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.

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

Systemic hypersensitivity vasculitis associated with bronchiectasis.

Systemic hypersensitivity vasculitis developed in a 53-year-old man during acute exacerbation of bronchiectasis infected with Pseudomonas aeruginosa. High grade fever, mononeuropathy multiplex, cutaneous vasculitis, and biopsy specimen-proved mesangioproliferative glomerulonephritis with crescent formation and leukocytoclastic vasculitis associated with circulating immune complex occurred. Corticosteroid and cyclophosphamide therapy was effective for vasculitis and bronchiectasis.

Effect of proton pump inhibitor alone or in combination with clarithromycin on mycobacterial growth in human alveolar macrophages.

The effect of omeprazole, a clinically used proton pump inhibitor, alone or in combination with clarithromycin was evaluated against Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium tuberculosis, using a human alveolar macrophage model of infection. Omeprazole exhibited no significant effect on the growth of the two M. avium complex strains or on the mycobactericidal activity of clarithromycin against them. In contrast, omeprazole significantly promoted the growth of Mycobacterium tuberculosis and the anti-mycobacterial activity of clarithromycin against it in human alveolar macrophages. It was speculated that intracellular acidic milieu around M. tuberculosis might be one reason for the lower activity of clarithromycin in the treatment of human tuberculosis.

Activity of KRM 1648 or rifabutin alone or in combination with clarithromycin against Mycobacterium avium complex in human alveolar macrophages.

SETTING: The activity of KRM 1648 (KRM), a new benzoxazinorifamycin, and rifabutin (RBT), alone or in combination with clarithromycin (CLA), was evaluated against Mycobacterium avium complex (MAC) that multiplied in human alveolar macrophages (AM). DESIGN: AM were recovered by bronchoalveolar lavage, incubated in RPMI 1640 medium with 10% human AB serum, infected with four strains of MAC (of non-acquired immune deficiency syndrome [AIDS] origin), and then treated with each drug alone or in combination. After incubation for 7 days, colony forming units in each well were counted on 7H10 agar. RESULTS: Although concentrations between 0.2 microgram/ml and 20 micrograms/ml of both rifamycins showed clear dose-dependent activities against all MAC strains tested, only 20 micrograms/ml of each drug had modest bactericidal effect. In combination with 2.0 micrograms/ml of CLA, however, 0.2 microgram/ml of both drugs caused a bactericidal response against two of the four MAC strains examined. CONCLUSION: According to this human alveolar macrophage model of MAC infection, KRM and RBT in combination with CLA was found to be a promising candidate against human pulmonary MAC infection, and deserves clinical evaluation.

Evaluation of Roche Amplicor PCR assay for Mycobacterium avium complex in bronchial washing.

SETTING: A commercially available polymerase chain reaction (PCR) test (Roche AmplicorTM Mycobacterium avium and M. intracellulare assay-MAC-PCR) designed to detect M. avium complex (MAC) in bronchial washing was evaluated. DESIGN: A total of 141 specimens from 127 patients with various pulmonary conditions were examined. Results were compared with acid-fast smears, cultures with Ogawa egg medium, as is still commonly used in Japan, and final diagnoses. RESULTS AND CONCLUSIONS: A total of 14 bronchial washing specimens yielded MAC. Six smear- and culture-positive specimens were all MAC-PCR positive. In eight smear-negative and culture-positive specimens, six were MAC-PCR positive. The overall sensitivity versus culture was 85.7% (12/14). However, sensitivity might be over-estimated, as there is a lower recovery rate of MAC with egg-based medium compared with liquid media. In 127 patients, 15 were identified as having pulmonary MAC disease, of whom 13 had positive MAC-PCR in bronchial washing. In the remaining 112 patients, MAC-PCR was negative, which suggests that positive MAC-PCR was not a contaminated result. However, in terms of sensitivity and speed, we were unable to show any additional clinical benefit for using MAC-PCR as opposed to liquid media, in which MAC can frequently be detected in 7 to 14 days.

