Significance of human beta-defensins in the epithelial lining fluid of patients with chronic lower respiratory tract infections.

Human beta-defensins (hBDs) are the most abundant antimicrobial peptides in epithelial cells, and function in the host immune system. Respiratory epithelial cells express hBDs to inhibit bacterial proliferation during respiratory tract infections. The aim of this study was to investigate the release of hBDs into the respiratory tract and their benefit as a host defence system in chronic Pseudomonas aeruginosa infections. The levels of four hBD peptides (hBD-1-hBD-4) were measured in the bronchial epithelial lining fluid (ELF) of nine patients with chronic lower respiratory tract infection caused by P. aeruginosa. Eight patients with idiopathic pulmonary fibrosis and eight volunteers free of pulmonary disease were recruited as controls. ELF was obtained by bronchoscopic microsampling and hBD levels were measured by radioimmunoassays. The antimicrobial effects of hBDs were studied individually and in combination using an in-vitro colony count assay for P. aeruginosa. Concentrations of hBD-1 and hBD-3 tended to be higher in patients with chronic lower respiratory tract infection than in the controls. hBD-2 and hBD-4 were detected in ELF from five and four of nine patients, respectively, but the hBD levels in controls were all below the limits of detection. All patients with infection caused by mucoid P. aeruginosa had detectable hBD-2 and hBD-4 levels in ELF. In-vitro colony count assays showed a potential synergism between hBD-2 and hBD-4 in inhibiting bacterial proliferation. The findings indicate that hBDs, especially hBD-2 and hBD-4, are pathophysiologically important in infections caused by mucoid strains of P. aeruginosa.

Identification of hBD-3 in respiratory tract and serum: the increase in pneumonia.

Human beta-defensin (hBD)-3, a 45 amino acid antimicrobial peptide, was originally isolated from human skin. hBD-3 mRNA has also been detected in the airways by RT-PCR. While hBD-3 may be involved in antimicrobial defences within the respiratory tract, the presence of hBD-3 peptide in the respiratory system has not yet been confirmed. The antimicrobial activity of the synthesised hBD-3 peptide was measured by a radial diffusion assay and a colony count assay. The present authors confirmed the presence of hBD-3 peptide in homogenates of human lung and serum using reverse-phase HPLC coupled with a highly sensitive RIA. The localisation of the hBD-3 peptide was investigated by immunohistochemistry. In addition, the serum concentrations of hBD-3 were measured by RIA. hBD-3 exhibited a strong antimicrobial activity, which was unaffected by increasing salt concentrations. Immunohistochemically, the current authors observed the expression of hBD-3 in bronchial and bronchiolar epithelial cells. The mean+/-sd serum concentration of hBD-3 in patients with bacterial pneumonia was 239.4+/-17.8 pg x mL(-1) in the acute phase and, decreased to 159.3+/-20.1 pg x mL(-1) after the completion of therapy. In conclusion, these findings will help elucidate the role of human beta-defensin-3 in host immune responses and identify the pathophysiological significance of this molecule in respiratory infections.

Increased concentrations of human beta-defensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis.

BACKGROUND: Human beta-defensin (HBD)-1 and -2 are antimicrobial peptides present in the respiratory tract. Recent reports have indicated reduced activity of beta-defensins in cystic fibrosis, suggesting that beta-defensins may play an important role in the pathological process of chronic respiratory tract infection. Diffuse panbronchiolitis (DPB) is a progressive disease characterised by frequent episodes of superimposed infection, typically caused by Pseudomonas aeruginosa. The aim of this study was to elucidate the role of these antimicrobial peptides in this disease. METHODS: The concentrations of HBD-1 and HBD-2 in plasma and bronchoalveolar lavage (BAL) fluid from 33 patients with DPB and 30 normal adults were measured by radioimmunoassay. Localisation of HBD-2 was investigated immunohistochemically in an open lung biopsy specimen obtained from a patient with DPB. RESULTS: High concentrations of HBD-1 and HBD-2 were noted in BAL fluid from DPB patients. Increased plasma concentrations of HBD-2, but not HBD-1, were found in patients with DPB compared with control subjects. In patients with DPB the HBD-2 concentration in BAL fluid correlated significantly with the numbers of cells recovered from the BAL fluid (total cells, neutrophils, and lymphocytes) and with the BAL fluid concentration of IL-1beta. Synthetic HBD-2, but not HBD-1, had dose dependent bactericidal activity against P aeruginosa. Treatment of 14 patients with macrolides significantly reduced BAL fluid concentrations of HBD-2 but not HBD-1 or plasma concentrations of HBD-1 and HBD-2. Immunohistochemistry of lung tissue showed localisation of HBD-2 in the epithelia of the distal bronchioles. CONCLUSIONS: These results indicate that beta-defensins, particularly HBD-2, participate in antimicrobial defence in the respiratory tract in DPB, and that the BAL fluid concentration of HBD-2 may be a useful marker of airway inflammation in patients with DPB.

