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We established a Southern blot hybridization using a DIG-labeled probe to detect monoclonal integration of HTLV-1 proviral genome. DIG was labeled by the PCR method and this probe was as sensitive as the 32P-labeled probe and able to detect only 1.6% of ATL cells. The clinical diagnoses of 44 patients with monoclonal band(s) were all ATL. In contrast, the clinical diagnoses of 39 patients without monoclonal band(s) were diseases other than ATL. We also performed a long PCR of HTLV-1 to characterize the integrated provirus. The method allowed us to find a defective provirus, which was frequently observed in aggressive forms of ATL; 14 of the 18 patients with acute type(78%), 6 of the 9 patients with lymphoma type(67%), and 2 of the 12 patients with chronic type(17%) had the defective provirus. We established a simultaneous PCR for each region of HTLV-1 for further examination of the defective provirus. To detect the monoclonality of IgH gene rearrangement of B-cells, we performed PCR according to the method described. None of 13 patients with T-lymphoproliferative disorders showed a monoclonal band. In contrast, 12 of 13 patients with CLL(92%), 22 of 25 patients with common ALL(88%), 18 of 24 patients with B-lymphoma(75%), and 3 of 3 patients with hairy cell leukemia(100%) showed a monoclonal band. We are now expanding this kind support system for the clinical diagnosis at a molecular biology level in the central laboratory.

Fas, also designated as Apo-1 and CD95, is a cell membrane receptor (mFas) involved in apoptotic cell death. A soluble form (sFas) lacking the transmembrane domain due to alternative splicing has been isolated. Abnormal expression of sFas and mFas is likely to be involved in lymphoproliferative disorders and auto-immune diseases. Adult T-cell leukemia (ATL) caused by human T-cell-leukemia virus type-1 (HTLV-1) is well known to be a T-cell neoplasm with strong mFas expression, suggesting a role of Fas in the pathology of the disease. We examined protein and mRNA expression of the 2 isoforms of Fas in fresh ATL cells and ATL cell lines. In general, mFas was strongly expressed in ATL cells, and sFas levels in sera were high, especially in malignant ATL. However, expression of the isoforms in some cases of ATL varied; there was no mFas expression on the cell surface and sFas levels were high in serum. In contrast, all ATL cell lines examined showed strong mFas expression and scarce production of sFas in the supernatant, corresponding to strong expression of full-length Fas mRNA and weak to negative expression of alternatively spliced mRNA lacking the transmembrane domain. Our findings indicate that the mode of expression of Fas isoforms in ATL cells is not always homogenous and that Fas may play a role in the malignant behavior and oncogenesis of ATL.

We document here in that two UDP-glucuronosyltransferase (UGT) isoforms sensitive to phenobarbital are involved in morphine glucuronidation in Wistar and Sprague-Dawley rats. The hepatic microsomal morphine UGT activity in untreated Gunn rats was significantly less than that of untreated Wistar rats. Although the morphine UGT activity in the liver of Gunn rats was increased by phenobarbital (PB) treatment, this was significantly less than that in the liver of PB-treated Wistar rats. UGT1.1r is an isoform of morphine UGT in rat, and UGT2B1 is also considered an isoform of morphine UGT, because UGT2B1 (stably expressed in V79 cells) exhibited morphine UGT activity. We prepared specific antipeptide antibodies against UGT1.1r and UGT2B1. Using isoform-specific antipeptide antibodies, both UGT1.1r and UGT2B1 in Wistar and Sprague-Dawley rats were inducible by PB treatment. However, UGT1.1r is not present in the liver from Gunn rats. This study is the first demonstration that protein levels of two morphine UGT isoforms, UGT1.1r and UGT2B1, in the liver of Wistar and Sprague-Dawley rats are inducible by PB treatment.

To characterize CD5+ B-cell neoplasms in Japan, where chronic lymphocytic leukemia (CLL) is rare and of different subtypes in comparison with Western countries, we collected 58 cases of CD5+ B-cell lymphomas/leukemias and analyzed their clinicopathologic features. According to the French-American-British (FAB) and standard histologic classification, the cases corresponded to small lymphocytic lymphoma (SLL, group I; n = 22, consisting of CLL, n = 10, CLL/PL, n = 3, and CLLmixed, n = 7); intermediate differentiated lymphoma/mantle cell lymphoma (IDL/MCL, group II, n = 18); and others with CD5-positive lymphomas (group III, n = 18). The CD5+ B-cell lymphomas showed morphologic and prognostic variability among the three groups. The clinical and immunophenotypic features were remarkably consistent in leukemic disease being seen in 73% of all cases, splenomegaly in 63%, and intense CD19, CD20, surface membrane immunogobulin M (SmIgM) or SmIgM and SmIgD, light-chain expression, and no CD10 expression. The median survival time of groups I, II, and III was 7.8, 3.3, and 0.8 years, respectively. These findings suggest that CD5 antigens may serve as valid markers for the prognosis and clinical features of B-cell lymphomas and that CD5+ B-cell lymphomas with an overall poor prognosis occurs at a relatively high frequency in Japan. This also suggests that a combination of immunophenotypic and morphologic features is of value for characterizing CD5+ B-cell neoplasms.

