Induction and treatment of anergy in murine leprosy.

Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial-specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG-inoculated and MLM-inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell-mediated immune response (CMI) that was temporary in the MLM-inoculated group and long-lasting in the BCG-inoculated group. DLE were prepared from the spleens of MLM- and BCG-inoculated mice at the peak of CMI. Independent MLM intradermally-inoculated groups were treated every other day with HLT-DLE, BCG-DLE or MLM-DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 10(6) splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy.

Presence of intestinal helminths decreases T helper type 1 responses in tuberculoid leprosy patients and may increase the risk for multi-bacillary leprosy.

Resistance to intracellular pathogens such as Mycobacterium leprae is dependent upon an effective T helper type 1 (Th1)-type immune response. On the other hand, intestinal helminths are known to subvert the host's immune response towards to either a Th2-type immune response or a regulatory T cell up-regulation, which may affect the host's ability to mount an effective response to mycobacteria. Here, we report a significant association between intestinal helminth infections and lepromatous leprosy [odds ratio (OR), 10.88; confidence interval (CI) 95%: 4.02-29.4; P<0.001]. We also observed that the frequency of intestinal helminths correlated strongly with the mycobacterial index (r=0.982, P<0.01). Corroborating with our hypothesis, intracellular levels of interferon-gamma were decreased significantly in leprosy patients co-infected with intestinal helminths when compared to leprosy patients without worms. Conversely, lepromatous leprosy patients with intestinal worms produced higher levels of both interleukin (IL)-4 and IL-10. Our results suggest that a pre-existing infection by intestinal helminths may facilitate the establishment of M. leprae infection or its progression to more severe forms of leprosy.

Leukocyte antimicrobial function in patients with leprosy.

Patients with lepromatous leprosy are unresponsive to lepromin skin-test material and possess defective lymphocyte function in vitro, including impaired mitogenesis in response to antigens of Mycobacterium leprae. It has been claimed that their macrophages cannot digest M. leprae in vitro; such a defect could explain both lepromin nonreactivity and impaired lymphocyte function on the basis of failure of the afferent limb of the immune response (i.e., defective macrophage "processing" of M. leprae). The present studies indicate that macrophages from patients with lepromatous and tuberculoid leprosy and from normal donors do not differ in their ability to digest heat-killed M. leprae in vitro, or in their ability to sustain the viability of M. leprae in tissue culture; that monocytes, macrophages, and polymorphonuclear leukocytes of leprosy patients and controls possess equivalent microbicidal activity against Listeria monocytogenes, Escherichia coli, Proteus vulgaris, Staphylococcus aureus, and Candida albicans; and that polymorphonuclear leukocytes from patients with lepromatous leprosy iodinate ingested bacteria normally. Whether the basic immune defect leading to the development of lepromatous leprosy resides in the lymphocyte or in the macrophage remains to be determined. However, the present study shows that phagocytic cells from patients with either principal form of leprosy function normally in a variety of sophisticated tests of antimicrobial function.

Utility of measuring serum levels of anti-PGL-I antibody, neopterin and C-reactive protein in monitoring leprosy patients during multi-drug treatment and reactions.