Elevated levels of thymus and activation-regulated chemokine (TARC) in pleural effusion samples from patients infested with Paragonimus westermani.

To investigate the pathogenic mechanisms of eosinophilic pleural effusion in patients with paragonimiasis, we measured the levels of various chemokines including thymus and activation-regulated chemokine (TARC), eotaxin, RANTES and IL-8 in pleural effusion samples. Samples were obtained from 11 patients with Paragonimus westermani infection, six patients with pleural transudate, eight with tuberculous pleurisy and five with empyema. High percentages of eosinophils were detected in pleural fluid (range 9-100%, median 81%) of patients with paragonimiasis. TARC concentrations in pleural effusions of paragonimiasis were markedly higher than those of other groups. Eotaxin levels were also higher in pleural effusions of paragonimiasis patients, although significant difference was noted only against transudate samples. There was a significant correlation between TARC concentrations and percentages of eosinophils, and between TARC and eotaxin concentrations in pleural effusion. There were also significant correlations between TARC concentration and the titre of anti-P. westermani IgG and between eotaxin concentration and the titre of anti-P. westermani IgG. Our findings suggest that TARC contributes to the pathogenesis of eosinophilic pleural effusion in paragonimiasis.

Raised plasma concentrations of alpha-defensins in patients with idiopathic pulmonary fibrosis.

BACKGROUND: Neutrophils are thought to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Human neutrophils contain antimicrobial and cytotoxic peptides in the azurophil granules which belong to a family of mammalian neutrophil peptides named alpha-defensins. A study was undertaken to investigate the role of alpha-defensins in the pathogenesis of IPF. METHODS: The concentrations of alpha-defensins (human neutrophil peptides (HNPs) 1, 2, and 3) in plasma and bronchoalveolar lavage (BAL) fluid of 30 patients with IPF and 15 healthy subjects were measured by radioimmunoassay. RESULTS: The concentrations of alpha-defensins in plasma, but not in BAL fluid, were significantly higher in IPF patients than in controls. BAL fluid concentrations of interleukin (IL)-8 in patients with IPF, which were significantly higher than in controls, correlated with those of alpha-defensins. An inverse relationship was seen between plasma alpha-defensin levels and the arterial oxygen tension (PaO(2)) and pulmonary function (vital capacity (%VC), forced expiratory volume in 1 second (FEV(1)), and carbon monoxide transfer factor (%TLCO)) in patients with IPF. Plasma levels of alpha-defensins also correlated with the clinical course in IPF patients with an acute exacerbation. Immunohistochemically, positive staining was observed inside and outside neutrophils in the alveolar septa, especially in dense fibrotic areas. CONCLUSION: These findings suggest that alpha-defensins play an important role in the pathogenesis of IPF, and that the plasma alpha-defensin level may be a useful marker of disease severity and activity.

[Beta-defensins in plasma and bronchoalveolar lavage fluid in patients with non-tuberculous mycobacterium infection].

