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)     

Determination of circulating IgG subclasses against lipoarabinomannan in the leprosy spectrum and reactions

IgG subclasses against lipoarabinomannan of mycobacteria were analyzed in the sera of leprosy patients. Patients with active leprosy [tuberculoid and lepromatous, patients undergoing erythema nodosum leprosum (ENL) and reversal reactions] and inactive cases (tuberculoid and lepromatous who were cured after chemotherapy) were included in this study. Active lepromatous patients had higher levels of IgG subclasses, except IgG4, compared to active tuberculoid patients. Some of the inactive cases (lepromatous patients cured after chemotherapy) were positive for the IgG1, IgG2 and IgG3 subclasses. However, their levels are lower than active lepromatous cases. On the other hand, no difference in the subclass levels between the active and inactive tuberculoid groups could be observed. While a significant fall in the level of IgG3 in ENL was observed as compared to lepromatous leprosy without ENL, higher levels of IgG1 and IgG2 were found in patients with reversal reactions compared to their active counterparts without reactions.     

Detection of Mycobacterium leprae DNA for 36kDa protein in urine from leprosy patients: a preliminary report

We have searched for Mycobacterium leprae DNA for 36kDa protein in urine using a M. leprae specific PCR technique. A limited number of 16 patients (of which 11 belonged to lepromatous leprosy and five to tuberculoid leprosy) and eight healthy individuals were included for the present study. The number of urine samples positive by PCR were 36.4% (4/11) in lepromatous patients and 40% (2/5) in tuberculoid patients. None of the samples from healthy individuals was positive. To our knowledge, the results indicate, for the first time, the presence of M. leprae DNA in urine from leprosy patients. Another important finding obtained out of the study is that amongst treated patients 66.6% (4/6) were positive whereas amongst untreated only 20% (2/10) were positive. From the present indicative data it appears that treatment improves the PCR results with urine as a sample. Thus, the approach could prove to be useful for monitoring the treatment response of individual patients and needs to be further evaluated with a large number of patients.     

Sero-immunoreactivity of cloned protein antigens of Mycobacterium leprae.

Sera from 173 leprosy patients with various types of disease (tuberculoid = TT, borderline tuberculoid = BT, borderline lepromatous = BL, and lepromatous = LL), 12 intrafamilial contacts, and 40 normal healthy individuals were assayed in an indirect enzyme-linked immunosorbent assay (ELISA) using Mycobacterium leprae antigens. Recombinant clones carrying M. leprae antigens, namely, Y3184 (12 kDa), Y3179 (18 kDa), Y3164 (28 kDa), Y3180 (36 kDa), and Y3178 (65 kDa) and a cell sonicate from armadillo-derived M. leprae were used for the study. A high degree of reactivity with the 65-kDa, 36-kDa, and 28-kDa protein lysates was observed in most of the sera from multibacillary patients, with a low degree of positivity with 18 kDa and 12 kDa. Only a few sera from paucibacillary patients showed positive reactions. The majority of the contacts' sera tested showed no reactivity with these antigens.     

Serum immunoglobulins in leprosy.

Serum immunoglobulins were quantitated by radial immunodiffusion in 25 cases each of tuberculoid and lepromatous leprosy. Immunoglobulins estimated from 50 normal healthy adults were the control. Serum IgG was markedly raised in both tuberculoid (mean 2420 mg/dl) and lepromatous leprosy (mean 2493 mg/dl) when compared with the controls (mean 1288 mg/dl) and the difference was significant (p less than 0.01). However the difference in serum IgM and IgA levels in cases as compared to controls were not statistically significant. Serum IgM was slightly raised, the mean values obtained being 222 mg/dl in tuberculoid leprosy, 221 mg/dl in lepromatous leprosy and 202 mg/dl in control. Serum IgA was reduced in lepromatous leprosy (mean 129 mg/dl) as compared to the controls (mean 168 mg/dl) and the cases of tuberculoid leprosy (mean 165 mg/dl). The range of values obtained in both groups of patients showed greater scatter than the controls and a few cases of both forms of leprosy showed very low values of both serum IgA and IgM.     

