARα protein expression and subcellular localization was determined via western blot and immunofluorescence, respectively. The activity of PPARα was analyzed by gel shift assay.ResultsIn lymphocytes, the expression of PPARα mRNA, but not of PPARβ, was reduced (-37%; p < 0.002) in CF patients compared with healthy persons and was therefore further analyzed. A similar reduction of PPARα was observed at protein level (-26%; p < 0.05). The transcription factor was mainly expressed in the cytosol of lymphocytes, with low expression in the nucleus. Moreover, DNA binding activity of the transcription factor was 36% less in lymphocytes of patients (p < 0.01). For PPARα and PPARβ mRNA expression in monocytes and neutrophils, no significant differences were observed between CF patients and healthy persons. In all cells, PPARγ mRNA levels were below the detection limit.ConclusionLymphocytes are important regulators of the inflammatory response by releasing cytokines and antibodies. The diminished lymphocytic expression and activity of PPARα may therefore contribute to the inflammatory processes that are observed in CF.

Expression of Human Beta Defensin (HBD-1 and HBD-2) mRNA in Nasal Epithelia of Adult Cystic Fibrosis Patients, Healthy Individuals, and Individuals with Acute Cold

Background: Lack or inactivation of defensins may facilitate chronic bacterial colonization in the cystic fibrosis (CF) lung. CF nasal epithelium exhibits typical biochemical abnormalities and can be used to study defensin expression in CF. Objectives: To evaluate the expression of beta defensin (HBD-1 and HBD-2) mRNA and the presence of inflammatory markers (percentage of neutrophils and IL-8 mRNA expression) in CF and non-CF nasal mucosa. Methods: Case-control study. Nasal brushing samples were obtained from 22 stable adult CF patients and 32 non-CF controls (25 healthy individuals and 7 individuals with acute cold). Samples were subjected to analysis involving mRNA expression (semiquantitative RT-PCR) and differential cell counting. Results: Defensins and inflammatory markers were expressed at low levels in healthy individuals and at high levels in subjects with acute cold. In non-CF controls, defensin expression correlated significantly with inflammatory parameters (p < 0.001). In CF, defensin mRNA expression was comparable to healthy individuals (p = 0.2). In contrast to non-CF controls, in CF patients high levels of inflammatory markers did not correlate with defensin mRNA levels. Conclusions: Defensin expression is not upregulated in CF epithelium in response to inflammatory stimuli. Further studies are necessary to elucidate whether this is a consequence of the CF gene mutation.

Effects of short-term inhaled fluticasone on oxidative burst of sputum cells in cystic fibrosis patients.

Inhaled corticosteroids have been proposed to decrease pulmonary inflammation in cystic fibrosis (CF). In this study the effects of therapy with inhaled fluticasone on clinical and sputum outcomes (leukocyte count, activity of myeloperoxidase, superoxide anion release) in adult CF patients were investigated in an open label design. Twenty-six stable patients (median+/-SD forced expiratory volume in one second (FEV1) 58.1+/-19.9% pred.) were randomly assigned to the study group (500 microg b.i.d., for three weeks) or the control group (n=14; nonsteroid medication). Sputum samples were obtained during inhalation of hypertonic saline (3%, 20 min), which was found not to alter the investigated sputum parameters. No significant changes in clinical parameters, sputum leukocyte count, activity of myeloperoxidase, and baseline superoxide anion release where observed following therapy. Surprisingly, stimulated superoxide anion release increased significantly after therapy (34.1+/-17.7 versus 25.2+/-17.4 nmol x hr(-1) x 10(6) cells, p<0.03) and exceeded spontaneous variability of this parameter (p=0.02 versus control group). In conclusion, in adult cystic fibrosis patients short-term fluticasone therapy had no evident effect on clinical and sputum parameters. Further investigations are necessary to evaluate whether the observed up-regulation of oxidative capacity of inflammatory cells is of concern or benefit in these patients.

TNF-α Promoter Polymorphism in Relation to TNF-α Production and Clinical Status in Cystic Fibrosis

The severity of lung disease in cystic fibrosis may be related to the genetic propensity of the host to produce tumor necrosis fector α (TNF-α). A polymorphism in the promoter region of the TNF-α gene at nucleotide 308 relative to the transcription start site may be important in determing the host’s TNF-α response. The aim of this study was to assess the correlation between a TNF-308 promoter polymorphism, ex vivo TNF-α production (before and after lipopolysaccharide (LPS) stimulation), and clinical status [FEV1, weight (z-score), BMI, Shwachman score, incidence of diabetes mellitus, and Pseudomonas aeruginosa infection). Genotyping for the biallelic TNF-308 polymorphism was performed by using a real-time PCR cycler. Patients (homozygous for Delta F 508) were grouped according to genotype (TNF2 carriers, n = 16, median age = 15 yr, female/male = 5/11; TNF1 homozygotes, n = 37, median age = 21 yr, female/male = 18/19). TNF-α was measured using a chemiluminescent immunometric assay. There was a trend toward higher TNF-α values [median TNF2 carriers vs. TNF1 homozygotes: x = 56 vs. 43.5 pg/ml, n.s. (Mann–Whitney U-test] in those carrying the polymorphism and better lung function results [FEV1 (%) 81 vs. 65, n.s.]. These differences equalized [TNF2 carriers vs. TNF1 56 vs. 51 pg/ml, n.s.; FEV1 (%) 84 vs. 79, n.s.] after age adjustment (± 2 yr, n = 15, median age TNF2 vs. TNF1-17/18 yr). There were no significant differences for TNF values after LPS stimulation and the incidence of diabetes mellitus. The TNF-308 promoter polymorphism does not seem to influence TNF-α release in whole blood cells and clinical status.

