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)     

Quantitative antibody ELISA for leprosy

Quantitative enzyme-linked immunosorbent assays (ELISAs) were established to measure IgM and IgG antibody levels to soluble Mycobacterium leprae sonicate (CD60) and to the synthetic disaccharide antigen based on the phenolic glycolipid-I antigen of M. leprae coupled to bovine serum albumin in 46 leprosy patients. Separate reference pools for IgM and IgG antibody were established. The reciprocal of the antibody titer was expressed as the number of arbitrary units in the reference pools which was subsequently used as the calibrator for assessment of units in individual test sera. The dose-response relationship for both IgM and IgG was highly specific and reproducible for both isotypes, as indicated by the intra- and inter-assay coefficients of variation. The distribution of antibody levels are in general agreement with the results from previous studies against different M. leprae antigens. The lepromatous group showed 10- to 100-fold higher IgM antibodies to both the soluble sonicate antigen and the disaccharide as compared to the control group. Very low to undetectable levels of IgM antibodies were observed in the tuberculoid group of leprosy patients. IgG antibodies, on the other hand, were not only present but showed considerable overlap with the lepromatous patient group. Optimized ELISAs, such as the one described in this study, would allow one to address issues such as antibody changes with treatment, antigen clearance, and correlation with other immune parameters associated with disease pathogenesis and protection.     

Serological response to purified mycobacterial phosphatidylinositol mannoside in healthy controls and in patients with tuberculosis and leprosy.

The serological response to a monoclonal antibody-defined phosphatidylinositol mannoside (L4-PIM) present in all mycobacteria was examined in patients with various mycobacterial diseases and healthy subjects from different populations. IgG but not IgM antibodies were detected in most patients with untreated lepromatous (84%) or borderline lepromatous (65%) leprosy, but in only a minority of those with disease at the tuberculoid end of the leprosy spectrum (< 17% positive). The response to L4-PIM was correlated with the IgM response to disaccharide octyl-bovine serum albumin (dBSA), and decreased with successful treatment. On the other hand, the test proved to be of little value in the diagnosis of untreated tuberculosis (4/15 positive) or atypical mycobacterial infection in patients with AIDS (0/11 positive). IgG antibodies to L4-PIM were also found in a significant proportion of healthy individuals, irrespective of their Mantoux status. These antibodies were shown to be specific for L4-PIM on immunoblotting, and their incidence increased with age in random donors from both urban Australia and rural Papua New Guinea. Despite the limited value of the assay in diagnosis of any particular mycobacterial disease, the presence of antibodies to L4-PIM appears to be a sensitive indicator of subclinical infection with environmental mycobacteria in subjects with an intact immune system.     

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.     

Humans respond predominantly with IgM immunoglobulin to the species-specific glycolipid of Mycobacterium leprae

The immunoglobulin classes of the antibody response to the species-specific phenolic glycolipid antigen of Mycobacterium leprae have been characterized for serum specimens from 78 patients with leprosy. These patients included the entire clinical spectrum from paucibacillary to multibacillary disease, including polar tuberculoid (TT; 11 patients), borderline tuberculoid (BT; 15), borderline (BB; 17), borderline lepromatous (BL; 13), and lepromatous (LL; 22)--clinical classifications according to Ridley-Jopling criteria. In each patient group, the levels of IgM antibody to phenolic glycolipid were significantly higher than levels of IgG or IgA. Inhibition experiments with purified antigen showed that antibodies to the phenolic glycolipid dominated the human IgM antibody response to the surface of M. leprae.     

Immunologically reactive <Emphasis Type="Italic">M. leprae</Emphasis> antigens with relevance to diagnosis and vaccine development

Background  

Leprosy is a chronic infectious disease caused by Mycobacterium leprae that can manifest a wide variety of immunological and clinical outcomes ranging from potent humoral responses among borderline lepromatous (BL) and lepromatous (LL) patients to strong cellular responses among tuberculoid (TT) and borderline tuberculoid (BT) patients. Until recently, relatively little has been known about the immune responses to individual proteins of M. leprae recognized during leprosy.     

