Preclinical anticancer effects and toxicologic assessment of hepatic artery infusion of fine-powder cisplatin with lipiodol in vivo.

We conducted an in vivo study to evaluate the anticancer effect and toxicity of fine-powder cisplatin suspended in lipiodol (fCDDP/LPD suspension) after a single administration of three different doses to rats via the intrahepatic artery after transplantation of rat ascites hepatoma cells. The toxicity of the fCDDP/LPD suspension was also assessed in the same protocol in noncancer-bearing rats and the observed toxicologic changes were compared among groups administered saline (Sal), an aqueous solution of fCDDP (fCDDP/Sal solution), and LPD alone. In parallel with the toxicity test, plasma CDDP concentrations were compared between the fCDDP/LPD suspension and fCDDP/Sal solution. The mean weight of the tumors in the fCDDP/LPD suspension groups was significantly less than in the LPD-alone group. The pathologic changes in the liver observed in the fCDDP/LPD suspension group increased with dose, were more marked compared with those in the fCDDP/Sal solution and LPD-alone groups, and were reversible. No other toxicologic effects were observed. The concentration of CDDP in the plasma in the fCDDP/LPD suspension group was slightly lower than that in the fCDDP/Sal solution group. In conclusion, the results indicate that the fCDDP/LPD suspension has sufficient anticancer efficacy and tolerability for use in the clinical treatment of hepatocellular carcinoma.

[Tuberculosis of the elderly (above the age of 75) in national hospitals].

In Japan tuberculosis is becoming rapidly the disease of the elderly. We studied the background, the type and level of hospital cares needed, and the outcome of patients with pulmonary tuberculosis (sputum smear and/or culture positive) above the age of 75 who were admitted to 8 national hospitals during the period from January 1 to December 31, 1997. The study included 150 patients (male: 109, female: 41, mean age: 81.6), of whom 25% needed care in a single-bed room, 84.3% had underlying diseases (cardiovascular diseases: 43.3%, malignant diseases: 20.9%, neuro-psychiatric diseases: 13.4%), and 47.6% needed cares mainly in feeding and excretions. 92 patients (62.6%) improved and 45 patients (30.6%) died, of whom the cause of death was directly related to tuberculosis in 42. The mean hospital stay was 4.7 months. However, in 42 patients whose cause of death was related to tuberculosis, 66% died within 3 months, while in 102 patients who were discharged 71% stayed more than 3 months. The same comparison was done in 508 patients with bacteriologically proven tuberculosis above the age of 75 admitted to National Tokyo Hospital during the period from 1990 to 1999. The result was almost the same, among 133 patients died in the hospital 60% died within 3 months, while in 375 patients who were discharged hospital stay was more than 3 months in 70%. In the near future, the elderly will occupy more than 25% of the beds of the tuberculosis ward in Japan and most of them have underlying diseases other than tuberculosis. Because tuberculosis, once the disease of the young, is becoming rapidly the disease of the elderly, it is imperative for us to make necessary adjustments to meet this inevitable trend.

[Proposal for a new method for evaluation of physical exercise capacity in patients with chronic respiratory disease].

We developed a new method of evaluating the tolerance for physical exercise in patients with chronic respiratory disease. Using a newly developed portable pulse oxymeter, with which we could measure kinetic energy (physical activity), calculated from the vertical acceleration involved in physical movements in the patient's daily life, we considered the correlation between the characteristics of the distribution of oxygen saturation (SpO2) and the degree of physical activity. The characteristics of SpO2 distribution in normal healthy subjects are uniform at all degrees of physical activity. In patients with chronic respiratory disease who complained of dyspnea on exertion, these characteristics became more uneven as physical activity increased. By comparing the characteristics of SpO2 distributions at rest with those at certain degrees of physical activity, we could quantitatively evaluate the exercise tolerance of patients with chronic respiratory disease, while monitoring their physical activity and SpO2 in daily life, without burdening the patients with stress such as would be imposed by the treadmill test. This new method is applicable for determining the indications for home oxygen therapy. Its application in home health care could offer a useful evaluation of a patient's activities of daily living and also early discovery of aggravation of chronic respiratory failure.

