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.     

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.     

Evaluation of bone and mineral metabolism in patients recently diagnosed with leprosy

This study was conducted to evaluate patients recently diagnosed with the tuberculoid and lepromatous forms of leprosy for bone mass, bone remodeling, and hormones related to mineral control. Eleven normal control individuals (CG) and 12 patients with leprosy (LG) matched for physical characteristics were submitted to evaluation of bone mass density (BMD) and to the determination of serum levels of PTH, 25-hydroxyvitamin D [25(OH)D], testosterone, LH, FSH, osteocalcin (OC), and urinary levels of deoxypyridinoline (DPD). The T score of lumbar spine and total radius (mean +/- SD) were significantly lower in leprosy patients (L1-L4: CG = -0.7 +/- 1.5 vs LG = -1.8 +/- 1.0 SD, P < 0.04, and total radius: CG = -1.43 +/- 0.6 vs LG = -2.1 +/- 0.8 SD, P <0.02), whereas no significant differences were observed in total hip or femoral neck T score. However, at all sites, the rate of low bone mass (T score < -1.0) was higher in LG (femoral neck: CG = 18% vs LG = 50%, total hip: CG = 27% vs LG = 42%). There was a significant difference in albumin and PTH levels between groups but not in serum 25(OH)D and OC levels or urinary DPD levels. The present results indicate that bone mass loss is an early event in leprosy patients and frequently is already present at diagnosis. Its etiopathogenesis is multifactorial, and further studies are needed to determine the most efficient way to prevent fractures in this condition. The data obtained in the present study need confirmation by the evaluation of a larger sample.     

Leucocytic alkaline phosphatase activity in leprosy: a possible guide in the follow-up of leprosy.

In order to investigate a possible involvement of phagocytic cells in the various types of leprosy, we undertook the study of enzymatic activities in circulating leucocytes. The activity of leucocytic alkaline phosphatase was studied by histochemical techniques on blood smears in 31 patients presenting with leprosy and aged between 4 and 73, and in 11 non infected people. The 31 patients suffering from leprosy were distributed as following: 14 lepromatous leprosy of which 6 had not yet been treated and 8 were under treatment, 9 cases of tuberculoid leprosy of which 7 had been treated and 2 had not yet, 3 cases of borderline leprosy which had all been treated, and 5 patients whose form of leprosy was indeterminate (before treatment). The distribution of the different values we obtain shows a very significant difference (p less than 0.001) between patients with and without leprosy (respectively 33.8 +/- 7.3 and 109.8 +/- 12.5). Moreover, the decrease of the alkaline phosphatase activity correlated with the severity of the disease (47.2 +/- 11.4 in tuberculoid leprosy and 20.6 +/- 9.3 in lepromatous leprosy) thus suggesting that the evaluation of leucocytic alkaline phosphatase activity should be advised as a possible prognosis guide in indeterminate leprosy.     

Human leukocyte antigen and leprosy: study in northern Louisiana and review

We examined the relationship of human leukocyte antigen (HLA) phenotype to leprosy in six sporadic cases in northern Louisiana and in the world literature through pooling of the results of several studies. We found that HLA antigens DR2 and DQwl were associated with leprosy in the six cases in northern Louisiana (relative risks, 4.57 for DR2 and 4.53 for DQwl), but the results are not statistically significant. We pooled the Louisiana study and other population studies of HLA and leprosy. The results of the pooling show DR2 and DQwl to be associated with leprosy (relative risks, 2.65 for DR2 and 2.73 for DQwl), and these associations are highly statistically significant (P less than 1 x 10(-8) for DR2 and P = 3.6 x 10(-8) for DQwl). Further, we pooled studies of lepromatous leprosy patients vs. controls and studies of tuberculoid leprosy patients vs. controls and found that DR2 and DQwl are associated with both the lepromatous and the tuberculoid forms of leprosy and that these associations are statistically significant. We consider the associations of DR2 and DQwl in these population studies to be evidence for an HLA-associated genetic influence on susceptibility to leprosy.     