Quantitation of IgM antibodies to the M. leprae synthetic disaccharide can predict early bacterial multiplication in leprosy

Quantitative enzyme-linked immunosorbent assays detecting IgM to the soluble Mycobacterium leprae crude sonicate (CD75) and the synthetic disaccharide antigen coupled to bovine serum albumin (ND-BSA) were assessed for their ability to determine early infection in families/household contacts of leprosy patients and employees of a leprosy center working in close contact with leprosy patients. Although IgM to both antigens (CD75 and ND-BSA) correlated with the bacterial index (BI) assessed histologically on skin-biopsy samples, the level of IgM antibodies to ND-BSA was a much more sensitive indicator of low bacterial loads. A 4.4-fold difference in antibody levels was observed between the mean group levels of endemic controls (N = 116) and tuberculoid leprosy patients with a BI of 0 (N = 88), increasing to sevenfold in tuberculoid leprosy patients with a BI of 1 (N = 20). Using a statistical cut off with endemic controls (mean + 2 S.D.), household/family contacts showed 30% seropositivity (N = 180) as compared to staff contacts who showed 17% seropositivity (N = 55). Percent seropositivity in family contacts was not related to the type of leprosy of the index case (lepromatous vs. tuberculoid) or the duration of treatment of the index case. Age of the individual in the family contact group had a significant influence on seropositivity. These results support the hypothesis that, in this community, factors other than the viable bacterial load of the index case, such as genetic susceptibility, may be influencing the high rate of seropositivity in family contacts. IgM ND-BSA antibodies seem to provide a good indicator of low antigenic loads and could prove to be useful in detecting subclinical infection before the onset of disease. Follow-up studies of these seropositive individuals are in progress to understand the relationship between seropositivity and the progress of clinical disease.     

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.     

Immunological assessment of sera of leprosy patients.

IgG levels were significantly high in sera of all types of leprosy. House-hold contacts of lepromatous leprosy (LL) cases also showed significantly higher values for IgG when compared to that of control. Except polar tuberculoid (TT) cases and house-hold contacts other types of leprosy revealed a significant rise in IgA levels in their sera. IgM was only raised in borderline tuberculoid (BT) cases. C-reactive protein (CRP) was present in the sera of all types of leprosy. Highest positivity (97%) was shown by sera from erythema nodosum leprosum (ENL) cases. Rose-Waaler antibody (RA) was noted in BT, borderline leprosy (BL), LL and ENL cases. Significance of these findings is discussed.     

Salivary anti-PGL-1 IgM may indicate active transmission of Mycobacterium leprae among young people under 16 years of age

Considering that the main route of Mycobacterium leprae transmission is the upper respiratory tract, detection of salivary antibodies can be a useful tool for diagnosing early infection. The study aimed to analyze salivary anti-PGL-1 IgA and IgM antibodies in 169 children aged 4–16 years old, who lived nearby or inside the house of multibacillary or paucibacillary leprosy patients in two endemic cities in Alagoas State – Brazil. Salivary anti-PGL-1 antibodies were quantified by modified ELISA method. The frequency of contact and clinical form of the index case were significantly associated with salivary antibody levels. High frequency of IgM positivity strongly suggests active transmission of M. leprae in these communities. We suggest in the present work that salivary anti-PGL IgA and IgM are important biomarkers to be used for identifying communities with probable active transmission of M. leprae.     

Serum IgA1 and IgM antibodies against Mycobacterium leprae-derived phenolic glycolipid-I: a comparative study in leprosy patients and their contacts

In order to evaluate the potentials of IgA1 versus IgM as well as of native phenolic glycolipid-I (PGL-I) versus PGL-I-disaccharide coupled to bovine serum albumin (D-BSA) as antigens in the serodiagnosis of leprosy, anti-D-BSA IgA1 and anti-PGL-I IgM were investigated and compared to anti-PGL-I IgA1 in sera from patients and contacts. Anti-D-BSA and anti-PGL-I IgA1 significantly correlate in patients and contacts. The higher IgA1 positivity rates obtained with D-BSA as compared to PGL-I may suggest D-BSA as the favorable antigenic material. In patients but not in contacts anti-PGL-I IgM and IgA1 correlate, IgM predominating over IgA1. In all three antibody systems, the mean values as well as the positivity rates increased from the tuberculoid toward the lepromatous disease pole. Also, the levels of all three antibodies significantly increased with the bacterial index (BI). However, anti-D-BSA (PGL-I) IgA1 appears to be preferable to IgM with respect to sensitivity, i.e., detection of disease activity, in paucibacillary or BI-negative patients. A number of contacts were detected as seropositive with anti-D-BSA and/or anti-PGL-I IgA1 but not with anti-PGL-I IgM. This suggests that IgA1 is a better tool than IgM for the detection of leprosy in its subclinical stage.     

