Interaction of Aspergillus with human respiratory mucosa: a study with organ culture model.

The interaction of Aspergillus fumigatus with human bronchial mucosa in an organ culture model, with an air-mucosal interface, was studied. A. fumigatus conidia were inoculated onto the organ culture tissues and incubated for 24 h. At each time point (1, 6, 12, 18 and 24 h), after the measurement of ciliary beat frequency (CBF) of bronchial epithelial cells, adherence and invasion of the epithelium by A. fumigatus conidia (and hyphae), as well as structural changes of the epithelium, were investigated by scanning and transmission electron microscopy. This study demonstrated that A. fumigatus caused damage to the bronchial epithelium that was associated with CBF slowing. In addition, the study demonstrated that some of the conidia of A. fumigatus were internalized within ciliated and non-ciliated epithelial cells, some conidia were located within the intercellular spaces of the epithelium, and that hyphae penetrated through both intercellular and intracellular spaces of the epithelium. These findings suggest there might be at least three pathways by which Aspergillus invades the bronchial mucosa: (1) penetration of hyphae through the intercellular spaces in the epithelium; (2) direct penetration of hyphae through epithelial cells; and (3) internalization of conidia within epithelial cells.

Regenerative repair of the mandible using a collagen sponge containing TGF-beta1.

INTRODUCTION: Alveolar bone resorption and atrophy of the mandible are a major challenge for regeneration medicine. In the present investigation, a collagen sponge that contained TGF-beta1 was placed at a mandibular defect and the osteogenic effects of collagen-TGF-beta1, complex were evaluated. MATERIAL AND METHODS: The Pm2, Pm3, and Pm4 teeth on both sides of the mandibles of 12 adult beagle dogs (9.0-12.0 kg) were extracted. After the extraction-site wounds healed, a bone defect (10.0 x 15.0 mm-wide, 10.0 mm-deep or 10.0 x 10.0 mm-wide, 10.0 mm-deep) was created on the mandible. A collagen sponge (10.0 x 10.0 x 10.0 mm) that contained TGF-beta1 (1.0 microg, 5.0 microg, or 10.0 microg, in physiological saline) was placed at the bottom of the defect and the overlying mucous membrane was sutured with 4-0 prolene. As a control, a collagen sponge that contained physiological saline only was placed in a defect on the opposite side. Two weeks after the surgery the wounds above the bone defects on both the control and TGF-beta1-treated sides had healed completely. RESULTS: At four, six, or eight weeks post-operatively animals were killed. Soft X-ray and bone-salt measurement analyses confirmed clearly that there was greater calcified bone formation in the defects into which TGF-beta1 had been incorporated than with the control defects. The implanted collagen sponges were fully resorbed and the bone tissue had regenerated from the bottom of the defects on the TGF-beta1, side by four weeks. On the control side, no such regeneration was observed. CONCLUSIONS: These results indicate that TGF-beta1, released slowly from a collagen sponge was effective in promoting bone remodeling when applied to mandibular defects in adult dogs.

Artificial trachea and long term follow-up in carinal reconstruction in dogs.

We have already reported successful carinal reconstruction of the trachea with an observation period of 1 - 2 years. In this study, we evaluate the long-term safety and efficacy of the reconstruction after 5-years of follow-up. The Y-shaped Marlex mesh tube was reinforced with a polypropylene spiral and coated with atelocollagen made from porcine skin. The prosthesis was 60 mm long with an outer diameter of 18 mm. Replacement of the tracheobronchial bifurcation was preformed through a right thoracotomy in a beagle dog. Bronchoscopical examination and sampling of the tracheal epithelium was performed periodically to check the function of cilia. The implanted prothesis was promptly infiltrated by the surrounding connective tissue and completely incorporated by the host trachea and bronchus. Bronchoscopically, sufficient epithelization was confirmed from the upper to the lower site of anastomosis. After 5 years neither stenosis nor dehiscence was observed. In spite of there being mesh-exposure at the luminal surface, the dog had no clinical symptoms until sacrifice for pathological examination. The bent frequency of the cilia was maintained within the normal range, indicating functional recovery of the regenerating airway. Our tracheal prosthesis is promising for clinical repair of the tracheobronchial bifurcation.

[A study on cases diagnosed as pulmonary tuberculosis after admission to the general hospital lacking tuberculosis ward].