A resected case of Mycobacterium szulgai pulmonary disease.

We present the first reported case of Mycobacterium szulgai pulmonary disease that needed surgical resection due to unsuccessful antimycobacterial chemotherapy. The patient was a non-immunocompromised 48-year-old male who presented with hemoptysis and whose sputum cultures repeatedly yielded M. szulgai. Antimycobacterial chemotherapy with isoniazid (INH) and rifampin (RMP)/ethambutol (EMB) for three years had been unsuccessful, and subsequent chemotherapy with RMP, EMB, ethionamide and kanamycin had to be discontinued due to liver dysfunction. Surgical resection was finally performed, and resulted in a favorable outcome. Although M. szulgai pulmonary disease is usually well controlled by antimycobacterial chemotherapy alone, surgical treatment may be necessary in some cases.

Nasal and paranasal sinus disease in patients with congenital velopharyngeal insufficiency.

OBJECTIVE: To investigate nasal and paranasal disease in patients with congenital velopharyngeal insufficiency (CVPI). DESIGN: Case series. SETTING: Patients with CVPI or cleft palate were investigated at Kyoto (Japan) University Hospital. PATIENTS AND OTHER PARTICIPANTS: Consecutive sample (60 patients with CVPI and 333 with cleft palate). No eligibility criteria or sociodemographic features. Random sample (normal subjects). MAIN OUTCOME MEASURES: The incidence of sinusitis and septal deviation, nasal airway resistance, nasal mucociliary function by saccharin test, and ciliary beating frequency were examined. These findings in patients with cleft palate and normal subjects were compared to clarify the features of nasal disease in CVPI. RESULTS: The incidence of sinusitis in CVPI was 46.7% and approximately equal to that in cleft palate (44.1%). The incidence of septal deviation in CVPI (26.7%) was low and nasal airway resistance was almost normal. In contrast, in patients with bilateral, unilateral, or incomplete cleft palate, the incidence of septal deviation was significantly more frequent (78.9%) than that in CVPI (P < 0.001), and nasal airway resistance was significantly higher than that in CVPI (P < .0001). In patients with submucosal cleft, septal deviation was found in 40.8% and nasal airway resistance was significantly higher than that in CVPI (P < .02). Although saccharin time was significantly prolonged (P < .001) and ciliary beating frequency was significantly lower (P < .005) in patients with cleft palate than in normal subjects, values for these tests in patients with CVPI were normal. CONCLUSIONS: The incidence of sinusitis was high in CVPI. However, the frequency of septal deviation and nasal airway resistance was almost normal. Nasal mucociliary function (saccharin time and ciliary beating frequency) was also normal.

The cause of nasal sinusitis in patients with cleft palate.

To determine the reason for the high incidence of nasal sinusitis in patients with cleft palate, the type of cleft, degree of nasal septal deviation, velopharyngeal function, and presence of pharyngeal flap were investigated in relation to nasal sinusitis. We also examined the relationship between the side of sinusitis and the cleft side in patients with unilateral cleft palate. Development of maxillary sinus, nasal mucociliary transport, and ciliary beating frequency of ciliated cells were also examined. The saccharin test indicated that nasal mucociliary transport was impaired. This impairment is believed to be one of the reasons for the high incidence of sinusitis in patients with cleft palate.

Rapid detection of Mycobacterium tuberculosis in sputa by the amplification of IS6110.