A study on the metabolism of etoposide and possible interactions with antitumor or supporting agents by human liver microsomes.

The metabolism of etoposide was investigated by using human liver microsomes and nine recombinant human cytochrome P450 (CYP) isoforms to identify the CYP isoform(s) involved in the major metabolic pathway (3'-demethylation) of etoposide as well as to evaluate the possible metabolic interactions with several antitumor or supporting agents. The 3'-demethylation of etoposide followed a Michaelis-Menten one-enzyme kinetic behavior in six human liver microsomal samples. The relationships were assessed with six different human liver microsomes between the 3'-demethylation of etoposide and metabolic activities for substrate probes of the respective CYP isoforms, showing a significant correlation (r = 0. 932, P < .01) only with 6beta-hydroxylation of testosterone, a marker substrate for CYP3A4. Inhibitor/substrate probes for CYP3A4, ketoconazole, troleandomycin, verapamil and cyclosporin, or supporting agents, vincristine and prednisolone, inhibited etoposide 3'-demethylation by human liver microsomes. p-Nitrophenol, a substrate for CYP2E1, also inhibited etoposide 3'-demethylation. Among the nine recombinant human CYP isoforms, CYP3A4 exhibited the highest catalytic activity with respect to etoposide 3'-demethylation, compared with the minor activities of CYP1A2 and 2E1. Collectively, these data suggest that etoposide 3'-demethylation is mediated mainly by CYP3A4 and to a minor extent by CYP1A2 and 2E1. Furthermore, some supporting agents (vincristine and prednisolone) and the substrates of CYP3A4, which may be coadministered with etoposide during the cancer chemotherapies, inhibit the etoposide 3'-demethylation activity in vitro. The results may provide clinical implications with respect to the possible metabolic interactions between etoposide and other drugs studied herein in patients with cancer undergoing etoposide concurrently with either of them.

Mutual inhibition between quinine and etoposide by human liver microsomes. Evidence for cytochrome P4503A4 involvement in their major metabolic pathways.

The mutual inhibition between quinine and etoposide with their major metabolic pathways (i.e. quinine 3-hydroxylation and etoposide 3'-demethylation) was examined in vitro by human liver microsomes. Etoposide inhibited quinine 3-hydroxylation in a concentration-dependent manner with a mean IC50 of 65 microM. The mean maximum inhibition by etoposide (100 micro) of quinine 3-hydroxylation was about 60%. Similarly, etoposide 3'-demethylation was inhibited by quinine in a concentration-related manner with a mean IC50 value of 90 microM. The mean maximum inhibition by quinine (100 M) of etoposide 3'-demethylation was about 52%. An excellent correlation (r = 0.947, p < 0.01) between quinine 3-hydroxylase and etoposide 3'-demethylase activities in six different human liver microsomes was observed. Two inhibitors of CYP3A4, ketoconazole (1 microM) and troleandomycin (100 microM), inhibited quinine 3-hydroxylation by about 90% and 80%, and etoposide 3'-demethylation by about 75% and 65%, respectively. We conclude that quinine and etoposide mutually inhibit the metabolism of each other, consistent with the previous finding that CYP3A4 catalyzes the metabolism of both substrates.

[Difference of new mutation rates in dystrophin gene between deletion and duplication mutation in Duchenne and Becker muscular dystrophy].

To clarify new mutational rates in the dystrophin gene between deletion and duplication mutations, carrier diagnosis was performed on 123 mothers of probands suffered from Duchenne (DMD) and Becker (BMD) muscular dystrophy. Quantitative Southern blot analysis with cDNA probes was applied in this study. Out of 108 mothers of DMD/BMD patients with deletion mutation in dystrophin gene, 69 were carriers and 39 were non-carriers. On the other hands, all of 15 mothers of probands with duplication mutation were carriers. The fact that no new mutation occurred in oogenesis in the families with duplication mutations in dystrophin gene indicates that duplications arise in spermatogenesis. The risk of the mother of an isolated case of DMD/BMD with duplication mutation of being a carrier is significantly higher than the estimated risk based on the equality of new mutation in oogenesis and spermatogenesis.