Serum calcium and magnesium in different types of leprosy

Serum calcium and magnesium were studied in 200 leprosy patients and 25 apparently healthy individuals. Serum calcium was found to be significantly decreased in all types of leprosy except tuberculoid. The decrease in serum magnesium was highly significant in tuberculoid, lepromatous and borderline lepromatous cases.     

Serum factors affecting the cell migration inhibition response to lepromin.

Cell migration inhibition of white blood cells in the presence of total protein lepromin (TPL) was studied in ten lepromatous patients, six tuberculoid patients, and ten normal controls; adding normal, tuberculoid, lepromatous, or no serum to the culture medium. Using normal or no serum, lepromatous patients and skin negative controls gave negative reactions, while tuberculoid patients and skin positive controls gave positive cell migration inhibitions. The addition of lepromatous serum gave a very significant overall increase of migration indices in all groups of subjects, turning to negative the positive reactions of lepromatous patients and positive controls. On the contrary, the addition of tuberculoid serum gave a decrease of migration index in all groups of subjects, turning to positive the reactions in lepromatous patients. The significance of these circulating factors, able to enhance or inhibit cell migration inhibition responses in patients and controls, is discussed.     

Cured tuberculoid patients have a greater life expectancy than cured lepromatous patients in Japan

Leprosy patients lack specific cellular immunity against Mycobacterium leprae, but other immunological functions are thought to be preserved. However, in a leprosy sanatorium in South Japan between 1982 and 2000, we found that the average age at death of cured lepromatous leprosy patients was about 5 yrs younger than that of cured tuberculoid patients; [male/lepromatous, 76.0 +/- 10.0 yrs old vs. male/tuberculoid, 79.7 +/- 9.4 yrs old, p = 0.026], and [female/lepromatous, 78.0 +/- 10.5 vs. female/tuberculoid, 85.3 +/- 9.8, p = 0.0001]. This trend was also observed in autopsy records of two other leprosy sanatoria in Japan. In a prospective study based on their age in 1982, among females in the age group between 60 and 69, lepromatous patients (75.3 +/- 6.0 yrs) died earlier than tuberculoid patients (81.0 +/- 5.1 yrs) (p < 0.01). These findings suggest that lepromatous patients have higher risk of death even in a post-chemotherapy era.     

In vitro studies on dermal granulomas of human leprosy: characterization of cells using monoclonal antibodies

Single cell suspensions from granulomas of leprosy cases were prepared to enable an in vitro study on the characteristics of infiltrating cells. In all, biopsies from 21 untreated cases of tuberculoid leprosy and lepromatous leprosy were analyzed. The granulomas were found to contain lymphocytes and macrophages. The numbers of lymphocytes were higher in the suspensions of tuberculoid granulomas in comparison to lepromatous granulomas. A high percentage of lymphocytes from tuberculoid granulomas expressed OKT11 and Ia-like antigens, thereby indicating the presence of activated T cells. The proportion of Leu3a+ cells was greater in comparison to OKT8+ cells in these granulomas. In lepromatous granulomas, only a few positive lymphocytes expressing OKT11 or OKT8 antigens were observed. The ratio of Leu3a+:OKT8+ cells (2.79 +/- 0.61) was higher in the tuberculoid granulomas than in the lepromatous granulomas. Most macrophages from both types of granulomas expressed Ia-like antigens.     

Are there subpolar tuberculoid cases?

The idea of the existence of subpolar tuberculoid cases is supported on the spectral conception of Ridley and Jopling who included the subpolar lepromatous patients between LL and BL cases. According with Languillon, we have studied 40 tuberculoid cases to find out clinical, bacteriological, immunological and histopathological parameters of subpolar T cases. The subpolar T cases do exist, they are unstable cases which can slide through the immunological spectrum towards the L pole. These cases are characterized by numerous, symmetrical, dysesthetic tuberculoid lesions with an important neural involvement. Bacilli can be found in a small number in the smears of nasal mucosae in some cases. Mitsuda reaction is always positive and histopathologically the granulomas, lymphocytes and epithelioid cells are in less number without the epidermotropism which is very often seen in polar T cases.     