Short-term effects of regular salmeterol treatment on adult cystic fibrosis patients.

Cystic fibrosis (CF) is characterized by chronic lung inflammation leading to airways obstruction. Bronchodilators, particularly short-acting beta2-agonists, are, therefore, often used by CF patients. The aim of this study was to evaluate, prospectively, the effects of the long-acting beta2-agonist salmeterol in adult CF patients. Twenty six patients with CF (10 males and 16 females; mean age (+/-SEM) 28+/-2 yrs) with mild-to-moderate airways obstruction (baseline forced expiratory volume in one second/forced vital capacity (FEV1/FVC) 56+/-2%) were monitored in an open, cross-over trial for 4 weeks by means of peak expiratory flow rates (PEFR), self-recorded symptom scores and body plethysmography. During a 2 week run-in period, all patients continued their treatment, including regular short-acting beta2-agonists. In weeks 3 and 4, short-acting beta2-agonists were replaced by the long-acting beta2-agonist, salmeterol (50 microg b.i.d.). Salmeterol produced a significant increase in PEFR compared to the run-in period (morning 375+/-23 vs 332+/-23 L x min(-1), deltaPEFR +15.1+/-3.1%, p<0.003; evening 384+/-24 vs 349+/-24 L x min(-1), deltaPEFR +11.7+/-2.4%, p<0.04). Similarly, patients reported lower symptom scores, e.g. less dyspnoea during the day, fewer nocturnal awakenings, less intense cough, and fewer unscheduled puffs of short-acting beta2-agonists. Thus, the long-acting beta2-agonist salmeterol provided clinical benefit to a majority of adult cystic fibrosis patients with airways obstruction. These short-term results are promising enough to set up long-term controlled studies.

Mediastinal lymph nodes demonstrated by mediastinal sonography : Activity marker in patients with cystic fibrosis

To evaluate the potential diagnostic role of mediastinal sonography in patients with cystic fibrosis (CF), we screened the mediastinum of adult CF patients with and without signs of infection and healthy controls.

Charcoal smoke causes bronchial anthracosis and COPD

ZusammenfassungHintergrund:Bronchopulmonale Erkrankungen, die durch die Inhalation von Rauch aus Holzfeuern verursacht sind, stellen ein bedeutendes Gesundheitsproblem in sog. Entwicklungsländern dar. Durch zunehmende Migration werden auch Ärzte in Europa mit diesen Erkrankungen konfrontiert.Fallbeschreibung und Diskussion:Eine 84-jährige afghanische Hausfrau, die nie geraucht hat und keine berufliche Exposition gegenüber anorganischen Stäuben aufweist, stellt sich mit dem Bild einer chronisch-obstruktiven Lungenerkrankung (COPD) in Verbindung mit bronchialer Anthrakose und Stenose eines Bronchus vor. Als ursächlich erweist sich die chronische Inhalation von Rauch aus einem zum Brotbacken verwendeten Ofen, der mit Holzfeuer in einem weitgehend geschlossenen Raum betrieben wurde. Das klinische Bild einer mit Holzkohlefeuer assoziierten Lungenerkrankung wird durch Husten und Dyspnoe mit bronchialer Obstruktion bestimmt. Radiologisch und bronchoskopisch finden sich Veränderungen, wie sie auch für Pneumokoniosen von Bergarbeitern charakteristisch sind, ohne dass eine entsprechende Exposition stattgefunden hat. Die Assoziation anthrakotischer bronchialer Stenosen mit einer Tuberkuloseinfektion gilt als häufig.Schlussfolgerung:Wegen fehlenden Risikobewusstseins geben die Patienten ihre Exposition gegenüber Holzfeuerrauch selten an. Deshalb ist eine gezielte Anamnese entscheidend, um die richtige Diagnose zu stellen und die Patienten einer adäquaten Diagnostik und Therapie zuzuführen.AbstractBackground:Bronchopulmonary disease due to inhalation of smoke from open woodfires represents a major health problem in developing countries. Due to increasing migration such patients also present to medical services in Europe.Case Report and Discussion:An 84-year-old Afghan housewife who never smoked nor has a history of exposure to inorganic dusts, presents with chronic obstructive pulmonary disease (COPD) in association with bronchial anthracosis and stenosis of a bronchus. The complaints are found to be caused by chronic inhalation of smoke from an open woodfire which was used for cooking. The main complaints of “woodsmoke-associated lung disease” are cough und dyspnea with bronchial obstruction. Radiology and bronchoscopy usually reveal changes which are similar to pneumoconiosis of miners but without patients’ relevant exposure. There is a frequent association of anthracotic bronchial stenosis and infection with tuberculosis.Conclusion:Since patients rarely recognize the risks of woodsmoke inhalation, they hardly report their exposure. Thus, the anamnesis is crucial to establish the right diagnosis and guide the patient to the appropriate diagnostic and therapeutic procedures.