[Serodiagnosis of tuberculosis by detection of antituberculous glycolipid antigen (TBGL antigen) antibodies in serum using enzyme-linked immunosorbent assay: clinical evaluation of anti-TBGL antibodies assay kit].

Kyowa Medex Co., Ltd. developed the kit for the sero-diagnosis of tuberculosis, which detects IgG antibodies against tuberculous glycolipids antigen containing cord factor (TBGL antigen) prepared from M. tuberculosis using the enzyme-linked immunosorbent assay technique. We evaluated the kit using clinical specimens and the results are as follows: 1) In total, 34 out of 39 cases (87.2%) with active pulmonary tuberculosis showed positive anti-TBGL antibody. 2) Patients with cavity, patients with extensive lesions and patients excreting large amount of acid fast bacilli tended to show high positivity rates. 3) The antibody titers increased in 7 out of 11 cases after starting the antituberculous chemotherapy. 4) The use of the antibody is unsuitable for the determination of the activity of tuberculosis since the antibody titers only slightly decreased even after chemotherapy for two years. 5) Two out of four nontuberculous mycobacteriosis cases showed high antibody titers 6) All three AIDS patients with tuberculosis showed low antibody titers. 7) The antibody was negative in almost all healthy controls showing a positive PPD skin test after vaccination with BCG, and it was therefore assumed that the antibody titer is not increased by BCG vaccination. 8) The antibody titers of the staff members working in the tuberculosis wards were not high compared with those of staff members working in the other wards.

[Home oxygen therapy (HOT) in patients with pulmonary tuberculosis sequelae--comparison between patients medically treated and those surgically treated].

In Japan there are about 40,000 patients under home oxygen therapy (HOT), of whom about 30 to 40% are pulmonary tuberculosis sequelae (TBS). These patients can be divided into three groups depending on the treatments they had, Group 1: those who had medical treatments only, Group 2: those who had artificial pneumothorax, and Group 3: those who had thoracoplasties or other surgical treatments. The purpose of this study was to observe the distributions and possible differences in the survival rates among these groups. The study included 1537 patients with TBS under HOT followed at National Hospitals and Sanatoriums nationwide in Japan. In 819 patients the treatments were specified and of those 354 were in Group 1, 29 in Group 2, and 436 in Group 3, so that the proportion of surgically treated patients in PTS was estimated between 28.4% (436/ 1537) to 53.2% (436/819). The ages at the onset of tuberculosis, at the start of HOT and the intervals in between were 36.6, 66.2 and 29.8 in Group 1, and 26.8, 65.5, and 38.1 in Group 3 respectively. Though the ages at the start of HOT were the same, those at the onset of tuberculosis were about ten years younger in Group 3 than in Group 1. Comparing Group 1 and 3, the survival rates after the initiation of HOT (Kaplan-Meier method) was better in Group 2 (surgically treated) than in Group 1 (medically treated). It is speculated that the reason could be a better preservation of the function of the remaining lung in the surgically treated and a higher incidence of obstructive impairments in the medically treated patients.

[Two cases of pulmonary disease caused by Mycobacterium chelonae subsp. abscessus].