Frequency and extent of thickening of the nucleated epidermis in leprosy lesions

Using 28 specimens of clinically normal skin from lepromatous leprosy subjects as a standard for comparison, the mean thickness of the nucleated epidermis was found to be significantly increased in untreated lesions from 16 borderline tuberculoid, 21 erythema nodosum leprosum (ENL), and 14 reversal reaction patients, but was unchanged in borderline lepromatous and lepromatous patients. Using specimens from 36 untreated lepromatous and borderline lepromatous lesions as the standard for comparison with the lesions of reversal reactions or ENL which these patients eventually developed, there was a significant thickening of the nucleated epidermis in both reactional states. In both comparison groups, there was a greater mean increase and a larger frequency of thickening in the ENL lesions than in those with reversal reactions. In the borderline tuberculoid and reversal reaction lesions the increase can be understood as secondary to the presence of gamma interferon or interleukin-2. The increase in thickness in the ENL lesions is more difficult to explain, but it is not inconsistent with a role for these same two cytokines.     

Comparative study of skin reactions in leprosy patients to M. leprae-lepromin and to ICRC-in, an antigen from cultivable acid-fast bacilli from M. leprae isolated from lepromatous nodules.

Skin test antigens (Dharmendra type) were prepared from fresh M. leprae (lepromin) and from a culture of strain C-44 ICRC bacilli (ICRCin) grown 'in vitro' from M. leprae isolate from lepromatous nodules. Comparative study of skin reactivity to lepromin and ICRCin--both "early" and "late" reactions in 76 leprosy patients was conducted. In 29 lepromatous (LL) cases, 25 exhibited totally negative reaction at the end of third week. In tuberculoid (TT) 22 and 23 out of 31 were positive (greater than 4.5 mm) at 3 weeks to lepromin and ICRCin respectively. In the 16 BB group, the reactions were comparable in the same patient. The cellular reaction in tuberculoid cases consisted of lymphocytic infiltration, epitheloid giant cells and Langhan type cells and indistinguishable from each other. These data with characteristic total lack of reaction in 25/29 lepromatous leprosy cases and identical cellular reaction in TT patients, provide strong evidence that ICRC bacillus strain C-44 is antigenically identical with M. leprae.     

OKT6+ epidermal Langerhans' cell numbers in DNCB reactions among leprosy patients

A study was made on the Langerhans' cells at the sites of contact sensitivity skin reactions in 45 untreated leprosy patients. The skin reaction was induced by 2,4-dinitrochlorobenzene (DNCB). Langerhans' cells were quantitated using OKT6 monoclonal antibody and indirect immunofluorescence. Clinically, the skin reaction in the tuberculoid patients was positive at 4, 24, and 48 hr, while the lepromatous patients failed to respond at any of the time intervals. Sequential histological analysis of the skin reaction showed predominantly mononuclear cell infiltrates around the blood vessels and neurovascular bundles in both the tuberculoid and lepromatous patients. Time kinetic assessment showed no difference in the numbers and distribution of OKT6+ epidermal Langerhans' cells at the site of the DNCB skin reactions among the tuberculoid and lepromatous patients. This, therefore, suggests that either there is a functional defect in Langerhans' cells or some other mechanism(s) such as a T-cell abnormality is responsible for the lack of clinical reaction in lepromatous patients.     

Biochemical, immunological and genetic studies in leprosy. II. Profile of immunoglobulins, complement components and C-reactive protein in sera of leprosy patients and healthy controls.

