(Vol.75, No.7 July 2000)


<1>Kekkaku Vol.75, No.7:463-469,2000

OPTIMAL CONDITIONS FOR ESTABLISHMENT OF EXPERIMENTAL 
TUBERCULOSIS MODEL USING AN AUTOMATED INHALATION 
EXPOSURE APPARATUS AND ITS APPLICATION 

*Isamu SUGAWARA, Hiroyuki YAMADA, Koji OTOMO, Toshiaki AOKI, 
Satoru MIZUNO, and Tadashi UDAGAWA 

*Department of Molecular Pathology, The Research Institute of Tuberculosis, 
Japan Anti-tuberculosis Association 

  Animal (mouse and guinea pig)pulmonary tuberculosis models were established, using 
an automated inhalation exposure apparatus (Glas-Col Corp., USA, Model 099CA-4212). 
This apparatus includes four steps-preheating, nebulization, cloud decay and decontami-
nation. The optimal conditions for M.tuberculosis H37Rv strain infection experiments 
were as follows:10(5-6) colony forming unit (cfu) tubercle bacilli;preheating for 15 min.; 
nebulization for 90 min.;cloud decay for 15 min. and decontamination for 5 min. When 10(4) 
cfu M.tuberculosis H37Rv strain were introduced into the lungs of interferon (IFN)-gamma 
knockout mice, using the inhalation exposure apparatus and were followed up for 9 months, 
the primitive cavitary lesions were observed. This apparatus was also useful for inhalation 
exposure experiments of guinea pigs. This apparatus can also be utilized for animal 
inhalation experiments of allergens. 

Key words:Automated inhalation exposure apparatus, 
Guinea pig, Mouse, M.tuberculosis 

*3-1-24, Matsuyama, Kiyose-shi, Tokyo 204-8533 Japan. 
(Received 9 Feb. 2000/ Accepted 3 Apr. 2000)
 





<2>Kekkaku Vol.75, No.7:471-476,2000

A STUDY ON THE EFFECT OF COMBINED CHEMOTHERAPY ON MYCOBACTERIUM AVIUM 
COMPLEX PULMONARY DISEASE 

1*Kazuhiro SATO and 2Tatsuo EBE 

1*Division of pulmonology, Department of medicine, Nagaoka Red Cross general hospital, 
2Ebe Clinic 

  Annual incidence of Mycobacterium avium complex (MAC) pulmonary disease has been 
gradually increasing in the last 10 years in Japan, however, the optimal therapeutic 
regimen for the disease has not yet established. We investigated the effect of our new 
regimen in twenty seven cases of pulmonary MAC infection without HIV infection, 
diagnosed according to the American Thoracic Society criteria during the period from 
January 1996 to October 1997 at our hospital. 
  These patients were treated with rifampicin (RFP), ethambutol (EB) and clarithromycin 
(CAM) for more than 12 months, together with streptomycin (SM) initially (first 2-3 
months), except one patient who was treated for 11 months only. Twenty-four months 
after the therapy, sputum cultures converted from positive to negative in 13 patients and 
the amount of bacilli in sputum reduced in two patietns. 
  The radiological findings improved in 10 patients showed no significant changes in 11 
paients, while worsened in the remaining 6 patients. As to adverse reactions 1 case of 
liver damage, 3 cases of skin disorders, 4 cases of gastrointestinal malfunctions, and 1 
case of optic neuritis were observed. This regimen was safe and tolerable even in the 
elderly outpatients, but not so effective against MAC pulmonary disease compared with the 
results of recent reports from the U.S. and Europe. Size of pulmonary lesions was 
closely associated with the effectiveness in this study. However, five bacteriologically 
converted cases did not show radiological improvement, and the reasons behind this fact 
remain to be investigated. 