We measured the levels of beta-defensin 1 and 2 (HBD-1, 2), novel antimicrobial peptides in plasma and in bronchoalveolar lavage fluid (BALF) from patients with with non-tuberculous mycobacterium infection (NTM). Plasma HBD-2 levels in NTM patients before treatment were higher than those in the controls, while the HBD-1 levels were similar to the control levels. High levels of HBD-2, but not of HBD-1, in BALF were also observed in NTM patients. In NTM, a positive correlation was found between HBD-2 levels in BALF and plasma, and also between HBD-2 and IL-1 beta levels in BALF. NTM patients with cavities or ectasia on chest radiography had higher HBD-2 levels in BALF than those without. Plasma HBD-2 levels in NTM patients were markedly decreased after successful treatment, while those of patients with an intractable mycobacterium infection maintained the same high plasma HBD-2 levels as those before treatment. These findings suggest that HBD-2 may participate in the host defense and plasma HBD-2 levels may reflect disease activity in pulmonary NTM.

Case report: Paragonimiasis westermani with seroconversion from immunoglobulin (Ig) m to IgG antibody with the clinical course.

A 66-year-old man visited our hospital with primary complaint of cough. Chest roentgenogram showed slight pleural effusion and pneumothorax in the left lung. Eosinophilia (22.8%) was also found in his peripheral blood. Multiple-dot enzyme-linked immunosorbent assay (dot-ELISA) for the detection of parasite-specific immunoglobulin (Ig) G antibody was used to screen his serum against various parasitic diseases, but no significant binding was observed with any of the 12 parasite antigens examined, including those of Paragonimus westermani and P. miyazakii. Although he seemed to have been spontaneously cured without treatment, a nodular shadow appeared in the right upper medial lung field on the chest roentgenogram 6 months later. This time, his serum was positive for anti-P. westermani IgG antibody by the same method. A reexamination of the first and second admission serum samples for parasite-specific IgM and IgG antibodies revealed significant level of IgM antibody in the serum of the first admission, which had decreased at the time of the second admission. Conversely, the level of IgG antibody, which was low at the first admission, became dominant in the second admission serum 6 months later. These results clearly show that although the dot-ELISA to detect IgG antibody is generally useful for screening and detecting paragonimiasis, detection of IgM antibody seems to be a useful aid and should also be included in immunoserological diagnosis, especially if the patient is considered to be in the early stage of infection.

[Clinical features in twenty-three patients with paragonimiasis westermani].

We assessed the clinical features in twenty-three cases diagnosed as paragonimiasis westermani from April 1988 to May 2000 in our clinic at Miyazaki Medical College Hospital. Twenty-one patients had eaten wild boar or crab meat. Their symptoms included productive cough, chest pain and hemosputum. The findings of radiography and computed tomography (CT) were nodules, masses, infiltrative shadows and pleural effusion. The percentages of eosinophils in the peripheral blood were elevated in 16 of 23 patients, and the serum IgE values were elevated in 12 of 18 patients. Serologic tests, which are very helpful for diagnostic purposes were performed on the blood sera of all patients, and on the pleural effusions of five patients. It is often difficult to diagnose paragonimiasis westermani in patients in whom eosinophilia or increased serum IgE is not present. However, paragonimiasis westermani should also be considered as a possible diagnosis when abnormal shadows are found in chest radiographs and chest CT.

Recombinant granulocyte colony-stimulating factor induces production of human neutrophil peptides in lung cancer patients with neutropenia.

Human neutrophil peptides (HNPs) 1, 2 and 3 are antimicrobial peptides localized in the azurophil granules of neutrophils. We investigated the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the biosynthesis of HNPs 1-3 using a sensitive radioimmunoassay and Northern blot analysis. Seven patients with lung cancer were first treated with various anticancer agents for 3 days (days 1-3) followed by treatment with rhG-CSF (2 microgram/kg weight/day) for 7 days (days 8-14). Chemotherapy caused neutropenia but the neutrophil count increased biphasically between days 8 and 14. Chemotherapy did not change the baseline plasma concentration of HNPs 1-3 (74.1+/-2.1 pmol/ml) but the concentration increased from day 12, 5 days after commencement of rhG-CSF therapy, to reach a peak value of 430.8+/-57.0 pmol/ml on day 15, 1 day after the last administration of rhG-CSF. Baseline HNPs 1-3 content per neutrophil was 0.59+/-0.02 fmol, decreased to 0.30+/-0.07 fmol on day 9, then increased to 0.78+/-0.07 fmol on day 15. Analyses of peripheral blood neutrophils by Northern blot and reverse-phase high-performance liquid chromatography showed that the amounts of HNPs 1-3 mRNA and precursors of HNPs 1-3 markedly increased in response to rhG-CSF. Our results indicate that recombinant hG-CSF does not only increase neutrophil count but stimulates HNPs 1-3 biosynthesis in neutrophils, thus enhancing the host defense system of compromised hosts with neutropenia.