A polymerase chain reaction (PCR) assay for the rapid detection of Mycobacterium tuberculosis in sputum samples was studied. The target DNA was a 123-base pair (bp) fragment of IS6110, which was repeated in the M.tuberculosis genome and was specific for the M.tuberculosis complex. Glass beads (2mm diameter) and lysozyme were used to lyse the mycobacteria and DNA was extracted by the phenol-extraction method. The amplified PCR product was detected by examination of ethidium-bromide-stained agarose gel and by hybridization with an oligonucleotide alkali-phosphatase-labeled probe. A total of 70 samples were tested. PCR was positive in all 13 smear and culture-positive samples, in 5 of 8 smear-negative and culture-positive samples, and in 1 of 49 smear and culture negative samples. The overall sensitivity and specificity were 85.7% and 98%, respectively. Thus, IS6110 as a PCR target was found to be very useful for the rapid diagnosis of M.tuberculosis infection and start of anti-tuberculous chemotherapy.

Diagnosis and management of endobronchial tuberculosis.

We examined the records of sixty-one patients (17 males and 44 females) with endobronchial tuberculosis (EBTB). Smear tests of acid-fast bacilli were positive in 42 cases and cultures of tubercle bacilli (TB) were positive in 57. The main findings of chest roentgenogram on admission were as follows: no abnormal findings in 8, atelectasis in 30, infiltration in 25, and cavitary lesions in 6. The localization and cross-sectional extension of lesions confirmed bronchoscopically were as follows: trachea in 15, with 3 circular lesions (CLs). Right (R-) main bronchus in 19 with 11 CLs, left (L-) main bronchus in 18 with 11 of CLs, R-truncus intermedius in 14 with 6 of CLs, R-upper lobar bronchus (UB) in 17 with 12 CLs, R-middle lobar bronchus in 14 with 11 of CLs, R-lower lobar bronchus (LB) in 6 with 2 CLs, L-UB in 10 with 7 CLs and L-LB in 3 with 2 CLs. All cases were treated by combination chemotherapy with isoniazid, rifampicin, streptomycin and/or ethambutol and the rate of negative conversion of TB was good, but most of circular lesions resulted in severe bronchial stenosis or complete obstruction during and after chemotherapy, and no improvement was seen in any of the atelectasis cases at the cessation of chemotherapy. We discuss the points of early diagnosis and management of EBTB.

Recurrent hemoptysis in tuberculosis with non-cavitary lung disease as a symptom of mild hemophilia A in a young adult.

Hemoptysis in patients with tuberculosis is usually associated with smear-positive and cavitary lung disease. The present case describes a patient suffering from recurrent hemoptysis associated with tuberculosis who had smear-negative and non-cavitary lung disease, and who was subsequently diagnosed as having mild hemophilia A. Although mild hemophilia A sometimes escapes detection until adolescence, there has been no reported case of mild hemophilia A detected by recurrent hemoptysis due to pulmonary tuberculosis. Here, we report a rare case of recurrent hemoptysis in a patient with tuberculosis who had smear-negative and non-cavitary lung disease and who was finally shown to have hemophilia A.

[A clinical study of pulmonary cryptococcosis. The Study Group of Respiratory Mycosis in Kyoto].

During the 7 years from 1990, thirty-two patients (20 in male and 12 in female, mean age; 53 years old) were diagnosed as having pulmonary cryptococcosis. To clarify the essential points for early diagnosis of pulmonary cryptococcosis, we reviewed the clinical records and chest images. Three patients had a past history of pulmonary tuberculosis and eleven patients had underlying disorders such as malignancy, chronic pulmonary diseases and so on, but no HIV infection, which would affect this disease. Eighteen patients did not have any past history nor complications. The symptoms such as cough, sputum, chest pain and fever were generally of low-grade, 14 patients had no symptom at diagnosis. Except of some patients with severe infections and severe underlying disorders, laboratory findings such as inflamatory and nutritious markers were almost within near the normal range. On plain chest X-ray films the distribution of lesions was almost in proprtion to the volume of the lobes. The multifocal nudular and/or infitrative shadows wer observed in about 2/3 cases and single lesion in about 1/3. The width of lesions were minimal except of one case with interstitial pneumonia and two cases with multifocal segmental pneumonia. The cavity lesions were observed in 7 cases and hilar lymphadenopathy in 3 cases. On CT images, the lesions were almost located in the outer zone, the lesions which were adjacent to the pleura were observed in 15 cases. Cavitary lesions were almost smooth in edge and ubiquitous, the walls were also thick. The peripheral air-bronchogram in the nodular/infitrative shadows were observed in three cases. Pulmonary cryptococcosis is air-borne and almost a chronic infection except in AIDS patients, so careful planning for examination is essential with considerations of the characteristics of clinical and imaging features of this infection.

