The effects of cyclophosphamide (CY) on reticuloendothelial system (RES) and immune responsiveness were studied in mice.
1) Single injection of sublethal dose of CY reduced the number of peritoneal cells, but the activity as measured by increased rates of carbon clearance and peritoneal macrophage spreading sustained almost normal immediately after CY injection. Thereafter the activity of RES increased slowly and reached its maximum about 10 days later.
2) CY-pretreatment increased delayed type hypersensitivity (DTH) and decreased the number of plaque forming cells (PFC) to sheep red blood cells immunized 3 days after injection of CY, while thereafter it was found that DTH decreased and the production of PFC increased and reached to a peak about 10 days later.
3) CY-pretreatment reduced a resistance against pseudomonas infection 3 days after injection, while the increased resistance was found thereafter and reached to a peak about 10 days later.

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Previously, the present authors observed that mycobacterial species showed various patterns of the distribution of radioactive spots in thin-layer chromatography of lipids after uptake of 35 S-methionine. The majority of the species showed their unique patterns for the species. However, M. scrofulaceum, M. gordonae, and M. flavescens showed two or more patterns. Further studies on M. avium, M. intracellulare, M. scrofulaceum, and M. gordonae showed that these organisms shared the same patterns, and that the patterns of the former three were found in those of M. gordonae.

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In our country 1% Ogawa's egg media are used for drug resistance test of tubercle bacilli. The report presents the results of experimental studies on decrease of drug acitivity during the storage of drug-containing media in diferent temperature.
The methods of examination and the results of studies are summarized as follows:
1) The drug-containing media of streptomycin, isoniazid, PAS, rifampicin, ethambutol, ethionamide, cycloserine, kanamycin, capreomycin, viomycin and enviomycin (a new drug produced in Japan and used for treatment of tuberculosis) were prepared and stored in 4-5°C refrigerator, 22°C, 30°C, 37°C thermoregulators. The drug resistance test of H₃₇Rv and strains isolated from previously untreated patients were performed at 1, 2 weeks and 1, 2 months after preparation of drug-containing media. The results of test were compared with that of test used drug-containing media before storage (used immediately after preparation).
The decrease of drug activity was most marked in ethionamide and rifampicin containing media stored outside of refrigerator and the decrease of drug activity was not observed in PAS, streptomycin, kanamycin, capreomycin, viomycin and enviomycin containing media. The degree of decrease of drug activity was as follows; rifampicin>ethionamide>cycloserine>isoniazid>ethambutol.
In the case of storage in refrigerator ethionamide showed the decrease of drug activity after two months storage, but other drug did not show the decrease of drug activity within 3 to 6 months.
2) The drug-containing media of streptomycin, kanamycin, capreomycin, viomycin and enviomycin were stored in 30°C thermoregulator and the drug resistance was examined by using these media after 3 and 6 months' storage. According to the test results, slight decrease of drug activity of streptomycin and capreomycin was observed but other drugs did not show the decrease of drug activity within 6 months.
Conclusively, the decrease of drug activity was marked in ethionamide and rifampicin containing media and was slight in ethambutol containing media during storage outside the refrigerator. The drug-containing media for drug resistance test of tubercle bacilli should be stored in refrigerator afer preparation and be used within 1 month.

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Sixty seven cases with pulmonary atypical mycobacteriosis (2 by M. kansasii, 62 by M. intracellulare, 2 by M. fortuitum and 1 by a possible new pathogen of group III mycobacteria) died in 9 Japanese national sanatoria up to October 1976.
Of these 67 cases, 40 died of atypical mycobacterial disease, and the remaining 27 cases died of other diseases. Roentgenological aggravation was found in 44 cases (1 by M. kansasii, 1 by M. fortuitum and 42 by M. intracellulare).
There were various types of roentgenological aggravation. Spread of non-cavitary foci, infiltrate and pneumonia were found most frequently (40/44, 90.9%). Enlargement of cavity was found in 12 cases (27.2%), appearance of pleural effusion in 5 cases (11.4%), and spontaneous pneumothorax in 3 cases.
Infection of bulla was found in 11 out of 22 cases with bullae as the underlying disease.
The first roentgenological aggravation was found in 20 (45.5%) out of 44 cases within 12 months; 13 cases (29.5%) between 13 to 24 months; and 11 cases (25%) over 2 years after the discovery of the disease.
From the results mentioned above, in the fatal cases, progression of the disease was predicted by the appearance of the roentgenological aggravation within 2 years after the discovery of the disease.
There were various courses of the progression of the lesions as shown in Figures 2a and 2b. One of the typical course of the progression was devided into the following 5 stages:
The first stage: localized cavitary lesion.
The second stage: spread of foci around cavity.
The third stage: spread of foci in contralateral lung.
The fourth stage: enlargement of cavity (appearance of giant cavity).
The fifth stage: extensive pneumonia in the lower lung field. Another typical course of the progression was the repeated infections of bullae.
Roentgenological aggravation found in patients with atypical mycobacterial disease was not rarely due to the mixed infection with various organisms (gram-negative bacilli, fungi and also human type tubercle bacilli).
The majority of the patients with underlying pulmonary disease (extensive emphysema, chronic bronchitis and bronchiectasis) died of pulmonary insufficiency in the relatively early stage of atypical mycobacteriosis.
The patients with the mixed infection have died, in spite of the negative conversion or the dicrease of the excretion of atypical mycobacteria.
There were two cases (M. intracellulare infection) complicated with pulmonary tuberculosis.

