Clofazimine-mediated augmentation of LPS-induced tumor necrosis factor production in macrophages.

Tumor necrosis factor (TNF) exerts multiple biological activities including immune response. It is also believed to play an important role in anti-bacterial response. In this study in vitro, we observed augmentation of LPS-induced TNF production from mouse macrophages by clofazimine treatment. Rifampicin, however, did not indicate such an activity. Clofazimine itself, on the other hand, did not have any TNF-inducing activity. Clofazimine is a well known anti-leprosy drug; in addition, from the results obtained here, this drug could induce anti-M. leprae response of host by way of the augmented immune response by enhanced cytokine production from macrophages.

Immuno-epidemiological studies on subclinical infection in leprosy. III. Yearly observations and follow-up studies of schoolchildren by using FLA-ABS and lepromin tests.

The schoolchildren in three regions of Miyako Islands, Okinawa Prefecture, were surveyed annually, from 1978 to 1984, by using the fluorescent leprosy antibody absorption (FLA-ABS) test and the lepromin reaction with the Dharmendra's antigen, for detecting the individuals at high risk of leprosy and for evaluating predictive value of these immunological tests. Constant potency of these tests was confirmed by the percentage of positive reactions among the children in the first grade of elementary schools (5 or 6 years old) surveyed from 1980 to 1984. A temporal rise or drop of the percentages during this period seemed to associate with the yearly reported number of new leprosy cases in each region. Among 1,168 schoolchildren tested with FLA-ABS once or twice or more, the percentage of positive reactions was significantly higher in the children with the enlargement of peripheral nerve without sensory loss than in those without this signs and symptoms. The longer the duration of this signs and symptoms, the higher the percentage of positive FLA-ABS tests. Lepromin test did not show any significant correlation with this signs and symptoms. A concordant persistence or change between the FLA-ABS tests and neural signs or symptoms was observed in 133 out of 331 children examined twice or more. Discordant changes in the remaining were mainly attributed to a conversion to seronegativity before the disappearance of neural signs and symptoms. Changes of FLA-ABS and lepromin reactivities between the initial and final tests suggested a spontaneous cure of subclinical infection with Mycobacterium leprae among the children who were FLA-ABS positive but lepromin negative at the initial test and therefore considered to be a group at high risk of leprosy. Although neural signs and symptoms were found in the majority of these children, none of them so far developed overt symptom of the disease. Based on these findings, predictive value of FLA-ABS test together with lepromin was discussed.

Epitope mapping of twelve monoclonal antibodies against the phenolic glycolipid-I of M. leprae.

Epitope mapping of 12 monoclonal antibodies (MAbs) directed to the trisaccharide part of the phenolic glycolipid-I (PGL-I) of Mycobacterium leprae was carried out by using the set of chemically synthesized sugar-BSA conjugates. The results can be summarized as follows: mAb (1-21), mAb (1-24) and mAb (1-25) recognized the outer (nonreducing end) monosaccharide of the trisaccharide chain of PGL-I. However, the affinity of these MAbs to the outer monosaccharide was weak. They required the contributions of some parts of the second sugar for enough affinity. MAbs ml 6A12, ml 8A2, ml 8B2, and PG2 B8F recognized the outer disaccharide. MAb F47-21-3 recognized the outer disaccharide and some parts of the third sugar. MAb SF 1 recognized the trisaccharide of PGL-I. MAb 3D1-A9 recognized the phenol group and the structure around the branching point on the carrier protein in addition to the trisaccharide. MAbs DZ 1 and 2G3-A8 had unique characters which recognized the inner part of the sugar chain. MAb DZ 1 recognized the inner (reducing end) disaccharide. MAb 2G3-A8 recognized the inner monosaccharide, phenol group and the structure around the branching point on the carrier protein. All of the MAbs tested, except for ml 6A12, recognized the anomeric configurations in the sugar parts they recognized; ml 6A12 recognized the anomeric configuration only within the outer disaccharide. This set of MAbs, which were well defined on their binding specificity, promises to be an effective tool for the immunological study of PGL-I and the clinical assessment of leprosy.