[Pulmonary dirofilariasis with cavity formation and pleural effusion].

A 61-year-old man visited a community hospital because of hemosputum. A solitary nodule in the left lower lung field was pointed out on a chest roentgenogram. The patient was treated with antibiotics, but the solitary nodule increased in size. He was referred to our hospital because of high fever and observations of cavity formation and pleural effusion on a chest roentgenogram. The pleural effusion showed no cytologic evidence of malignancy, and cultures were also negative for bacteria. An increased percentage of lymphocytes was detected in the pleural effusion, but slight eosinophilia was found in blood samples. Dot enzyme-linked immunosorbent assay and Ouchterlony's double-diffusion test yielded a diagnosis of pulmonary dirofilariasis. After drainage of the pleural effusion by thoracentesis, spontaneous regression was observed. Cavity formation, pleural effusion, and spontaneous regression are in general rare in patients with pulmonary dirofilariasis.

Paragonimiasis westermani with multifocal lesions in lungs and skin.

We report a case of Paragonimus westermani infection with a reticulonodular lesion in the right lung, left pleural effusion, and a mobile subcutaneous mass. Analyses of pleural effusion and bronchoalveolar lavage fluid (BALF) showed marked eosinophilia and high levels of eosinophil cationic protein and interleukin (IL)-5. Transbronchial lung biopsy revealed the presence of pneumonia with mild eosinophilic infiltration but remarkable lymphocytic infiltration. In this patient, high IL-5 levels in both BALF and pleural effusion could explain the remarkable eosinophilia.

[Bronchial atresia with atelectasis of the left upper lobe].

A 69-year-old woman visited her physician on October 1 complain of dry cough. However, the chest radiograph revealed no abnormalities. She was later admitted to our hospital because a radiograph taken by another physician on November 26 revealed a massive lesion in the right upper mediastinum. Computed tomographic findings showed a massive lesion containing a branching structure with a few calcifications, suggesting a case of atelectasis of the left upper lobe with mucus plug. Bronchoscopic examination revealed complete obstruction of the orifice of the left upper lobe bronchus, and so a diagnosis of bronchial atresia was made. However, since the patient had a history of tuberculous peritonitis and the mass lesion was somewhat calcified, the possibility that this was an acquired case could not be ruled out. After treatment with oral antibiotics, the size of the atelectasis was decreased. Therefore, we considered that the expansion of the atelectasis could have been due to superimposed bacterial infection.

[Thymic cyst with elevated SLX levels in serum and cystic fluid].

A 24-year-old man was admitted to our hospital in June 1996 with complaints of anterior chest discomfort. Chest X-ray films on admission showed an abnormal mediastinal shadow with well-defined margin. Chest X-ray examinations about 6 weeks earlier had not detected any abnormalities. Laboratory tests on admission showed a high serum concentration of Siaryl Lewis X-i antigen (SLX). A computed tomographic scan of the chest showed a large (6 x 6 x 12 cm) homogeneous mass in the right anterior mediastinum. The mass was removed completely and histologically diagnosed as a thymic cyst. Biochemical analysis of fluid from the cyst revealed remarkably high levels of SLX, CA 19-9, and CEA. In immunohistochemical studies, epithelial cells from the cystic walls stained positive for SLX, CA 19-9, and CEA. After the operation, the level of serum SLX returned almost to normal.

Granulocyte-colony stimulating factor levels in bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis.