[Chest radiograph of atypical pneumonia: comparison among Chlamydia pneumoniae. Pneumonia, ornithosis, and Mycoplasma pneumoniae pneumonia].

No report has been found comparing Chlamydia pneumoniae (C. pneumoniae) pneumonia radiographically with other atypical pneumonias, Chlamydia psittaci (C. psittaci) pneumonia and Mycoplasma pneumoniae (M. pneumoniae) pneumonia. We described the chest radiographs of three kinds of pneumonia cases: 46 cases of C. pneumoniae pneumonia, 39 cases of C. psittaci pneumonia, and 131 cases of M. pneumoniae pneumonia. Radiographic shadows were categorized into main shadows and sub-shadows. The main shadows are classified from the viewpoint of the characteristics; air space consolidation(AS), ground-glass opacity(GG), reticular shadow(RS), bronchopneumonia(BP), and small nodular shadows (SN). The size, the site, and the number of the main shadows were also analyzed. In comparison among the three pneumonias, BP was the most frequent in M. pneumoniae pneumonia (0.40/case). AS predominated in C. pneumoniae pneumonia (0.67/case), and GG in C. psittaci pneumonia (0.62/case). The number of main shadows was equal, about 1.4/case in three pneumonias. Large shadows were less frequent in M. pneumoniae pneumonia than C. pneumoniae pneumonia (p = 0.02) and C. psittaci pneumonia (p = 0.01). Main shadows were more frequent in the outer zone in M. pneumoniae pneumonia than C. psittaci pneumonia (p = 0.01), and in the middle zone in C. psittaci pneumonia than in M. pneumoniae pneumonia (p = 0.02). Cases with bilateral main shadows were less common in M. pneumoniae pneumonia (9%) than C. pneumoniae pneumonia(33%, p = 0.001) and C. psittaci pneumonia(30%, p = 0.005). Thickening of bronchovascular bundles as a sub-shadow was most frequently noted in M. pneumoniae pneumonia. Some differences among the three atypical pneumonias were seen in the chest radiograph. However, no specific findings of C. pneumoniae pneumonia were shown radiographically in this study.

[New drugs against tuberculosis and nontuberculous mycobacterial infections: a review].

The number of cases with tuberculosis is again increasing in many countries, and recently several nosocomial outbreaks of multidrug-resistant tuberculosis have occurred in the United States. The number of patients with disseminated Mycobacterium avium complex (MAC) infections in AIDS population, and patients with MAC pulmonary disease unassociated with HIV seem to be also increasing. It takes at least 6 to 9 months for an initial treatment of active tuberculosis due to drug-sensitive strains with the standard regimen which includes isoniazid (INH) and rifampicin (RFP). Treatment for the diseases caused by drug-resistant M. tuberculosis and MAC is much more time-consuming and more toxic than for the diseases caused by drug-sensitive strains, and often unsuccessful. For the reasons described above, the developments of new agents with potent antimycobacterial activities are highly desired. The new agents should also be useful for treating patients who have acquired resistance to many of the currently available drugs. In this review the new antimycobacterial drugs are summarized. Some of them have already been used clinically, but many are still in experimental evaluations. 1) Rifamycin derivatives: rifabutin (RBT), KRM-1648 (KRM), rifapentin (RPT), FCE-22250, FCE-22807, CGP-7040, SPA-S-565 and other rifamycin derivatives. New rifamycin derivatives including RBT, KRM have increased in vitro antimycobacterial activities. RBT and KRM are much more active in vitro and in vivo than RFP against both M. tuberculosis and MAC. KRM seems to be more potent than RBT against MAC in experimental studies.(ABSTRACT TRUNCATED AT 250 WORDS)

[Multidrug-resistant tuberculosis].