We analyzed retrospectively the clinical data of 12 patients diagnosed as pulmonary tuberculosis after admission to the National Himeji Hospital during the past 5 years. Ten out of 12 patients were male and were of elder age-groups (mean age: 65.5 years, range: 32-76 years). Seven patients at first visited the department of respiratory medicine, and the remaining three patients were admitted without consulting the department of respiratory medicine before admission. Only patient had a history of pulmonary tuberculosis. Two patients were suspected of pulmonary tuberculosis on admission. Tubercle bacilli were positive sputum smear in 6 patients, sputum culture in 1, smear of bronchial washing in 4, and smear of BALF in 1. It took 12.7 days on the average from the admission to make a final diagnosis as pulmonary tuberculosis patients into a general hospital lacking TB ward were as follows: (1) As pulmonary TB was not suspected, Chest X-ray and sputum examination were not performed. (2) The admission was done due to another disease and respiratory symptoms were scarce. (3) Tuberculosis lesions on chest X-ray were harbored by pleural effusions and diffuse shadows. (4) Though pulmonary tuberculosis was suspected, a patient was admitted because of general prostration as sputum smear was negative. After admission, however, repeated sputum culture revealed positive results. (5) Pulmonary tuberculosis developed after the admission.

[Successful treatment of allergic bronchopulmonary aspergillosis with erythromycin and fluconazole].

A 30-year-old woman was admitted to our hospital because of productive cough, wheezing, and the disclosure of abnormal shadows on chest X-ray films. The patient was given a diagnosis of allergic bronchopulmonary aspergillosis (ABPA) based on eight findings: asthma, eosinophilia, elevated serum IgE concentrations, immediate skin reactivity to Aspergillus antigen, the presence of precipitating antibodies against Aspergillus antigen, lung infiltration, central bronchiectasis, and repeated culture of Aspergillus fumigatus in sputum. Because she refused steroids, we administered erythromycin. The volume of her sputum subsequently decreased, her symptoms were brought under control, and her serum IgE fell, but the lung infiltrates did not clear. Discontinuation of erythromycin resulted in exacerbation of the patient's asthmatic symptoms, with high fever, increased sputum volume and IgE levels, and worsening lung infiltrates. These symptoms responded well to oral prednisolone medication, but sputum culture was still positive for Aspergillus fumigatus. Following discontinuation of prednisolone, the patient was treated with erythromycin, to which oral fluconazole was added for 16 months. Subsequent sputum cultures were negative for Aspergillus fumigatus, and for 7 years thereafter the patient remained in remission. Erythromycin and anti-fungal drugs may be worth trying in cases of allergic bronchopulmonary aspergillosis.

[Serrapeptase-induced lung injury manifesting as acute eosiniphilic pneumonia].

An 84-year-old man was referred to our hospital because of fever, cough, and hemoptysis. The patient had acute respiratory failure (PaO2 < 40 mmHg) on admission, with diffuse interstitial infiltration and bilateral pleural effusion. The bronchoalveolar lavage fluid was bloody, and contained a high percentage of eosinophils (32%). A diagnosis of acute eosinophilic pneumonia was established, and the patient made a rapid recovery after corticosteroids were administered. When the DLST (drug lymphocyte stimulation test) was performed after the corticosteroid therapy was stopped, it was positive for serrapeptase, which had been prescribed for chronic cystitis for 3 months before the onset of the pneumonia. This was a case of drug (serrapeptase)-induced pneumonitis manifesting as acute eosinophilic pneumonia.

Effects of Aspergillus fumigatus culture filtrate on antifungal activity of human phagocytes in vitro.