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 leprosy

Serum calcium and magnesium were studied in 70 leprosy patients and 25 normal healthy individuals. An attempt has been made in this study to find out if there is any correlation between the clinical and pathological status of the disease and serum calcium and magnesium levels in the blood. Serum calcium was found to be significantly decreased in lepromatous leprosy (Ca=8.42+/-0.7 mg%, P less than 0.001) and dimorphous leprosy (Ca=8.68+/-0.94 mg%, P less than 0.05) while it was normal in tuberculoid leprosy (Ca=9.14+/-2.12, P less than 0.05). The decrease in serum magnesium level was highly significant in all clinical types of leprosy (Mg=1.08+/-0.29, P less than 0.001).     

Human leukocyte antigens in forms of leprosy among Japanese patients

Human leukocyte antigens (HLA) class II alleles were analyzed among Japanese leprosy patients to ascertain whether immunogenetic differences exist among the leprosy classification forms of Ridley and Jopling. Ninety-three unrelated Japanese leprosy patients (21 lepromatous, 24 borderline lepromatous, 17 mid-borderline, 26 borderline tuberculoid, 5 tuberculoid) and 114 healthy control subjects were investigated. The frequencies of HLA-DRB1*1501, -DRB5*0101, -DQA1*0102 and DQB1*0602 were significantly increased in all of the Japanese leprosy patients. The frequencies of HLA-DRB1*0405, -DQA1*03 and -DQB1*0401 were significantly decreased in the Japanese patients after correction of the p value. Conversely, there were no significantly different distributions of the HLA-DRB1, -DRB5, -DQA1, DQB1 alleles in the five subgroups of these patients. We conclude that HLA class II alleles were not associated with the form of leprosy. Other HLA, a non-HLA gene, and/or environmental factors may play a critical role in the different manifestations of leprosy.     

Lymphocyte transformation test in healthy contacts of patients with leprosy. II. Influence of consanguinity with the patient, sex, and age.

The study was carried out in the Gurage area of Ethiopia, where 53 household contacts of lepromatous patients, 37 household contacts of tuberculoid patients, and 91 control persons were examined with the lymphocyte transformation test (LTT) for their responses to whole and sonicated antigen preparation from M. leprae to BCG, M. avium, M. gordonae and phytohemagglutinin. The potential influence of host factors, namely the state of consanguinity with the leprosy patient, sex and age on the LTT responses was evaluated. In the 35 household contacts of "active," i.e., highly bacilliferous, lepromatous patients, consanguinity with a lepromatous patient was not associated with a significant depression of the LTT responses to M. leprae antigens. Male household contacts of active lepromatous patients showed significantly greater LTT responses to M. leprae antigens than female household contacts. Possible confounding factors for this finding are discussed. Sensitization of M. leprae antigens was present already in a high proportion of the 6 to 14 year old household contacts of active lepromatous patients, which was the youngest age group examined in our study. No significant results were found in any of the other patient contact groups with regard to the host factors examined.     

Blastogenic response with autologous plasma and Mitsuda's reaction in leprosy patients and their unaffected sibs

Many investigators have evaluated the influence of genetic constitution on the susceptibility to leprosy in studies linked to different types of research. To determine the possible existence of a family trait linked to the lymphoproliferation and to lepromin reactivity we studied the blastogenic response to phytohaemagglutinin, lepromin and M. leprae and the Mitsuda's reaction in leprosy patients and their unaffected sibs. Sixty-eight individuals were studied, 34 were leprosy patients (17 lepromatous and 17 tuberculoid leprosy) and the remaining were their sibs previously matched by sex and age. The indices of blastogenesis and lepromin reactivity were lower in lepromatous than in tuberculoid patients, that confirmed the immunological polarity of the two types of leprosy. Both the lymphoproliferation and Mitsuda's reaction results suggest different cell immune responses in leprosy patients and their unaffected sibs, so that the hypothesis of a family trait favouring the similarity of responses to these tests among sibs becomes unlikely.     

Defective blood mononuclear phagocyte function in patients with leprosy.