Immuno-epidemiological studies on subclinical infection in leprosy. I. Clinical and immunological findings in schoolchildren and adults in Okinawa.

For the purpose of understanding subclinical infection with Mycobacterium leprae among the inhabitants in an endemic area, 3,547 schoolchildren and 1,487 adults in several regions of Okinawa were surveyed by using clinical examinations and immunological tests, i.e. the fluorescent leprosy antibody absorption (FLA-ABS) test and the lepromin test using Dharmendra's antigen, during a period from 1978 to 1984. The enlargement of peripheral nerves, especially that of lateral or bilateral auricular and/or ulnar nerves, without loss of sensation was found in 8.4% of the schoolchildren and in 9% of the adults. A frequency of these signs and symptoms was significantly higher in male than in female and tended to increase with age. The percentage of positive reactions in FLA-ABS tests for schoolchildren and adults was 21.8 and 22.5, respectively, the values suggesting a minimum frequency of subclinical infection with M. leprae among these inhabitants. The percentage of positive FLA-ABS tests was significantly higher in those with neural signs and symptoms than in those without. Such a correlation was not found in the lepromin test. Ill-defined depigmentation of the skin without loss of sensation was found in a few percentage of both schoolchildren and adults. However, this sings and symptoms did not correlate with the FLA-ABS test nor with the lepromin test. A history of tuberculin test and BCG vaccination in the schoolchildren seemed to influence the lepromin-reactivity. Among 770 schoolchildren tested with both FLA-ABS and lepromin, 70 (9.1%) were the FLA-ABS positive but lepromin non-positive responders. Neural signs and symptoms were found in 31 of these children.

Immuno-epidemiological studies on subclinical infection in leprosy, II. Geographical distribution of seropositive responders with special reference to their possible source of infection.

The percentage of positive fluorescent leprosy antibody absorption (FLA-ABS) tests showed significant differences among the inhabitants of different regions in Okinawa. Provided that this percentage indicates the frequency of subclinical leprosy infections, the numbers of inhabitants with subclinical infection per new case with leprosy in the same region ranged from 723 to 3,039. The FLA-ABS test was positive in 16.2% of adults in Minami daito Island where no new case with leprosy was found during the survey for seven years. The differences in the percentages could not be explained by the different prevalence and incidence rates of leprosy in each region nor by the differences in age and sex of the individuals examined. A significant correlation between the FLA-ABS tests and neural signs or symptoms was found in 3 regions. None of adults in Minami daito Island showed such signs or symptoms. The distribution of FLA-ABS positive and negative responders in two hamlets where the incidence of leprosy was relatively high suggested the localization of positive responders surrounding houses in which a leprosy case had recently been found and also the distribution of positive responders in the remote houses. These facts seem to indicate that a possible source of infection to the majority of positive responders might be from the environment rather than from direct contact with leprosy patients.

Down regulation of Ia expression in macrophages following incubation with mycobacteria.

Macrophages are known to release cytokines in response to various kinds of stimulators. In the present study, peritoneal macrophages from C3H/He or C3H/HeJ mice were incubated in vitro with heat-killed M. lepraemurium, M. intracellulare or M. gordonare for 3 days followed by harvest culture supernatant to analyze cytokine activities. It, therefore, seems that macrophages phagocytizing these mycobacteria, released interleukin-1 (IL-1) and tumor necrosis factor (TNF) in culture media. The amount of release was dose dependent on mycobacteria employed. In addition, macrophages, as already have reported elsewhere, treated with IFN for 2 to 3 days showed enhanced expression of surface Ia; although the expression was inhibited if the cells phagocytized mycobacteria. Similarly, the reduced expression of Ia was observed in peritoneal macrophages from MRL/lpr mice after 3 day-culture with mycobacteria in vitro. More importantly, in the presence of the supernatant obtained from macrophages incubated with mycobacteria, IFN gamma-treated normal macrophages exhibited suppressed expression of Ia. These results demonstrate that cytokine release and reduced expression of surface Ia in macrophages are simultaneous phenomena after phagocytosis of mycobacteria. Suppression of Ia may be in part induced by Ia suppressive factor(s) released from mycobacterium-phagocytized macrophages.