We re-evaluated several antibiotics including newer ones, for their in vitro killing activity, as well as their inhibitory activity, against clinical isolates of periodontopathic bacteria. Tetracyclines were active against Porphyromonas gingivalis, and were highly active against Prevotella intermedia, but demonstrated only a low killing activity against Actinobacillus actinomycetemcomitans. Rokitamycin, a new macrolide, and clindamycin were highly active against P. gingivalis and P. intermedia, but showed very weak killing activity against A. actinomycetemcomitans. Quinolones demonstrated excellent bactericidal activity against A. actinomycetemcomitans, and good inhibitory and bactericidal activity against P. gingivalis and P. intermedia. Metronidazole had an activity almost equivalent to quinolones against P. gingivalis and P. intermedia; but it was the least active against A. actinomycetemcomitans.

The subgingival microflora of 42 pubertal children (aged 12-15 years) and 18 young adults (aged 21-25 years) was investigated by anaerobic culture and phase contrast microscopy. Motile rods, spirochetes and Prevotella intermedia were elevated proportionately in pubertal children with gingivitis (median GI > or = 1); however, no statistically significant differences in enumerated organisms on selective media were observed between pubertal children and young adults. These organisms were positively correlated with the index of bleeding on probing and the gingival index of pubertal children. Pubertal children were divided into 3 distinct subject clusters according to the similarity of subgingival microbial features, and the subjects who were then selected for the gingivitis group were distributed into clusters 1 and 2. The proportions of motile rods, P. intermedia and Eikenella corrodens were significantly higher in cluster 2 than in cluster 1. These results suggest that these 2 species and motile rods which differentiated features of clusters, are useful for screening of high-risk subjects for worsening of inflammation.

A rare case of blood coagulation disorder, congenital factor XII deficiency, detected in a patient with mandibular osteomyelitis is presented. Routine laboratory tests showed prolonged clotting and activated partial thromboplastin time. Detailed investigations for the intrinsic and extrinsic pathways of coagulation, fibrinolytic system, kinin-kallikrein system, and complement system were performed because factor XII is known as an activator of these systems. No hemorrhagic or thromboembolic complication occurred during and after surgery. Substitution therapy with fresh frozen plasma was not necessary. Magnetic resonance imaging and electrocardiography were used to examine the possible occurrence of postoperative cerebral hemorrhage or myocardial infarction.

To study bone loss relationships to aging and menopause, cross-sectional bone mass measurements by digital image processing (DIP method), and menopause information collected by questionnaire, were analyzed on 291 women who live in Tsukude village. The results are as follows. 1) The mean DIP values (sigma GS/D, MCI) by age-stratified groups decrease with age after menopause. The rate of bone loss in sigma GS/D is almost constant, but in MCI it increases with aging. 2) In 30-year old and 40-year old age groups, the frequency distribution of DIP values is symmetrical and bell-shaped. But after the fifties the distribution is asymmetrical, with the mode of distribution deviated toward low bone mass. The change of mode with aging is larger than that of mean. This fact suggests that change of mean bone mass substantially underestimates actual bone loss from aging. 3) The change of the mean DIP values stratified by years elapsed since menopause is not especially large at start of menopause but becomes almost constant after menopause. DIP values reflect the bone loss from the aging rather than from menopause, and are beneficial to the study of bone loss in elderly women.

For the purpose of clarifying functions expected of primary care physicians (PC physicians) in Japan, a study of the types and frequencies of health problems seen in primary care clinics located in three areas, together with consultation/referral rates of the patients to other institutes. The study was conducted using ICHPPC-2 (Japan version) which had been compiled by WONCA as a classification of diseases. In order to obtain much more generalized characteristics of the primary care, clinics located in the city, suburban district and remote places in the mountains were studied. The results were as follows: 1) The health problems treated by the clinics in each of the three areas were respectively: 162 types/7,207 items/4 months: 303 types/17,519 items/2 years: and 280 types/61,916 items/2 years. 2) The consultation/referral rates were under 2%. 3) PC physicians treated over 98% of the health problems encountered. This was found by the investigation, which had been intended to disclose a range of so-called COMMON DISEASES treated by PC physicians themselves, based on a relation between the referral rates and the types of health problems. The above findings suggested that the COMMON DISEASES should include at least 95% of the health problems which had been treated by the PC physicians. This corresponded to approximately 100 types of diseases.