Key words:Mycobacterium avium complex, Drug treatment, Clarithromycin, 
Sputum conversion, Radiological improvement, Safety 

*297-1, Terajima-machi, Nagaoka-shi, Niigata 940-2101 Japan.
(Received 14 Oct. 1999/ Accepted 14 Apr. 2000)
 





<3>Kekkaku Vol.75, No.7:477-482,2000

CHANGES IN ANTIBACTERIAL ACTIVITY OF MURINE PERITONEAL 
MACROPHAGES AGAINST MYCOBACTERIUM TUBERCULOSIS
AFTER PROLONGED IN VITRO PRECULTIVATION 

1,2Tatsuya AKAKI 1Katsumasa SATO, 1Toshiaki SHIMIZU, and 1*Haruaki TOMIOKA 

1*Department of Microbiology and Immunology, Shimane Medical University, 
2Department of Dermatology, Shimane Medical University 

  We examined profiles of intramacrophagial growth of M.tuberculosis (MTB) when 
mouse peritoneal macrophages (MƒÓs) were infected with the organisms at day 0 or day 7 
after in vitro precultivation, and obtained the following results. First, the growth rate 
of the virulent MTB H37Rv strain as well as attenuated H37Ra strain was slower in MƒÓs 
which had been precultured for 7 days (MƒÓs [day 7])than in freshly prepared MƒÓs 
without precultivation (MƒÓs [day 0]). The doubling time of MTB H37Rv was 2.2 and 
2.9 days in MƒÓs [day 0] and MƒÓs [day 7], respectively, and that of MTB H37Ra was 
2.9 and 3.6 days in MƒÓs [day 0] and MƒÓs [day 7], respectively. Second, MTB-mediated 
cytotoxicity in terms of the LDH release from infected MƒÓs was less marked in MƒÓs 
[day 7] than in  MƒÓs [day 0], when they were infected with MTB of either the H37Rv 
or H37Ra strain. MTB H37Ra strain exhibited much weaker cytotoxic effects on host 
MƒÓs than did H37Rv strain. Third, when MƒÓs [day 7] were infected with MTB of 
either the H37Rv or H37Ra strain, they showed markedly lowered levels of reactive oxy- 
gen intermediate (ROI) production than did MƒÓs [day 0]. In contrast, the reactive 
nitrogen intermediate (RNI) producing ability of MƒÓs in response to MTB infection 
was not so markedly reduced in MƒÓs [day 7] from that of MƒÓs [day 0]. As mentioned 
above, the MƒÓs [day 7] did not permit accelerated growth of infected MTB, compared 
to the MTB growth in the MƒÓs [day 0]. It thus appears that ROI played a trivial role 
in the antimicrobial activity against MTB of murine peritoneal MƒÓs which had been 
precultured for long periods. Although it is regarded that RNI played more critical roles 
in MƒÓ anti-MTB activity than did ROI, the present results also suggest that other kinds 
of antimicrobial effectors are required in MƒÓ antimicrobial activity against 
MTB organisms, particularly in the case of MƒÓs after prolonged in vitro cultivation. 

Key words:Mycobacterium tuberculosis Macrophages, 
Reactive nitrogen intermediates, Reactive oxygen intermediates, 
Cytotoxicity 

*89-1, Enya-cho, Izumo-shi, Shimane 693-8501 Japan.
(Received 28 Jan. 2000/ Accepted 28 Apr. 2000)






<4>Kekkaku Vol.75, No.7:483-491,2000

The 75th Annual Meeting Opening Lecture 

FIFTY YEARS OF RESEARCH ON TUBERCULOSIS 

Lessons I Have Learnt during 50 Years and Topics to be Investigated in the Future 