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) is known as a potent neutrophil chemotactic glycoprotein in vitro but its contribution to chemotactic activity in neutrophil mediated lung diseases is not yet known. The aims of this study were to determine whether G-CSF is present in high concentrations in bronchoalveolar lavage (BAL) fluid of patients with idiopathic pulmonary fibrosis (IPF, also called cryptogenic fibrosing alveolitis), a neutrophil mediated lung disease, and to what extent G-CSF in BAL fluid contributes to neutrophil accumulation in the lung of patients with IPF. METHODS: G-CSF concentrations in BAL fluid samples from 16 healthy volunteers, 24 patients with IPF, and 73 patients with non-IPF lung disease were measured by enzyme linked immunosorbent assay. The relationship between G-CSF concentrations and neutrophil count in BAL fluid was also examined. Neutrophil chemotactic activity (NCA) was measured in BAL fluid in healthy volunteers and patients with IPF. The contribution of G-CSF to overall NCA in lungs with IPF was assessed by repeating the measurement of NCA after a complete neutralisation of G-CSF bioactivity by anti-human G-CSF antiserum. RESULTS: Detectable levels of G-CSF were found in BAL fluid of 83% of patients with IPF while the levels in all healthy volunteers were below the detection limit. In patients with IPF a significant correlation was observed between the BAL fluid neutrophil count and the concentration of G-CSF in the BAL fluid. The neutrophil count also correlated significantly with percentage forced vital capacity. In BAL fluid samples from patients with IPF the mean NCA value was reduced by 35% after neutralisation with an anti-human G-CSF antiserum. CONCLUSIONS: G-CSF may be involved in enhancing neutrophil accumulation in the lungs of patients with IPF.

[A study of human beta-defensin-1 and human beta-defensin-2 in airway mucosal defense].

Human beta-defensin-1 (hBD-1) and human beta-defensin-2 (hBD-2) are new members of the defensin family. In this study, we investigated their gene expressions in the respiratory epithelial surface of the human lung and their bactericidal activities against Escherichia coli. Both hBD-1 and hBD-2 gene transcripts were detected in human lung tissue and cellular component of bronchoalveolar lavage fluid by reverse transcription-polymerase chain reaction. Synthetic hBD-1 and hBD-2 had dose-dependent bactericidal activities against E. coli. The concentration for the 50% colony reduction of hBD-2 was 0.46 nmol/ml, that for HNP-1 2.15 nmol/ml, and hBD-1 99.3 nmol/ml under conditions nearly the same as in human bronchial airway surface liquid. We prepared an antiserum against hBD-2 and established a highly sensitive radioimmunoassay and quantified plasma hBD-2 concentrations in normal subjects and patients with pneumonia. The plasma concentration of hBD-2 in normal individuals was 8.3 +/- 0.9 fmol/ml (mean +/- SE). The mean hBD-2 plasma concentration for the 12 patients with bacterial pneumonia in the acute stage was 34.2 +/- 3.4 fmol/ml, significantly higher than normal individuals, and returned to the normal range after recovery. The presence of hBD-1 and hBD-2 in the airway tract and their bactericidal activity suggest that they function in innate airway mucosal defense.

Pulmonary Nocardia otitidiscaviarum infection in a patient with chronic respiratory infection.

We report a case of pulmonary Nocardia (N.) otitidiscaviarum infection in a 76-year-old man with chronic respiratory infection. The patient responded poorly to intravenous imipenem and oral minocycline, but later improved after treatment with trimethoprim-sulfamethoxazole. Pulmonary infection with N. otitidiscaviarum should be considered in the differential diagnosis of chronic respiratory infections. Further studies are needed to evaluate the correlation between species and drug susceptibility.

[Idiopathic interstitial pneumonia exacerbated by alveolar hemorrhage].

A 60-year-old man, in who idiopathic interstitial pneumonia (IIP) was diagnosed in 1990, was admitted to our hospital in March 1995 with exertional dyspnea, hemosputum and fever. On chest X ray, new infiltrates in the bilateral middle lung fields were seen, in addition to reticulonodular shadows in the bilateral lower lung fields, which had been noted in 1990. Intubation was reformed because of hypoxemia, and bronchoalveolar lavage (BAL) was performed in right B6. The BAL fluid was bloody, and a diagnosis of alveolar hemorrhage was made based on the presence of many hemosiderin-laden macrophages in the fluid. After pulse therapy with methylprednisolone, the hypoxemia and chest X ray findings improved, and he was extubated. Alveolar hemorrhage may be one possible complication leading to an exacerbation of IIP.