We encountered two-cases of pulmonary disease caused by M. chelonae subsp. abscessus, [Case 1] A 72-year-old man was admitted to the hospital because of fever. He had been observed for one year after being given a diagnosis of pulmonary disease caused by Myocobacterium avium complex. Sputum examination revealed acid-fast bacilli (Gaffky 9). He recovered after administration of clarithromycin (CAM) and other drugs. [Case 2] A 61-year-old man was admitted to the hospital because of coughing and sputum production. He had been observed for 4 years after being given a diagnosis of pulmonary M. fortuitum disease. Sputum examination revealed acid-fast bacilli (Gaffky 7). His symptoms deteriorated even though he received anti-tuberculosis agents and CAM. After measurement of minimal inhibitory concentration (MIC), he was given amikacin (AMK). In both cases, the bacilli found in sputum obtained on admission were identified as M. chelonae subsp. abscessus by DNA hybridization. They were completely resistant to all anti-tuberculosis agents. However, the disk method show that they were sensitive to AMK, imipenem and CAM. The MIC value of those strains to CAM was 0.78 microgram/ml in case I and more than 100 micrograms/ml in case 2. The results obtained by MIC measurement were consistent with the clinical outcome. AMK, cefoxitin (CFX), and CAM had been used to treat M. chelouae subsp. abscessus in Europe, but the MIC value differed from strain to strain within a species. Thus the present data suggest that measurement of the MIC value of CAM would be necessary to predict its therapeutic effect.

[Evaluation of a new detection method of Mycobacterium tuberculosis in clinical specimens by amplification of ribosomal RNA and its clinical application].

A new rapid M. tuberculosis detection method, the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD), which allows direct detection of M.tuberculosis in clinical specimens by amplification of M.tuberculosis ribosomal RNA (rRNA), was evaluated. In the present study, MTD and conventional smear and culture examinations were performed on 225 sputum, 36 pleural fluid (PF), nine cerebrospinal fluid (CSF) and 41 bronchoalveolar lavage fluid (BALF) specimens. Among 225 sputum specimens from 132 patients, 20 culture-negative specimens from 14 patients and one specimen which was culture-positive for M.fortuitum on Ogawa's egg medium were MTD-positive. On the other hand, there were 6 M.tuberculosis culture-positive but MTD-negative specimens from 6 patients. The amount of bacilli in all six cases, however, was very few. The sensitivity of M.tuberculosis detection by MTD, as compared with Ogawa's egg medium and MB-Check, was 93 and 92%, respectively, in sputum specimens, and the specificity of MTD was 80 and 85%, respectively. Taking into account not only culture-positive specimens but also MTD-positive specimens from patients clinically diagnosed as active tuberculosis, the sensitivity and specificity of MTD were 93% and 99%, respectively. Among 36 PF specimens from 31 patients, of which 20 specimens from 16 patients were clinically diagnosed as tuberculous, only 4 from 4 patients were culture-positive for M.tuberculosis while 7 from 5 patients were MTD-positive. The results of MTD in 9 CSF specimens coincided well with those of MB-Check, but one MTD-positive specimen yielded a false-negative result with Ogawa's egg medium. Among 41 BALF specimens from 39 patients, only one was culture-positive while one culture-positive and 7 culture-negative specimens were positive on MTD. All these MTD-positive patients were later verified as having pulmonary tuberculosis either by transbronchial lung biopsy and other examinations, or by the clinical course of the disease, particularly the response to anti-tuberculosis drugs. Inconsistent results were seen in 21 sputum specimens from 15 patients, 4 PF specimens from 3 patients and 7 BALF specimens from 7 patients, all of which were culture-negative for M.tuberculosis but MTD-positive. None of these patients had any clinical findings inconsistent with diagnosis as tuberculosis. It was noted that MTD was very useful for rapid detection of M.tuberculosis in sputum as well as other clinical specimens. We observed the clinical course of 20 pulmonary tuberculosis patients by monthly examinations of sputum, using smears, Ogawa's egg medium, MB-Check and MTD. MTD-positivity rates fell down in parallel with decreased pulmonary tuberculous activity. During the clinical course, out of 61 MTD-negative specimens only 4 (6.6%) were culture-positive for M.tuberculosis, while out of 63 smear-and-culture-negative specimens 6 (9.5%) were MTD-positive. These data suggest that MTD is useful to assess the clinical activity and course of tuberculosis.

Cell-associated IL-8 in human blood monocytes: analysis by flow cytometry.