Foot pad enlargement as a measure of induced immunity to Mycobacterium leprae.

Foot pad enlargement (FPE) has been used as a measure of induced immunity to M. leprae, FPE peaked at 2-3 days, but it sometimes persisted for 4 weeks or more. Both as the inducing and eliciting antigen, heat-killed M. leprae were effective, and the optimum dose was about 1 x 10(7) bacilli. Higher doses were associated with flattening of the dose-response curve. Disrupted bacilli were not effective in immunizing mice, but they elicited FPE responses in mice immunized with intact bacilli. Cord factor was not found to have adjuvant activity for M. leprae. In immunization, the intradermal route was confirmed to be more effective than the foot pad route; the subcutaneous route was effective in providing protection against infection. FPE tests were used to investigate the steps of standard purification procedures for M. leprae in armadillo livers. A trypsin-chymotrypsin digestion step was found to be harmful to immunogenicity in one of two experiments.

[Epidemiological immunological studies on leprosy in Okinawa. 6. Lepromin, lymphocyte transformation, FLA-ABS and leproagglutination tests in the in-and out-patients with leprosy and the relationships among these tests and personal or family history or clinical findings of the patients].

In- and out-patients with leprosy in Okinawa were surveyed by using the following immunological tests: cutaneous reaction to Mitsuda's lepromin (40 million bacilli/ml) (LPR), lymphocyte transformation test with Dharmendara's antigen (DL-LTT), that with phytohemagglutinin (PHA-LTT), fluorescent leprosy antibody absorption test (FLA-ABS) and Leproagglutination test with cardiolipin-lecithin antigen (LAT). A correlation between two tests and a relationship between the test and personal or family history or clinical finding of the patients were evaluated by non-parametric statistics. Spearman's rank correlation coefficient was significant between LPR and DL-LTT, while a reverse correlation was found between LPR and FLA-ABS, DL-LTT and FLA-ABS, and DL-LTT and LAT, respectively. High reactivity in PHA-LTT was found in almost all of patients, irrespective of type and stage of leprosy, suggesting general immune responsiveness of leprosy patients in Okinawa. The other tests showed a significant difference in the mean rank of reaction values according to clinical findings such as type and stage of leprosy, property of skin lesion, loss of eyebrow, and bacteriological test. The use of LPR and DL-LTT for the study of cell-mediated immunity in leprosy was therefore confirmed. FLA-ABS was found useful for early serodiagnosis, while LAT for the study of autoimmunity in leprosy. None of these immunological tests showed significant difference among the subgroups of patients classified by the following items: presence or absence of consanguineous patient with leprosy, living place, place of onset, presence or absence of neural symptoms such as the enlargement of peripheral nerve, claw hand and facial palsy, ocular and nasal involvement, ENL in lepromatous and borderline leprosy and conditions in the treatment. On the other hand, LPR showed higher reactivity in female than in male, in the patients at home than those discharged, and in the patients with drop foot than those without. Higher reactivity of DL-LTT was also found in male than in female and in the patients with plantar ulcer than those without. FLA-ABS reactivity was significantly higher in the discharged patients than the inpatients, in the period of 5 to 9 years after onset than that of 20 or more years, but the reactivity was lower in the patients with drop foot than those without. LAT reactivity was significantly higher in the 30-39 years old patients than 0-19 years old, and in the patients with physical work than those with mental.(ABSTRACT TRUNCATED AT 400 WORDS)

Power spectral analysis of heart rate and arterial blood pressure oscillation in brain-dead patients.