Patients with lepromatous leprosy possess a defective lymphocyte function in vivo and in vitro that is less evident in the tuberculoid form. Data concerning their macrophage ability to digest Mycobacterium leprae are controversial. The purpose of this study was to determine whether monocytes from patients with either tuberculoid or lepromatous leprosy were altered in their enzyme systems, that is myeloperoxidase-dependent and myeloperoxidase-independent systems. The ability of adherent blood monocytes to ingest and kill Candida pseudotropicalis after 30 and 60 min of incubation with yeast cells was tested. Mononuclear phagocytic cells from patients with either principal form of leprosy functioned similarly to normal monocytes in phagocytosis while their fungicidal activity for C. pseudotropicalis was statistically significantly altered and was more evident in the lepromatous than in the tuberculoid type. The results indicate that peripheral blood monocytes from patients with leprosy possess an impaired enzymatic candidacidal activity.     

The association of leprosy and pulmonary tuberculosis.

The association of leprosy with pulmonary tuberculosis was seen in 20 cases during January 1972 to October 1977. Of these 20 cases, 15 were of lepromatous, 3 of dimorphous and 2 of tuberculoid leprosy. Maximum number of cases were from 3rd decade of life with male to female ratio of 3 : 1. Patients were suffering from the disease for a very long time (8 cases of lepromatous leprosy with duration of illness 10-15 years). The symptoms of leprosy seemed to have preceded to the symptoms of pulmonary tuberculosis. Duration of tuberculosis in most of them was within 6 months (55%). The predominent presenting symptom was cough with expectoration (100%) and fever (80%). Anaemia (75%) and crepitations (60%) were the presenting physical sign mainly. Radiologically bilateral extensive pulmonary lessons were seen in 14 cases (70%). Sputum for acid fast bacilli was positive in 80% of cases. The general condition and nutritional status was poor and death was resulted in 4 cases (3 of lepromatous and 1 of dimorphous leprosy).     

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.     

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.     

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.     

Enzyme immunoassay of phagocytosis stimulating tetrapeptide "tuftsin" in normal and leprosy sera.

The serum concentrations of the phagocytosis stimulating the tetrapeptide, tuftsin, were determined by competitive enzyme immunoassay in borderline tuberculoid/tuberculoid (BT/TT, 16 cases), borderline lepromatous/lepromatous (BL/LL, 16 cases), and in healthy controls (20 cases). Using checkerboard titration, 10 ng/well of diphtheria toxoid-p-aminophenylacetyl tuftsin (DTPT) conjugate when incubated with tuftsin antisera at 1:15,000 dilution with a preincubation time of 60 min with the competitor (tuftsin) followed by a further 60-min incubation onto the DTPT-coated wells gave consistent results with a sensitivity of 5 ng/well tuftsin. The mean serum tuftsin concentration was significantly lower in BL/LL patients (134.42 +/- 48.7 ng/ml, p less than 0.01) than in healthy controls (262.86 +/- 59.8 ng/ml), while BT/TT sera (210.94 +/- 75.5 ng/ml) showed slightly decreased levels than did normals, which was not statistically significant. The mean serum IgG levels in BL/LL and BT/TT patients (37.26 +/- 10.99 mg/ml; 28.08 +/- 6.57 mg/ml, respectively) showed significantly (p less than 0.001) higher concentrations than did healthy controls (12.3 +/- 3.6 mg/ml). These observations on the serum concentrations of tuftsin and IgG in leprosy individuals suggest that there is splenic dysfunction in BL/LL patients in terms of the processing of leukokinin to release the free, active molecule tuftsin.     

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)     

Nucleation time and age in patients with gallstones

The relationship between nucleation time (NT), total lipid concentration (TLC) and age was studied in a group of 45 gallstone patients (10 male, 35 female, age 50.1 +/- 14.5 yrs). Bile was obtained by direct fine needle puncture of the gallbladder under local anaesthesia and sonographic monitoring. There was a positive correlation between age and nucleation time (r = 0.626, p less than 0.001), in addition to a negative correlation between age and total bile lipid concentration (TLC) (r = -0.414, p = 0.005). The negative correlation between age and TLC indicates that gallbladder's ability to concentrate the bile decreases significantly with age. It is possible that the decreased concentration is a result of the chronic pathogenetic effects of gallbladder stones. Practically all patients showed a prolonged nucleation time after the 60th year (11.54 +/- 5.50 vs. 2.65 +/- 2.52 days). This would seem to indicate that these patients suffered primarily from bilirubin or calcified stones, currently unsuited for conservative therapy methods.     

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.     

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.