Several cell-associated cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor, exist on the cell surface and are biologically active. Although extracellular IL-8, a potent chemotactic factor for primarily neutrophils, has been studied extensively, cell-associated IL-8 has barely been studied. In this study, we analyzed the intracellular and cell-surface IL-8 in human blood monocytes in vitro by using flow cytometry and predicted the biological activity of the cell-associated IL-8 in vivo. After fixation with paraformaldehyde, mononuclear cells were divided into two subgroups. One subgroup was left untreated to study cell-associated antigens, and the other subgroup was permeabilized with saponin to detect intracellular antigens. In lipopolysaccharide (LPS)-stimulated monocytes, IL-8 was detected solely intracellularly, whereas both the intracellular and cell-surface IL-1 beta was detectable. In a time-course study, the intracellular IL-8 increased in response to LPS stimulation, but the cell-surface IL-8 was undetectable throughout the course. In an LPS-stimulated monocytic cell line, both ELISA and flow cytometry detected the quantitative change of the intracellular IL-8. The dissimilar localization between IL-8 and IL-1 beta within cells was confirmed by the immunohistochemical analysis. In summary, LPS stimulation induced a time-dependent increase in intracellular but not cell-surface IL-8 in monocytes. Thus, it is unlikely that the cell-associated IL-8 is functioning physiologically. The semiquantitative flow cytometric procedure may be useful for simultaneous examination for cell-surface and intracellular cytokines.

[A case of AIDS with bronchial tuberculosis].

A case is 48 years-old Japanese man who had a history of frequent sexual contact with prostitutes in Thailand and the Philippines. He presented with chief complaint of chest discomfort in April 1995. His chest X-ray film showed right mediastinal lymph node swelling in other hospital and the sputum smear was strongly positive for acid fast bacilli. In May 1995, he was admitted to our hospital and serological tests for HIV were positive both by EIA and Western blot methods. The CD4 lymphocyte count was 167/microliters. He was diagnosed as a case of AIDS according to the criteria proposed by the AIDS surveillance committee of the Japanese Ministry of Health and Welfare. Although numerous tubercule bacilli were detected in sputum, the chest X-ray did not show abnormal shadow in lung fields. So the diagnosis of bronchial tuberculosis was suspected by these apparently contradictory findings and the bronchoscopy was performed. Biopsy specimen of the bronchial mucous membrane obtained by bronchoscopy confirmed the presence of acid fast bacilli by Ziehl-Neelsen's staining method, however, histological findings were atypical of tuberculosis. A month after the initiation of treatment with isoniazid, rifampicin and ethambutol and AZT, his clinical symptoms improved and the sputum smear and the culture tests for tubercule bacilli converted to negative. Complications of AIDS, (Pneumocystis carinii infection, Cytomegalo virus infection, Kaposi's sarcoma, etc) other than tuberculosis have not developed to date. In the past reports, we could not find reports of bronchial tuberculosis with AIDS. Tuberculous granuloma formation was scarce in this case, and it was suspected that bronchial tuberculosis with AIDS would show characteristic sign as same as pulmonary tuberculosis with AIDS.

Detection of Mycobacterium tuberculosis in clinical specimens other than sputum by a specific DNA probe with amplification of the ribosomal RNA.

OBJECTIVE: To evaluate the Gen-Probe Amplified Mycobacterium tuberculosis Direst Test (MTD) for detection of Mycobacterium tuberculosis in clinical specimens other than sputum, especially cerebrospinal fluid (CSF) and pleural fluid (PF) specimens. DESIGN: Six CSF, 17 PF and 23 bronchoalveolar lavage fluid (BALF) specimens that were submitted to our clinical laboratory for detection of mycobacteria were subjected to MTD and conventional smear and culture examinations. RESULTS: Of 6 smear-negative CSF specimens, two were positive in MTD and culture examination. In PF specimens, two of 15 smear- and culture-negative specimens were positive in MTD, while one smear-negative but culture-positive specimen was MTD negative. Three of 20 smear- and culture-negative BALF specimens were positive in MTD. Three BALF specimens in which mycobacteria other than the M. tuberculosis complex detected by culture were negative in MTD. CONCLUSION: MTD is very useful for rapid detection of M. tuberculosis in clinical specimens other than sputum as well as sputum, and should be especially valuable for rapid diagnosis of tuberculous meningitis which requires prompt initiation of appropriate anti-tuberculosis drug treatment.