To clarify the mechanism of the slow arterial blood pressure oscillation seen in brain-dead patients, we investigated the frequency of fluctuations in arterial blood pressure and heart rate using power spectral analysis. The electrocardiogram, arterial blood pressure and respiration were recorded simultaneously from 9 brain-dead patients and 8 vegetative patients. Power spectral analysis of these data revealed a very slow fluctuation (0.002-0.01 Hz) in arterial blood pressure in brain-dead patients, the frequency of which was equal to that of the low-frequency spectrum of heart rate, indicating vasomotor sympathetic activity. Neuropathological examinations of the medulla and spinal cords of 4 autopsied brain-dead patients revealed that the spinal cord, ventral and dorsal nerve roots, and the nucleus intermediolateralis of the lateral horn below the level of C3/4 were virtually intact. These findings suggest strongly that the slow oscillation of arterial blood pressure in brain-dead patients originates from the vasomotor tone controlled by spinal sympathetic nerves.

Assessment of autonomic nervous activity by heart rate spectral analysis in patients with variant angina.

The purpose of this study was to assess the role of the autonomic nervous system in the pathogenesis of coronary artery spasm in patients with variant angina. We evaluated cardiac sympathetic and parasympathetic activity from the power (logarithmic scale) of the low-frequency (approximately 0.04 to 0.12 Hz) and the high-frequency (approximately 0.22 to 0.32 Hz) spectral components of heart rate variability with Holter monitoring in seven patients with nocturnal variant angina and in 11 healthy men who served as control subjects. None of the patients had organic coronary artery stenosis as determined by angiography. Low-frequency and high-frequency logarithmic values were calculated for each 5-minute period from 30 minutes before to immediately before each angina attack. The logarithmic low-frequency value during the 5-to-0-minute period was greater than the low-frequency values during most of the other periods (p < 0.05 - p < 0.01). The logarithmic high-frequency values during the 10-to-5-minute and 5-to-0-minute periods were greater than those during the 30-to-25-minute period (p < 0.05 and p < 0.01, respectively). These data indicate that parasympathetic activity increased during the 10 minutes before attacks of nocturnal variant angina, whereas sympathetic activity with vagal modulation increased during the 5 minutes before such attacks. The same pattern of changes in heart rate variability was found in the absence of ST-segment elevation in patients and in control subjects. So this phenomenon was not just associated with coronary spasm and variant angina. It is suggested that circadian variation in disease activity is also associated with spontaneous attacks.

Salivary immunoglobulins and antibody activities in leprosy.

The technics of immunodiffusion and the fluorescent leprosy antibody absorption (FLA-ABS) test were used to determine the levels of immunoglobulins and their antibody activities against Mycobacterium leprae in the serum and the saliva collected from a total of 110 patients with leprosy (50 lepromatous, 24 borderline, and 36 tuberculoid). The average levels of serum IgG, IgM, and IgA were not significantly different among these patients. In saliva, however, IgM was detected in only two cases with lepromatous leprosy and three tuberculoid cases. Salivary IgG and IgA levels and their ratios to those in the sera were not significantly different according to the classification of leprosy. The percentages of positive FLA-ABS tests in the sera and saliva were compared by using fluorescent antibodies specific for IgG, IgM, and IgA, respectively. The results indicated that M. leprae-specific antibodies in the serum were mainly found in IgG and IgM and, less frequently, in IgA. IgG antibodies were found more frequently in lepromatous and borderline patients than in tuberculoid cases. On the other hand, salivary IgA antibodies against M. leprae were found in a significant number of specimens; whereas IgG and IgM antibodies were scarcely found. However, the percentage of positive FLA-ABS tests caused by salivary IgA antibodies was higher in the patients with tuberculoid or borderline leprosy than in those with lepromatous leprosy. A significant number of patients with tuberculoid or borderline leprosy secreted M. leprae-specific IgA antibodies into saliva without detection of circulating IgA antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)