Various classes of immunoglobulins (IgA, IgM, IgG, IgD and IgE), complement components (C3 and C4) and C-reactive protein (CRP) were estimated in sera from normal healthy controls and leprosy (lepromatous and tuberculoid) patients from Ethiopia. Higher levels of IgA, IgM, IgG and IgD were found in lepromatous leprosy compared with normal healthy people while in tuberculoid leprosy only IgM, IgG and IgD levels were increased. Borderline leprosy patients showed increase in IgG level only. Although an increase in IgE was noted in lepromatous leprosy, it was not significant; the variations in IgE levels could be due to different socioeconomic background and exposure to intestinal parasites. C3 component was significantly reduced in leprosy patients compared with healthy controls while no difference in C4 component was observed. The results point towards an involvement of the "alternate pathway". A positive test against C-reactive protein antiserum was given by about 20% of the normal healthy controls while more than 60% lepromatous and tuberculoid leprosy patients were CRP positive. The results are discussed in relation to the status of immunoglobulins and complement components in leprosy and possible factors (environmental and genetic) which might affect them.     

Biochemical, immunological and genetic studies in leprosy. I. Changes in serum lactate dehydrogenase isoenzymes, creatine phosphokinase and aldolase activity in different forms of leprosy.

Serum lactate dehydrogenase isoenzymes, creatine phosphokinase and aldolase activity were determined in healthy control subjects and in lepromatous and tuberculoid leprosy patients from Ethiopia. Sera from lepromatous patients showed a higher total LDH activity compared with control subject. The values for tuberculoid leprosy patients were similar to those of controls. Sera from normal healthy controls showed a higher proportion of LDH-H form (72%) while lepromatous leprosy patient's sera exhibited a higher proportion of LDH-M form (55%). Tuberculoid leprosy patients showed a pattern similar to that of healthy controls. A possible significance of these observations is discussed. No significant variations were observed in fructose-1,6-diphosphate aldolase activity within the different types of disease and controls. Although creatine phosphokinase levels in different types of leprosy decreased significantly from those of normal healthy, it falls within the reported variation of the activity in normal sera.     

Histologic responses in sixty multibacillary leprosy patients inoculated with autoclaved Mycobacterium leprae and live BCG

Sixty lepromatous or borderline lepromatous patients were submitted to immunotherapy with a mixture of autoclaved Mycobacterium leprae and BCG. The histopathologic findings in skin biopsy specimens taken before and after immunotherapy were evaluated independently by six histopathologists in a workshop setting. Their pooled observations on diagnosis and classification were analyzed to assess the histopathologic changes following various periods of immunotherapy. Expressing the results as the average value of five to six independent observations, there were changes in classification of reversal or upgrading toward the tuberculoid end of the leprosy spectrum in 90.5% of the patients initially classified as lepromatous (LL), and in 83.3% of those initially classified as borderline lepromatous (BL). The histopathologic findings amply support the clinical, bacteriologic and immunological changes following immunotherapy from LL or BL, to BL, mid-borderline (BB) or even borderline tuberculoid (BT) leprosy.     

Immune responses to recombinant proteins of Mycobacterium leprae

Identification of antigenic determinants of the polar immune response in leprosy may illuminate both protection and pathogenesis. Thirty subjects were studied (22 with polar disease and 8 healthy controls who were heavily exposed but disease-free) by assaying the proliferative, interferon (IFN)-gamma, and antibody responses to recombinant antigens of Mycobacterium leprae (10, 28, 36, and 65 kDa). The 10-kDa antigen elicited IFN-gamma production from all tuberculoid (TT) and borderline tuberculoid (BT) patients but little from controls, lepromatous (LL), or borderline lepromatous (BL) patients (P<.05). Production of 65-kDa-specific IFN-gamma was higher in TT/BT than in controls or LL/BL patients (P<.006). All subjects produced 65-kDa-specific antibody, but it was higher in LL/BL patients than in healthy controls, whose responses were higher than in TT/BT subjects (P=.035). The 36-kDa antibody responses were selectively increased in LL/BL subjects (P<.02). The intermediate phenotype of the controls suggests that M. leprae-specific production of IFN-gamma may contribute to pathology and to protection in leprosy.     

