菌陽性のらい腫らい患者144例と健康志願者20名とにHBO療法(加圧及び減圧を含めて3気圧で1時間半)を2日から21日間続けて行った。代謝係数(すなわち組織による酸素消費),アデノシン三燐酸塩の血液濃度,血清リパーゼ濃度は,抗菌薬剤治療下の患者では主として,正常化への傾向を示した。超微構造検査によるらい腫の衰退像と臨床症状の改善とが見出された。HBOはらい腫らいの化学療法に役立つ補助手段に発展するかも知れない。

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An outline of the Draper's collection method (protocol 1/79) is illustrated in Fig. 1. Infected armadillo liver is homogenized with blender, and the bacterial fraction is collected by centrifugation for 2 times. The sediment is treated with DNase and filtrated through stainless steel mesh. The sediment after centrifugation is suspended in 30% percoll solution and is again centrifuged at 27, 000×g. Bacterial fraction is separated with 2 polymer system such as polyethyleneglycol and dextran. Leprosy bacilli are condenced in upper phase.
A summary of the Mori's collection method is illustrated in Fig. 2. A homogenous suspension of leprosy bacilli is prepared by filtration through 2 sheets of gauze to prevent an agglutination of leprosy bacilli by centrifugation. Percoll gradient is made with 90% percoll 4ml, 80% 5ml, 70% 5ml, 60% 10ml, 50% 5ml and then 8ml of bacterial suspension is placed on the top layer. The tubes are centrifuged with beckman Swinging Bucket SW 27 roter at 27, 000rpm (about 100, 000×g) for 1 hour. Top clear red zone consists of soluble tissue fraction and haemoglobin. Next white zone is insoluble tissue fraction. Middle white zone is rich in leprosy bacilli. Bacterial fraction collected in the bottom of centrifuge tube by Draper's method is more brown than our fraction (Fig. 5 and 6). It may be tissue contaminants. The findings in Ziehl-Neelsen staining of both smear preparations are not different so much. Every bacterial fractions obtained only by centrifugation or 2 polymer system are contaminated with liver tissue components which are stained by anti-armadillo liver rabbit serum and second fluorescent antibody. Host liver tissue contaminant is not found in our alkali treated fraction by the immunofluorescence test. Since the armadillo liver components adhere to the leprosy bacilli fraction, we cannot obtain pure leprosy bacilli without the drastic treatment such as 1 N sodiumhydroxide treatment.
Enzyme treatment was not used in Mori's method, but DNase treatment was used in Draper's method, then it may be unconvenient to extract the deoxynucleic acid from M. leprae. Mori's method finishes in one day, while it takes two days in Draper's method. Yield of leprosy bacilli is 8 % by the Draper's method. On the other hand 56.8 % of leprosy bacilli are collected by Mori's method. A lot of loss in the yield may happen in the 2 polymer system, which is trobublesome, expensive and dangerous to infection. In some improper armadillo livers, brown granules precipitate to the same place of percoll gradient in which the leprosy bacilli precipitate. This armadillo may have some other liver diseases. Therefore, armadillo liver should be treated one by one.