Two-dimensional echocardiography (2DE) was used in the mass screening of 3,017 participants (1,195 males, 1,822 females, mean age 59) living in 13 areas in Japan. The 2DE findings of left ventricular hypertrophy (LVH) and LV function were compared with the presence and severity of hypertension (Ht). Out of 1,100 patients who had an evident history of Ht above 160/95 mmHg (Ht group), 298 patients (27.4%) were complicated by LVH. In contrast, LVH was also recognized in 60 (11.2%) of 535 borderline Ht cases and in 87 (6.3%) of 1,382 normotension cases. LVH cases in the Ht group were divided into 3 groups: mild (220: 73.8%), moderate (63: 21.2) and severe (15: 5.0). Asymmetric septal hypertrophy (ASH) was recognized in 111 patients (37.2%) of the 298 LVH cases. The prevalence of ASH in these LVH patients was higher according to the severity of the LVH: mild LVH = 31%, moderate LVH = 51% and severe LVH = 67%. The prevalence of LVH was higher in males than in females. This was especially apparent in the moderate to severe LVH groups and also noted to be higher according to the increase of mean blood pressure. The prevalence of mild to moderate LVH was significantly higher in the poorly-controlled group than in the well-controlled group. In contrast, no significant difference in prevalence of severe LVH was noted between the well-controlled and poorly-controlled groups. The prevalence of LV dysfunction was significantly greater in moderate or severe LVH groups than in non or mild LVH groups. It tended to be higher in the poorly-controlled group compared with the well-controlled group. The regression of LVH was frequently detected in the well-controlled group by the follow-up study. We conclude that 2DE observation of LV performance in mass screening will be extremely valuable in the long term follow-up of Ht patients.

Developmental changes in salivary proteins secreted by the submandibular glands of male rats from 2 to 10 weeks of age in response to the beta 1- and alpha 1-adrenoceptor agonists, isoproterenol, methoxamine, dopamine, phenylephrine, epinephrine, norepinephrine and alpha-methylnoradrenaline, were studied by different electrophoreses and amino acid analysis. The electrophoretic patterns of proteins secreted by the glands in response to both stimuli changed dramatically every week up to 7 weeks of age, but thereafter appeared similar to those of adult submandibular salivary proteins. At 2 and 3 weeks of age, a few differences were found between the types of proteins secreted in response to two stimuli. These findings were also confirmed by an analysis of amino acid composition. The glandular levels of cGMP from 2 weeks and cAMP from 3 weeks of age were significantly elevated in response to methoxamine and isoproterenol. We conclude that functional maturation precedes morphological maturation in the submandibular glands of male rats.

We used two-dimensional echocardiography (2-D echo) for the mass screening of 2,673 subjects for cardiovascular disorders (1,032 men, 1,641 women, aged 23-95 years with a mean age of 59), who lived in 12 rural areas in Japan. In 1,665 of the 2,673 subjects (62.3%), cardiac abnormalities were recognized. Three major abnormalities included sclerotic aortic (33.0%) and mitral valves (21.8%), and left ventricular (LV) hypertrophy (15.9%). Apparent pericardial effusion (54 cases), mitral valve prolapse (44 cases), and dilated cardiomyopathy (4 cases) were incidentally found in this study. Prevalence of the abnormalities insidiously increased with age (below 49 years: 39%, and above 80 years: 97%), and this was apparently higher in men than in women. The abnormal findings which were significantly higher in men were the sclerotic aortic valves, LV hypertrophy and dysfunction, the prevalence of which was particularly high in ages 40-79, but it became nearly equal beyond 80 years of age. These results may indicate a sexual difference in the influence of age on the cardiovascular system. It was concluded that 2-D echo is helpful for primary screening and prevention of cardiovascular disorders.

The present study involved cultural and microscopic characterization of the subgingival microorganisms of pubertal children with gingivitis. Samples were obtained from eight children 12-14 years of age. Gram-positive organisms made up 56% of the isolates and included mainly Actinomyces israelii, Actinomyces naeslundii. Gram-negative anaerobic rods constituted 25% of the isolates and Bacteroides intermedius (9.7%) was most predominant species. The selective media permitted significantly lower recovery of Actinomyces spp. and Capnocytophaga spp. than nonselective blood agar medium. However, sixty-nine percents of isolates were cultured on the selective media employed in the present study. These results suggest that the subgingival microflora of gingivitis in pubertal children may be similar to those of adult gingivitis and that the use of these six selective media makes it possible to enumerate predominant cultivable microorganisms in subgingival area of pubertal children with gingivitis.