In vivo responses to Mycobacterium leprae: antigen presentation, interleukin-2 production, and immune cell phenotypes in naturally occurring leprosy lesions

To investigate the immune defect in lepromatous leprosy we studied immune cell phenotypes, lymphocyte activation states, and interleukin-2 (IL-2) production in naturally occurring leprosy skin lesions. Mouse hybridoma monoclonal antibodies reacting with the IL-2 receptor (anti-Tac), unbound IL-2 (DMS-1), antigen-presenting Langerhans' cells (OKT6) and the OKT4-Leu3 and OKT8 T-lymphocyte subpopulations were used with indirect horseradish peroxidase and alkaline phosphatase techniques on frozen biopsy sections. The percentage of Tac+ lymphocytes and the number of OKT6+ cells in the epidermis and dermal granuloma were significantly correlated in naturally occurring lesions (correlation coefficient 0.79) and were higher in tuberculoid than in lepromatous lesions. Leu3 antigen was expressed by 70-90% of Tac+ cells in tuberculoid lesions. Although the percentage of cells producing IL-2 was low in lesions of both lepromatous and tuberculoid patients, it was about 15 times greater in tuberculoid than in lepromatous lesions (0.032 +/- 0.037 tuberculoid vs 0.0019 +/- 0.023 lepromatous). There was an association between the number of OKT6+ cells and the percentage of IL-2-producing cells, but the association was weaker than that of OKT6+ cells and the percentage of IL-2 receptor-bearing cells (r = 0.2), implying that IL-2 production is not an intervening variable in the latter association. The absolute number of OKT4-Leu3+ lymphocytes was significantly different in different clinical leprosy groups and was positively correlated with host resistance (mean OKT4-Leu3+ cells/mm2 in 6 micron sections; 1412 +/- 288 tuberculoid, 400 +/- 93 borderline lepromatous, 200 +/- 100 polar lepromatous; r = 0.95). Absolute numbers of OKT8+ cells/mm2 in lesions were not significantly different. We conclude that there is a relative paucity of OKT4-Leu3+ cells as well as IL-2-producing cells at the local level in lepromatous leprosy lesions. Possible functional relationships between these findings and the failure of macrophage activation and destruction of Mycobacterium leprae in lepromatous leprosy are discussed.     

HORMONAL CHANGES ASSOCIATED WITH TESTICULAR ATROPHY AND GYNAECOMASTIA IN PATIENTS WITH LEPROSY

Basal LH, FSH, 17 beta-oestradiol and testosterone and the gonadotrophin responses to luteinizing hormone releasing hormone (LHRH) were studied in male patients with leprosy (twenty-four with lepromatous and six with tuberculoid leprosy). The mean basal LH and FSH was significantly elevated in the lepromatous group and was associated with an excessive response of both gonadotrophins following LHRH administration. The mean basal testosterone and 17 beta-oestradiol values in the lepromatous group were significantly lower than those of the tuberculoid and control groups. The abnormal gonadotrophin and sex steroid values in the lepromatous group are in keeping with the testicular atrophy and gynaecomastia accompanying this form of leprosy. However, the lack of a significant correlation between basal FSH and testicular atrophy should be noted. In addition, no correlation between any of these hormonal values and gynaecomastia could be demonstrated. The patients with tuberculoid leprosy had essentially normal hormonal profiles (except for two who had raised 17 beta-oestradiol values). This is compatible with the lack of gonadal involvement in these patients.     

A histopathological study of lymphnodes in 43 cases of leprosy

Eighty cases of leprosy including 60 cases of lepromatous type and 20 cases of tuberculoid type, during the period of 1974-75, have been examined for evidence of lymphnode enlargement. Of the 52 cases of enlarged lymphnodes, lymphnode biopsy was done in 43 cases including 38 cases of lepromatous type and 5 cases of tuberculoid type. The lymphnodes have been studied for evidence of any pathological changes and presence of acid-fast bacilli. In cases of lepromatous leprosy, lepromas and acid-fast facilli were seen in 92.2 per cent of the cases and patchy fibrosis was noted in 23.6 per cent of the cases. No amyloid could be demonstrated. In cases of tuberculoid leprosy, only non-specific reticular hyperplasia was noted. No specific granuloma or acid-fast bacilli could be demonstrated. The findings have been described in detail and discussed in the light of previous published data.