*Tadao SHIMAO 

*Japan Anti-Tuberculosis Association 

  I have engaged in the research on tuberculosis for 50 years, and lessons I have learnt 
during this period could be summarized in the following ten topics. First is great re- 
search achievements by our predecessors on the establishment of so-called primary infec- 
tion theory on the pathogenesis of TB, planning of TB control principles based on the 
theory and development of new technologies used for TB control, such as mass miniature 
X-ray examination and BCG vaccination in 1920s and 1930s. TB control law was en- 
forced in 1951, and the modern TB programme was initiated. 
  Second, the field is a treasure house of interesting data. Several interesting data on 
TB soon after the World War ‡U in Tokyo and a rural area were collected and analysed from 
the mass health examination. Third, looking at the increase of tuberculin positivity with 
age, it was found that the tuberculin negativity decreased as the exponential function of 
age, and the current concept of the annual risk of TB infection was already developed in 
late 1940s. It was 18.1% in male and 11.6% in female in Tokyo in late 1940s. Based on 
this concept, age specific TB mortality was analyzed by the type of TB, and the rates of 
miliary TB and TB meningitis were similar to the rate of newly infected to the total 
population, while the rate of all forms could be divided into early and late death as 
shown in Fig.1. 
  Fourth, I suffered from TB by myself from 1951 to 1953, receiving first thoracoplasty 
in two stages under local anaesthesia, then right upper lobectomy and segmentectomy of 
superior segment of right lower lobe. From this experience, I learnt a lot about the psy- 
chology and suffering of TB patients. 
  Fifth, the importance of recognition of real magnitude of the problem in such a disease 
as TB in which many TB cases did not aware of their disease. The answer to this was the 
first TB prevalence survey in 1953 using stratified random sampling method, and based 
on the results of the survey, the mass health examination originally focussed on youth 
was expanded to the total adult population of Japan. Sixth, TB could be reduced rapidly 
by applying appropriately planned control programme. In big enterprises, the application 
of intensive case-finding programme brought about the rapid decline of severe TB cases, 
contributed to the increase of the productivity of the enterprises, thus to the rapid 
increase of GDP of whole Japan, and the growing spiral between the improvement of 
health and the economic development was formed by successful TB control. In addition to 
the mass health examination, BCG vaccination and spread of appropriate treatment in the 
original TB control law, the registration and case management system and the more 
extensive application of hospitalization for infectious cases were introduced in early 
1960s. 
Observing the proportion of TB care expenditure to the national medical expendi- 
ture, it was 28% in 1954, and it dropped down to 0.4% in recently as shown in Fig.2. The 
decline of TB in Japan during 1950s and 1960s was one of fastest in the world. 
  Seventh, there had been marked differences in the prevalence of TB as well as the cover- 
age and quality of TB programmes in several areas of Japan though it was often said 
that Japan is homogeneous country. To know the real status in various areas of Japan, 
a chart to express graphically the magnitude of TB and coverage and quality of TB pro- 
grammes was developed (Fig.3), and it was finally refined to the current form. Eighth, 
difficulty in changing existing programmes, and we are grateful for kind cooperation on 
Niigata Prefecture for making several new attempts. 
  Ninth, it has been needed to observe TB problems from global standpoint, and it was 
actually done through participation to the bilateral cooperation projects on TB control 
and conducting the international training courses sponsored by JICA. Tenth, TB is a per- 
tinacious disease. As shown in Fig.4, positive correlation is seen between the TB mortal- 
ity rate in 1950 and the incidence of smear (+) PTB in the age group 65 years of age 
and over, and comparing the age-specific incidence of TB in whole Japan and Ishikawa 
Prefecture, the rates in Ishikawa below 70 years were lower than the rates in whole 
Japan(Fig.5), while the rate in the age group 70 years and over was markedly higher 
in Ishikawa, where the prevalence of TB was extremely high in younger generation 50 
years ago. These facts indicate that efforts to control TB for at least several decades 
are needed to eliminate TB. 
  Topics for future priority research could be summarized as follows:First to recon- 
struct phthisiology based on the concept of EBM to adapt to the changing situation of 
TB. Second, it is urgently needed to elucidate mechanisms of the onset of TB among 
elderly population, in particular endogenous reactivation, and to develop methods to 
prevent it. Third, how to control TB in urban district with high incidence and many 
vulnerable populations such as homeless. 

Key words:Primary infection theory, TB prevalence survey, 
A chart to evaluate magnitude and quality of TB and its control, 
Pertinacity of TB 
 
*1-3-12, Misaki-cho, Chiyoda-ku, Tokyo 101-0061 Japan.
(Received 6 Jun. 2000)