Multidrug-resistant tuberculosis (MDR-TB) is defined as one that is resistant to both isoniazid and rifampicin regardless of its resistance to any other antituberculosis drugs. According to "Study on the incidence of drug resistance for new admissions" reported by TB Sanatorium Council in 1992 in Japan, no increase of incidence was observed in either the first treatment or re-treatment as compared with former reports. However, U.S.A. study indicates a significant increase of MDR-TB, which is supposed to have been caused by a primary drug resistance prevailed in an infected area, or an acquired (secondary) drug resistance due to incomplete and/or faulty treatment for active TB. Many incidences were also reported for mass nosocomial infection of MDR-TB with HIV patients. In spite of these serious issues in U.S.A., MDR-TB has not yet been a major concern in Japan, while Japan should work out countermeasures in advance with careful observation of its tend. One of the causes of mass nosocomial infection of MDR-TB observed in U.S.A. is reported due to a delayed treatment after long procedures of TB identification and susceptibility tests followed specimen sampling. Rapid tests of identification and susceptibility for TB, MDR-TB in particular, are long expected. The introduction of recent molecular genetics technology will help to develop new rapid tests. While a relationship between drug resistance and TB gene is recently known to certain extent, total mechanism of TB resistance cannot be fully explained with only certain gene identified in the connection with drugs. Early treatment is critical for MDR-TB with HIV patient, as their prognosis is far worse than MDR-TB with non-HIV. Aside from HIV infection, very limited drugs are available for the treatment of MDR-TB. Drugs should be carefully selected based on the resistance patterns of each strain as well as its side effects anticipated.

[Clinical features of the patients with "secondary infection" of Mycobacterium avium complex--Radiographic pattern of progressions in the patients with and without underlying pulmonary conditions].

We reviewed the radiographic features of 42 patients with Mycobacterium avium complex (MAC) infection. Six cases were definite "primary", 20 were "secondary", and 15 were indeterminate (probably "primary"). In the definite and probable "primary" cases, and some of the "secondary" cases, pulmonary lesions slowly progressed following a common and characteristic sequence from a cluster of small nodules or fibro-productive nodules to those with subpleural thickening, or with thickening of the draining bronchi, or with both subpleural and bronchial thickening, and to cystic bronchiectatic changes associated with collapse of the segment or the lobe, in the final stage. Cases of airspace pneumonia appeared rarely. In these cases, neither apical-subapical region prevalence, pleural effusion, nor mediastinal lymphadenopathy were found. On the contrary, in five cases of "secondary" infection, MAC lesions located at the same place with the predisposing lung disease and did not progress during more than five years of observation. In the progressive cases of "secondary" infection, however, the appearance of new lesions and the progressions thereafter showed the same pattern as in "primary" infection. These features would suggest that MAC infection would occur and progress slowly among patients who had unknown pathogenetical factor, whether they had underlying predisposing lung conditions or not.

[The need for new antituberculosis agents in Japan].