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Enviomycin (EVM, Tuberactinomycin) is a new anti-tuberculous agent isolated from the culture of Streptomyces griseoverticillatus var. tuberacticus N6-130.
This report presents the result of a co-operative clinical study at 11 tuberculosis hospitals in Osaka region on therapeutic effect and side-effects of Enviomycin in 189 retreatment cases of drug resistant pulmonary tuberculosis with cavities. The duration of EVM treatment has been fixed for six months and the drug was administered by intramuscular injections at the dose of 1 gram once daily for the first three months and twice weekly thereafter combined with other antituberculous drugs which had been used orally just prior to EVM treatment.
The results are summarized as follows:
1) Background factors of 189 cases are shown in Table 1. The majority of cases (71%) were far-advanced tuberculosis and 47% of total cases were resistant to four or more anti-tuberculous drugs. The time course of EVM therapy is summarized in Table 2 including 143 completed cases of six months treatment.
2) Negative conversion of bacilli in sputa by smear and culture are shown in Tables 3 and 4. The sensitive group which has been treated with EVM and other unused drugs gives the higher negative conversion rate at sixth month by culture (67%) than the resistant group in which only already-used drugs were combined with EVM (27%).
3) Changes in basic lesions and cavities on chest radiograms are shown in Table 5. Improvements on chest radiograms were scarcely observed, as the most cases had chronic lesions and cavities with sclerotic wall to which no chemotherapeutic agents had been expected to react effectively.
4) The resistance against EVM was measured at 25 and 100μg/mlglml EVM containing Ogawa's egg media. The emergence of EVM resistance to 100μg/mlg/ml EVM at sixth month was 27.6% in EVM sensitive group. On the other hand, in another group which had shown 25μg/ml resistance against EVM at the start, all cases showed resistance to 100μg/ml EVM at sixth month (Table6).
5) Side-effects such as tinnitus, hearing drop, dizziness and headache which were assumed to be caused by EVM were observed in 27 cases (14.3%), and 15 cases of them (7.9% of total 189 cases) had dropped out from the therapy.
6) Hearing drop over 20 db at 8, 000 cis was observed in 12 cases (6.5%), however, only one case dropped out by this reason.
In conclusion, enviomycin (EVM, Tuberactinornycin) has been proved to be one of usefulanti-tuberculous drugs which can be used daily in combination with other effective oral drugs in retreatment of resistant pulmonary tuberculosis, as the incidence of side-effects, especially hearing impairment was relatively low.

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The purpose of present study is to analyse the clinical picture and the state of cell-mediated immunity on pulmonary atypical mycobacteriosis.
Cell-mediated immunity was examined by the methods of tuberculin skin test and in vitro thymidine uptake of lymphocytes stimulated with PPD and PHA.
The study subjects were 26 cases of pulmonary atypical mycobacteriosis, who were admitted to Keio University Hospital from 1966 to 1976.
The results of the clinical analysis of 26 cases was closely similar to those previously reported in Japan, except the fact that the frequency of subjective complaints of this disease at the first medical examirration was higher than that of pulmonary tuberculosis in the anther's material. Tuberculin tests were examined for 21 patients of which twelve reacted positive, five negative and four doubtful positive. Among cases with negative and doubtful tuberculin reactions and showed clinical improvement, their tuberculin reaction turned to positive, and in one case who showed worsening, tuberculin reaction converted to negative.
Lymphocyte response to PPD and PHA was carried out by the following methods. Blood was taken from 11 cases of pulmonary atypical mycobacteriosis, from 13 adults patients with pulmonary tuberculosis and from 10 healthy hospital employees in whom the skin reactivity to PPD was positive. All donors had not been tuberculin tested for some weeks prior to the examination. Preparation of lymphocytes was carried out by the method of gravity sedimentation. Culture were set up in glass tubes containing 1×10⁶-lymphocytes in 2ml of Eagle' medium supplemented with 10% fetal-calf-serum and with 100 us PC and 100μg SM/ml, and then 2μg/ml of PPD or 10-3/ml of PHA solution were added to these tubes.
It was found that the concentration of PPD of 2μg/ml in culture suspension gave the maximal stimulation for lymphocytes from tuberculin positive donors. The duration of culture was 5 days for PPD-stimulated and 3 days for PHA-stimulated cultures and 0.2μCi of 2-¹⁴C-thymidine was added to those cultures at 4 hours before harvesting of cultures and the harvesting was performed using a modification of the method described by Dutton and Page (1964). The samples were counted in a liquid scintillation counter, and these results were shown as disintegrations per minute after correction for quenting and efficiency. The thymidine uptake after stimulation with PPD and PHA was shown as the subtracted value of the thymidine uptake of unstimulated lymphocytes from that of stimulated lymphocytes.
The thymidine uptake afer stimulation with PPD was studied in the following three groups, and it was 1, 565±2, 541 DPM (mean±S. D.) for pulmonary atypical mycobacteriosis, 5, 231±2, 070 DPM for tuberculosis and 5, 377±2, 487 DPM for healthy donors. Thymidine uptake after stimula tion with PHA was also studied in the above mentioned three groups, and it was 18, 617±12, 276 DPM, 33, 081±9, 385 DPM and 36, 197±5, 658 DPM, respectively.
The value of these thymidine uptake after stimulation both by PPD and PHA in pulmonary atypical mycobacteriosis was significantly lower than that in the other twc groups. Accordingly it could be said that there were general depression of cell-mediated immunity in cases with atypical mycobacteriosis.

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