[Severe bronchorrhea accompanying alveolar cell carcinoma: treatment with clarithromycin and inhaled beclomethasone].

We report the efficacy of oral clarithromycin and inhaled beclomethasone against severe bronchorrhea in a patient with alveolar cell carcinoma. A 54-year-old man produced about 500 to 900 ml of clear and egg-white-like sputum each day. Anti-cancer chemotherapy and erythromycin therapy did not reduce the volume of sputum. After administration of clarithromycin and inhaled beclomethasone, sputum volume decreased to about 300 nl each day and the patient's ability to perform daily activities improved. Two months later, clarithromycin was stopped and the patient was treated with inhaled beclomethasone alone. Sputum volume did not increase for 6 months, although the chestroentgenographic findings gradually worsened. Then the sputum volume gradually increased. Five months after the sputum volume began to increase, he was producing about 2 liters of sputum each day and died of respiratory failure. Although the levels of CA 19-9, SLX, and CEA in serum were all within the normal range, the sputum contained high levels of CA 19-9 (1,133,620 U/ml), SLX (3,000 U/ml), and CEA (283 ng/ml). In patients with bronchorrhea, measurement of tumor markers in sputum may be useful for the diagnosis of alveolar cell carcinoma.

Identification of human beta-defensin-2 in respiratory tract and plasma and its increase in bacterial pneumonia.

Human beta-defensin-2 (hBD-2), a novel antimicrobial peptide, was originally isolated from human skin. We found that synthetic hBD-2 has high bactericidal activity against Escherichia coli under conditions nearly the same as in human bronchial airway surface liquid. We prepared an antiserum against hBD-2 and established a highly sensitive radioimmunoassay (RIA). Reverse-phase high-performance liquid chromatography coupled with the RIA showed that hBD-2 in patients with human lung, bronchoalveolar lavage flid, and plasma. The plasma concentration of hBD-2 in patients with bacterial pneumonia was 32.1 +/- 3.7 fmol/ml (mean +/- SE), 3.9-fold that of normal individuals. By reverse transcription-polymerase chain reaction, the hBD-2 gene transcript was detected in the respiratory epithelial surface of human lung. Human beta-defensin-2 seems to function in airway mucosal defense. Our findings provide a clue to elucidate its pathophysiological significance in respiratory infection.

Elevated concentrations of defensins in bronchoalveolar lavage fluid in diffuse panbronchiolitis.

Human neutrophils contain three isoforms of antimicrobial and cytotoxic peptides in the azurophil granules, which belong to a family of mammalian neutrophil peptides named defensins. Here we investigate the role of these peptides in diffuse panbronchiolitis (DPB). Defensins (human neutrophil peptide-1, -2 and -3) were measured by radioimmunoassay in bronchoalveolar lavage fluid (BALF) of 30 patients with DPB, 16 patients with idiopathic pulmonary fibrosis (IPF) and 15 healthy adults. The concentration of defensins was higher in BALF of patients with DPB than in patients with IPF and healthy subjects. DPB and IPF patients also had significantly higher plasma concentrations of defensins than controls. In patients with DPB, BALF concentration of defensins correlated significantly with neutrophil count or BALF concentration of interleukin (IL)-8. Immunohistochemistry of open-lung biopsy specimens from four DPB patients showed localization of defensins in neutrophils and mucinous exudate in the airways, and on the surface of bronchiolar epithelial cells. In vitro studies showed an enhanced extracellular release of defensins following stimulation of neutrophils with phorbol myristate acetate, N-formyl-methionyl-leucyl-phenyalamine, and human recombinant IL-8. Treatment of DPB with macrolides for 6 months significantly reduced neutrophil count and concentrations of defensins and IL-8 in BALF. Our results indicate accumulation of neutrophil-derived defensins in the airway in diffuse panbronchiolitis, and suggest that defensins may be a marker of neutrophil activity in this disease.