We discussed the need of new antituberculosis agents in Japan concerning drug adverse reactions in particular as well as drug interactions and drug resistance. We reviewed medical charts of hospitalized patients receiving standard antituberculosis chemotherapy (INH + RFP + EB/SM +/- PZA), and analyzed all symptoms, signs and abnormal laboratory data presumably caused by the chemotherapy. About 48% of 228 cases analyzed had at least 1 episode of adverse reactions: 77 episodes of hepatotoxicity, 15 episodes of ototoxicity, 14 episodes of eruption, 14 episodes of ocular toxicity and so on. About 23% cases treated with RFP, 20% cases with SM, 12% cases with INH and 7.7% cases with EB had at least 1 episode of adverse reactions due to the corresponding agent. In 59 cases, at least one agent was stopped to administer because of adverse reactions. Finally, standard chemotherapy could not be completed in 23% of all cases, and in stead, the other agents, mainly new quinolones, were administered. RFP induces cytochrome P450 3A in the liver to decrease the activities of many drugs, such as cyclosporin A, tacrolimus, protease inhibitor, itraconazole, and clarithromycin. In some clinical settings, RFP cannot be administered in combination with such drugs. In conclusions, new antituberculosis agents that have strong activities with less adverse reactions and drug interactions are needed in Japan, even without considering drug resistance.

[In vitro activities of new rifamycin derivatives against Mycobacterium tuberculosis and M. avium complex].

The in vitro anti-M. tuberculosis and anti-M. avium complex activities of five new rifamycin derivatives, KRM1648, KRM1657, KRM1668, KRM1674 and KRM2312, provided by Kanegafuchi Chem. Ind. Co. Japan were evaluated and compared with those of rifampicin (RFP) and rifabutin (RBU). Antimycobacterial activity was tested by broth dilution method using Kirchner's liquid medium supplemented with 10% bovine serum. The MICs 90 (micrograms/ml) of all five KRMs and RBU for 20 clinical isolates of M. tuberculosis were 0.035-0.07, whereas that of RFP was 1.25. The new rifamycin derivatives showed 16 to 32 times lower MICs than those of RFP against M. tuberculosis. All five KRMs inhibited 100% of 20 clinical isolates of M. avium complex at a concentration of 1.25 micrograms/ml, while only 35% and 10% of the strains were inhibited by the same concentration of RBU and RFP, respectively. The MICs 90 (micrograms/ml) for the strains tested were 0.07-0.3 for all five KRMs, and 5 and 40-80 for RBU and RFP, respectively. The new rifamycin derivatives were 16 times more active than RBU, which was 8 times more active than RFP. The new rifamycin derivatives were far more effective against M. tuberculosis in vitro than RFP, and their superiority to RBU which showed the effect superior to RFP was notable in in vitro anti-M. avium complex activities.

[In vivo activities of new rifamycin derivatives against mycobacteria].

Therapeutic effects of new rifamycin derivatives, 3'-hydroxy-5'-(4-alkylpiperazinyl) benzoxazinorifamycins, KRM 1648, 1657, 1668, 1674 and 2312 (kindly supplied by Kanegafuchi Chem. Ind. Co. Japan), were evaluated on experimental tuberculosis and Mycobacterium avium complex infection in mice. I. Experimental tuberculosis in mice Male ddY mice were inoculated via tail vein with ca. 1 x 10(9) CFU of M. tuberculosis H37Rv suspended in 0.2 ml medium. Treatment of the mice with the new rifamycin derivatives or rifampicin (RFP: as a control drug) was performed by daily oral administration of 10 mg/kg of the drugs, starting at the 24th hour of infection and continuing until the 40th day of infection. Therapeutic effect of each drug was assessed by mortality of the treated mice. All control mice which did not receive any drug died within the 20th day (in Exp. 1) and the 22nd day (in Exp. 2) of infection, while 25% (in Exp. 1) and 40% (in Exp. 2) of RFP-treated mice and 100% (in Exp. 1 and 2) of mice treated with any of the KRMs survived on the 40th day of infection. II. Experimental M. avium complex infection in mice Female beige mice (8-12 weeks old) were inoculated via tail vein with ca. 1 x 10(8) CFU of M. avium complex strain 31F093T, a mouse-virulent strain, suspended in 0.2 ml medium. Treatment of the mice with each drug (daily oral administration of 20 mg/kg) was started 24 hours after the inoculation, and was continued throughout 12 weeks of infection.(ABSTRACT TRUNCATED AT 250 WORDS)