[Respiratory failure: respiratory disorder during sleep in patients with myotonic dystrophy].

Myotonic dystrophy (MyD) patients have been reported to show severe nocturnal desaturation related to sleep respiratory disorders. However, the reason of respiratory failure in MyD has remained unclear. In this study, ten patients with MyD underwent overnight polysomnography to evaluate the mechanisms which would cause respiratory failure, compared with Duchenne muscular dystrophy (DMD) patients in these three views: 1) sleep-related nocturnal desaturation, 2) histopathological evaluation of respiratory muscles, and 3) abnormalities of respiratory center. Nocturnal desaturation was more prominent in MyD rather than DMD. Apnea-hypopnea index (AHI) was higher in MyD than DMD. Type of respiratory disorder during sleep was mainly central apnea-hypopnea pattern, including Cheyne-Stokes respiration. In histopathological findings, central core change in respiratory muscle related to respiratory muscle fatigue was found less frequently in MyD than DMD. In respiratory center function, MyD showed hyporesponse to both alveolar hypercapnic and hypoxic stimulation. However, DMD showed normal response to both stimulations. We concluded that respiratory failure in patients with MyD would be attributed to respiratory center disorder rather than respiratory muscle weakness, which is the main cause of respiratory failure in patients with DMD.

[Chronic respiratory failure--survival for nine years with home mechanical ventilation].

We report the case of a woman, now 58 years old, with chronic respiratory failure due to spinal progressive muscular atrophy. She first noticed gradual progressive muscular weakness in her extremities in 1973. She started to complain of dyspnea on exertion in 1978. Chronic respiratory failure due to spinal progressive muscle atrophy was diagnosed in 1983. Home oxygen therapy was begun, but CO2 narcosis and exacerbation of chronic respiratory failure occurred at the end of that year. A tracheotomy was done and mechanical ventilation was begun. As her general condition improved and she could breathe without the ventilator for a few hours each day, home mechanical ventilation was begun. Seven years later, her general condition is still good and she can live without any life-threatening distress. There are few reports of patients in Japan who have survived for long periods of time with home mechanical ventilation. We believe that improvement in her respiratory care and in her social situation contributed to her long standing clinical course.

[Two cases of tuberculous meningitis diagnosed rapidly by amplified mycobacterium tuberculosis direct test (MTD)].

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) is a rapid direct specimen assay method for identification of M. tuberculosis by amplification of ribosomal RNA and detection of the product with a specific chemiluminescent DNA probe. We report two cases of tuberculous meningitis which were positive in the MTD. Since the cerebrospinal fluid showed negative smears and positive cultures in both cases, we could diagnose tuberculous meningitis by MTD about 2 months earlier than by conventional smear and culture methods. In one patient, the fever didn't subside immediately after staring chemotherapy. In the other patient, an intracerebral focus appeared after administration of antituberculosis drugs. However, we did not hesitate to continue the therapy because of the MTD positive findings. MTD is considered to be a very useful test especially in diseases like tuberculous meningitis which need rapid diagnosis and rapid therapy, and is useful for improvement of convalescence and is economical.

Mycobacterium avium-intracellulare pleuritis with massive pleural effusion.

Atypical mycobacterial infection is seldom accompanied by pleural involvement. We report a very rare case of Mycobacterium avium-intracellulare pleuritis with massive pleural effusion. The patient was a non-immunocompromised 35-year-old Japanese male with insidious onset of fever, chest pain and anorexia. The pleural effusion gradually resolved with empirical antimycobacterial treatment, leaving considerable pleural adhesion and thickening.