IgM antibodies against phenolic glycolipid I from Mycobacterium leprae in leprosy sera: relationship to bacterial index and erythema nodosum leprosum

Serum IgM antibodies against Mycobacterium leprae-derived phenolic glycolipid I (PG) were determined in 121 leprosy patients, in contacts and controls by an enzyme-linked immunosorbent assay technique. Anti-PG IgM levels correlated with disease classification, increasing from the tuberculoid towards the lepromatous pole of the disease spectrum. There was a linear correlation between serum IgM PG-antibody levels and bacillary index (BI), a measure of bacterial load. Elevated anti-PG IgM in bacillary negative patients was usually indicative of active disease, undetected by BI. We conclude that anti-PG IgM levels are valuable for monitoring the degree of disease activity. Serum anti-PG IgM levels were significantly lower in patients with erythema nodosum leprosum (ENL) as compared to those without ENL, suggesting that IgM PG-antibodies are also involved in the pathogenesis of ENL.     

Immunologic aspects of leprosy with special reference to the study of immunoglobulin E.

The serum levels of IgG, IgM, IgD and IgE have been determined in normal subjects, individuals suffering from ascariasis and filariasis, and in leprosy patients. Allergic and parasitic diseases were excluded in these normal subjects and in leprosy patients before they were taken for the study of their serum IgE. The circulating IgG was significantly raised in both tuberculoid and lepromatous forms of leprosy and also in filariasis; IgM was significantly elevated in only the lepromatous form of leprosy, ascariasis as well as in filariasis; while IgA was exclusively raised in both forms of leprosy. IgD was detected in the sera of more subjects with ascariasis and filariasis than in normal individuals and leprosy patients. The mean level of serum IgE in 35 normal Indian subjects was 1,025 I.U. per ml, 9 of them (25%) having serum IgE concentrations above 700 I.U. per ml. The highest mean level of serum IgE was found in ascariasis (7,328 I.U. per ml), followed by leprosy (5,180 I.U. per ml), and filariasis (4,244 I.U. per ml). Furthermore, no significant difference between the mean serum IgE levels of tuberculoid and lepromatous leprosy patients was observed. Although the rise of serum IgE level in these parasitic diseases, as well as in leprosy, was spectacular, the augmented synthesis of this unique class of immunoglobulins was not invariably present in all patients. The results have been discussed on the basis of recent ideas on immunoglobulin synthesis.     

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 antibodies against peripheral nervous system antigens in leprosy.

Since antibodies against peripheral nervous system (PNS) antigens may play a pathogenetic role in the mechanism of nerve damage in leprosy, sera from leprosy patients and contacts were investigated for anti-PNS antibodies by ELISA and immunoblot. In ELISA, elevated anti-PNS antibody levels were detected in 4 of 98 (4.1%) leprosy patients (4 of 52, 7.7%, lepromatous leprosy patients), in 1 of 28 (3.6%) contacts, and in 1 of 18 (5.6%) normal controls. There was no correlation between anti-PNS antibody levels and the bacterial index or neuropathy in leprosy. Immunoblot with a sample of six leprosy and five control sera showed that the antigenic binding pattern (mainly within the 100-200-kDa region) was very similar in patients and controls. Staining intensity, however, appeared to be higher with the leprosy sera than with the control sera. IgM and IgG were found to contribute to the staining pattern: IgM in the 150-200-kDa range, IgG with multiple bands between 25 kDa and 200 kDa. Thus, the presence and levels of serum anti-PNS antibodies in leprosy appear to be unrelated to parameters of disease activity, neuropathy in particular, and do not seem to be critically involved in the pathogenesis of nerve damage.     