BACKGROUND: Aspergillus fumigatus can colonise the airways and the lungs with localised underlying conditions and occasionally invade the surrounding lung tissues even in subjects without systemic predisposing factors, presumably by escaping the local host defences. The aim of this study was to investigate the effects of A fumigatus culture filtrate (ACF) on the activities of human phagocytes--inhibition of germination of A fumigatus spores by alveolar macrophages (AMs) and hyphal damage by polymorphonuclear leucocytes (PMNs)--which are the critical host defences against A fumigatus. METHODS: Spores were incubated with AMs at a ratio of 1:1 in a medium containing different concentrations of ACF for 10 hours at 37 degrees C. Spore germination was visualised with light microscopy and the inhibition rate was calculated. The percentage of hyphal damage caused by PMNs pretreated with various concentrations of ACF was measured by a colorimetric tetrazolium metabolic assay. RESULTS: The inhibition rate of spore germination by AMs cultured with medium alone (control) was 90 (0.8)% whereas that by AMs cultured with the medium containing 10% ACF was significantly (p < 0.05) reduced to 41.7 (4.6)%. ACF suppressed the inhibition of spore germination in a dose dependent manner without altering the phagocytosing activity against the spores. The percentage of hyphal damage caused by PMNs pretreated with medium-199 (control) was 78.1 (2.3)% compared with 65.3 (2.8)% when PMNs were pretreated with 50% ACF (p < 0.05). CONCLUSIONS: A fumigatus releases biologically active substance(s) which suppress the inhibition of spore germination by AMs and also suppress PMN mediated hyphal damage, and thus may contribute to the pathogenicity of this fungus.

[Two probable cases of adult Williams-Campbell syndrome--advocation of Williams-Campbell-type bronchiectasis].

Williams-Campbell syndrome is a unique type of bronchiectasis which shows ballooning during expiration and collapse during inspiration. Its etiology is thought to be a congenital decrease in bronchial cartilage. We experienced two adult cases of Williams-Campbell syndrome. Since the mucociliary transport system was normal in both cases and neither case had chronic sinusitis, these cases differed from sino-bronchial syndrome. Cases of Williams-Campbell syndrome reported in Japan show characteristic bronchography, but a decrease in bronchial cartilage may or may not be present and its congenital nature is very difficult to demonstrate. We therefore advocate referring to such cases of bronchiectasis with these bronchographic characteristics Williams-Campbell-type bronchiectasis, even if a decrease in bronchial cartilage is not observed.

[A case of minocycline-induced pneumonitis with bronchial asthma].

We report a case of minocycline-induced pneumonitis. A 30-year-old woman was treated with minocycline for mycoplasma pneumonia of the right upper lobe. About 15 days after starting treatment, she developed a productive cough, stridor, and dyspnea. The chest X-ray film showed pulmonary infiltration in the left middle lung field. Based on the clinical history and the detection of eosinophilia in the bronchoalveolar fluid, drug-induced pneumonitis was suspected. Treatment with minocycline was discontinued and prednisolone (20 mg/day) was started, after which her symptoms subsided and there was marked regression of the pulmonary infiltrates on chest X-ray films. The lymphocyte stimulation test for minocycline was negative, but the diagnosis was confirmed by a positive oral provocation test.

Effects of alkaline protease or restrictocin deficient mutants of Aspergillus fumigatus on human polymorphonuclear leukocytes.

Several substances including proteases and restrictocin have been suggested as candidates for virulence determinants in invasive pulmonary aspergillosis. However, the roles of such substances are not well understood. This study compared the in vitro suppressive effects of Aspergillus fumigatus culture filtrates (ACFs), on the functions of human polymorphonuclear leukocytes (PMNLs), the principal cells in the host defence against aspergillus hyphae, from a clinically isolated wild-type and isogenic mutant strains which lack production of elastolytic alkaline protease (Alp) and/or restrictocin. ACFs were obtained by culturing conidia of each strain in Medium- 199 at 37 degrees C for 5 days. ACFs of the wild-type significantly (p<0.01) suppressed chemotaxis, superoxide anion (O2-) release and PMNL-mediated hyphal damage, compared with the control (Medium-199). ACFs of the mutant strains that lack Alp or restrictocin significantly (p<0.01) suppressed chemotaxis and O2(-)-release, but did not suppress hyphal damage, compared with the control. The wild-type significantly (p<0.01) suppressed chemotaxis of PMNLs compared with the mutant strains lacking Alp or restrictocin, whereas there were no significant differences in suppression of O2(-)-release and hyphal damage by PMNLs. ACF of a mutant strain that lacks both Alp and restrictocin had much less activity, but significantly (p<0.01) suppressed chemotaxis of PMNLs compared with the control. In conclusion, alkaline protease and restrictocin may play roles in the suppressive effect of Aspergillus fumigatus culture filtrates on the functions of human polymorphonuclear leukocytes. Other antiphagocytic substances produced by Aspergillus fumigatus remain to be identified.