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Convit used the mixture of heat-killed leprosy bacilli and live BCG as a leprosy vaccine. It seems that some devices could be applied to inactivate the bacilli instead of heat-killed bacilli of Convit's vaccine. When heat-killed bacilli were inoculated to mouse, many stainable acid-fast bacilli were detected in the injected site for a long time. However, in the case of live leprosy bacilli, stainable acid-fast bacilli disappeared comparatively ear-lier in the injected area. Leprosy bacilli of live but defected growth ability may be more efficient for vaccine than heat-killed bacilli.
Purified leprosy bacilli were prepared from leproma produced in nude mouse foot pad infected with Mycobacterium leprae, Thai-53 strain, by ficoll gradient centrifugation me-thod and treatment of 1 N sodiumhydroxide. The bacillary counts were 1.6×1010 in 16.3ml of 0.1M P. B. (pH 6.4). The first group of nude mice were inoculated with live bacilli into their right hind foot pads, the second group were injected with heat killed bacilli, the third group were injected with formalin treated bacilli, the fourth group were inoculated with rifampicin treated bacilli, the fifth group were injected with Co⁶⁰ irradiated bacilli and the sixth group were injected with bacilli cultivated on Ogawa egg yolk medium for 4 months. The first to the third groups consisted of 5 mice and the fourth to sixth groups consisted of 8 mice. Bacterial suspension of 0.05ml were injected in right hind foot pad. For the first to the third groups, observation was carried out at 14 months after inocula-tion and for the fourth to the sixth groups, one mouse was killed every 2 months. Bacterial numbers in the foot pad were counted. In the first group injected with live bacilli, all 5 mice produced lepromas in the injected sites of right foot pads. In the second group injected with heat-killed bacilli, bacterial count was not done because of some trouble. In the third and fifth groups injected with the formalin treated and Co⁶⁰ irradiated bacilli respectively, bacterial counts were almost the same in the 0 time and after 14 months as seen in Table 1. In the sixth group injected with the cultivated bacilli, the bacillary counts after 14 months were less than that of 0 time and their acid-fastness were weak. In the fourth group, the bacilli treated with rifampicin grew gradually in the foot pads at 8 months after injection and reached to 10 times growth after 14 months.
Leprosy bacilli kept in the ordinary medium at 33°C for 4 months may be regarded as dead bacilli. Moreover, if they have been treated with rifampicin, they are probably inactive in growth. If nude mice could not produce leproma in foot pad after injection of leprosy bacilli kept in culture media for a long time, the cultivation method would be hopeless.

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Personal and family histories of the totally 864 patients with leprosy admitted in the National Leprosaria Okinawa Airakuen and Miyako Nansei-en were surveyed since 1973, for the purpose of understanding epidemiological and immunological characteristics of leprosy in Okinawa. Frequency of intermarriage in the parents of index cases was not significantly higher than that in the public near Miyako Nansei-en. The intermarriage did not correlate with the classification of leprosy in the index cases nor with the incidence of leprosy in other members of their family. It is therefore unlikely that leprosy is associated by recessive gene. Consanguineous patients with leprosy were more frequently found in the index cases with younger age than those in the elder and in the cases with lepromatous leprosy than those with tuberculoid. The frequency in 778 siblings of index cases was 19.9%, indicating a significant difference with that (8.9 %) in 563 parents and children, and these percentages were significantly higher than the incidence rate of leprosy (2.1%) in 191 spouses of index cases. A coincidence in the classification of leprosy was found in 37 out of 53 (69.8%) pairs of consanguineous inpatients, but the percentage was not significantly different among three groups of consanguinity, i. e. parent and child, siblings and the others. These facts seem to indicate that the susceptibility to leprosy is not so largely associated by genetic factors as expected previously.
On the other hand, a long period of household contact with index cases did not necessarily increase the frequency of affected cases in the family. The percentage of lepromatous leprosy in the index cases was highest in those who had household contact of 10-15 years and their age at onset was significantly younger than those without household contact. The percentage was also significantly different according to the place at onset. These acts seem to indicate that the susceptibility to leprosy is greatly influenced by postnatal factors such as the household contact at childhood, physiological, immunological and living conditions.

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The frequencies of various symptoms in the totally 864 patients with leprosy admitted in the National Leprosaria Okinawa Airaku-en and Miyako Nansei-en were surveyed ac-cording to their sex, classification of leprosy and stage of symptom. The results were compared with those seen in literatures. Among initial symptoms, dermal symptom was more frequently found in the younger patients than in the elder, whereas neural symptom was more frequent in the elder than in the younger. However, the frequency of initial neural symptom in Okinawa was far lower than that in the mainland. The frequency of lepromatous leprosy in Okinawa was significantly higher in male than in female, but it was also far lower than that in the mainland. The enlargement of peripheral nerve, fall of eyebrow, ocular and nasal symptoms were more frequently found in lepromatous cases than in non-lepromatous. However, the frequencies of alopetia leprosa, ocular and nasal symptoms and the reactions including ENL were significantly lower in Okinawa than in the mainland. On the other hand, the frequencies of claw hand, drop hand, drop foot, facial palsy and plantar ulcer were significantly higher in non-lepromatous cases than in lepromatous and not significantly different from those in the mailand.
Based on these findings, it is concluded that the frequency and severity of lepromatous leprosy in Okinawa are significantly lower than those in the mainland of Japan and rather similar to those in tropical regions. Several differences in the conditions of treatment and the bacterial index were also discussed by the findings as described above.

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