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.     

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.     

Antigenic protein from Mycobacterium leprae released in macrophages in vitro as indicator of viability of bacteria

Peritoneal macrophages from randombred, Swiss white mice, when cultured and infected with Mycobacterium leprae for 24 hours, are able to show the presence of antigen(s) with binding affinity to antibodies present in the sera of bacteriologically positive, lepromatous leprosy patients. Such antibodies are not seen in sera from normal and healthy persons, tuberculoid leprosy patients, or long-term-treated, bacteriologically negative, lepromatous leprosy patients. The production of the antigen(s) is blocked by the anti-M leprae drug rifampin. Other mycobacteria when incubated with macrophages from mice show very little antigens in the lysate but the antigens have an equal affinity for antibodies in sera from both normal individuals and lepromatous patients. Only the lysates from macrophages exposed to live M. leprae could discriminate and could exhibit differential binding to sera from leprosy patients compared to sera from normal individuals. This antigen(s) does not have any binding ability to the monoclonal antibodies available to the antigens of M. leprae identified at present and shown to be specific to M. leprae. This indicates a separate identity of this product which has potential for further exploitation in exploring host-pathogen interactions related specifically to the leprosy infection and the tolerance of M. leprae inside cells.     

Use of human skin to demonstrate antinuclear antibodies in lepromatous leprosy patients

A common finding in the sera of leprosy patients is the presence of antinuclear antibodies (ANA), but their specificity for autologous antigens is unknown. The aim of this work was to investigate the reactivity of these ANA toward the cell nuclei of human skin. ANA were investigated in the sera of 35 patients with lepromatous leprosy by immunofluorescence reactions performed with sections of human skin biopsies (autologous from each patient and healthy human skin obtained from plastic surgery procedures), and compared with the results obtained when rat liver was used as substrate. ANA titer, immunofluorescence pattern, immunoglobulin classes (IgG and IgM) and complement-binding capability were also investigated. When human skin sections were used as substrates, 30 out of 35 patients (85.7%) gave positive ANA tests; most of them gave a 1:4 to 1:16 titer for IgG with an annular pattern and 1:4 for IgM with an annular or a granular pattern. ANA of 30 patients bound C1q and 14 bound C3. However, when rat liver sections were used as substrates only 9 out of 35 cases (27.1%) gave positive ANA tests. These results show that human skin sections are a better substrate to demonstrate the ANA present in the sera of patients with lepromatous leprosy. Their significance in the pathogenesis of tissue damage remains to be investigated.     

Antibodies to a synthetic analog of phenolic glycolipid-I of Mycobacterium leprae in healthy household contacts of patients with leprosy

Fifty-four household contacts of lepromatous patients, 39 household contacts of tuberculoid patients, and 99 control persons were examined with an enzyme-linked immunosorbent assay for their antibody responses to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae using a synthetic analog (PGL-ISA) with the same terminal sugar epitope, namely, O-(3, 6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(alpha-L-rhamnopyranosyl )-(1----9)-oxynonanoyl-BSA. This study was conducted in the Gurage area of Ethiopia in 15 households with a leprosy patient and 15 matched control households. Household contacts with more than 1 year of exposure to a lepromatous patient had antibodies to PGL-ISA significantly more often (19 of 34 persons) than did household contacts with less than 1 year of exposure to a lepromatous patient (4 of 20 persons), household contacts of tuberculoid patients (8 of 39 persons), and persons without exposure to leprosy in the household (33 of 99 persons). No significant association was found between the prevalence of antibodies to PGL-ISA in the household contacts and disease activity in the lepromatous index patients at the time of examination; nor was there a significant association between antibody responses and age or sex of the contacts. The increased prevalence of antibodies to M. leprae antigen in healthy persons with more than 1 year of contact with a lepromatous patient provides further evidence that subclinical infection in leprosy is common, and is related to the type of leprosy in the index patient. The fact that antibodies to PGL-ISA were detected in one third of the persons without household exposure to leprosy emphasizes the necessity to always include comparable controls from the same endemic area in studies of leprosy contacts.