OBJECTIVE: To verify the validity of measuring the levels of Mycobacterium leprae-specific anti-phenolic glycolipid (PGL)-I antibody, neopterin, a product of activated macrophages, and C-reactive protein (CRP), an acute phase protein, in serial serum samples from patients for monitoring the leprosy spectrum and reactions during the course of multi-drug treatment (MDT). METHODS: Twenty-five untreated leprosy patients, 15 multi-bacillary (MB) and 10 paucibacillary (PB), participated. Eight patients developed reversal reaction and five developed erythema nodosum leprosum (ENL) during follow-up. The bacterial index (BI) in slit-skin smears was determined at diagnosis and blood samples collected by venipuncture at diagnosis and after 2, 4, 6 and 12 months of MDT. PGL-I antibody and neopterin were measured by enzyme-linked immunosorbent assay, whereas the CRP levels were measured by the latex agglutination method. RESULTS: The levels of PGL-I antibodies and neopterin were higher in the sera of MB than PB patients, which correlated with the patients' BI. The serum levels of CRP did not differ significantly between the MB and PB patients. The serum levels of PGL-I and neopterin were no higher in reactional patients than non-reactional patients prone to such reactions. However, ENL patients had higher serum CRP levels than non-reactional MB patients. The serum PGL-I antibody levels declined significantly during MDT, in contrast to neopterin and CRP levels. CONCLUSION: Measuring the serum levels of PGL-I antibodies and neopterin appeared to be useful in distinguishing MB from PB patients, whereas monitoring the levels of PGL-I antibodies appeared to be useful in monitoring MB patients on MDT. Measuring serum CRP, although not useful in monitoring the patients, has limited significance in detecting ENL reactional patients.

Serum adenosine deaminase levels in reactional and non-reactional leprosy.

BACKGROUND AND AIMS: Altered serum adenosine deaminase (ADA) levels have been recorded in various diseases involving lymphocytes and/or lymphoreticular system including leprosy. The study was planned to evaluate alterations in serum ADA levels, if any, in reactional and non-reactional leprosy. METHODS: Eighty patients of leprosy, comprising 60 patients of non-reactional leprosy and 20 patients of reactional leprosy were studied along with 20 normal healthy controls. Five milliliters of venous blood was collected and ADA levels were estimated by the method of Giusti (1974). RESULTS: There were 54 males and 26 females. The age of the patients ranged from 5 years to 62 years. The duration of leprosy ranged from 15 days to 3 years. The mean serum ADA level in normal controls was 10.31 +/- 0.58 u/L. The serum ADA levels were raised in leprosy patients, significantly so in multibacillary patients. The serum ADA levels were higher in patients of leprosy with reaction. CONCLUSIONS: The study showed significantly high serum ADA levels in multibacillary leprosy and this was further increased in patients of leprosy with reaction. This may be because of increased lymphoreticular activity during the reactional phases.

Detection of Toll-like receptor 2 (TLR2) mutation in the lepromatous leprosy patients.

Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacterial infections and the mutations in the TLR2 have been shown to confer the susceptibility to severe infection with mycobacteria. To define this, we screened the intracellular domain of TLR2 in 131 subjects. Groups of 45 lepromatous and 41 tuberculoid leprosy (TT) patients and 45 controls were investigated. Ten subjects among the lepromatous leprosy (LL) patients had a band variant detected by single-stranded conformational polymorphism. DNA sequencing detected a C to T substitution at nucleotide 2029 from the start codon of the TLR2. The mutation would substitute Arg to Trp at amino acid residue 677, one of the conserved regions of TLR2. In our results, the mutation was involved in only LL, not TT and control. Thus, we suggest that the mutation in the intracellular domain of TLR2 has a role in susceptibility to LL.

Production of host-protective (IFN-gamma), host-impairing (IL-10, IL-13) and inflammatory (TNF-alpha) cytokines by PBMC from leprosy patients stimulated with mycobacterial antigens.

The production of IFNgamma, IL-10, IL-13 and TNFalpha was determined using PBMC from 7 tuberculoid (TT) and 7 lepromatous leprosy (LL) patients, after stimulation with several mycobacterial antigens, in an attempt to characterize the cytokine responses to these antigens. The results showed that TT patients displayed higher IFNgamma levels than LL patients with the mycobacterial antigens tested, but no differences in IL-10 production were observed between the two groups. MLSC antigen was associated with the lowest IFNgamma production in TT and LL groups. Only BCG could be identified with stimulation of IFNgamma production in some LL patients. The mycobacterial antigens SP+, SP- and BCG were associated with higher TNFalpha production in patients and controls, suggesting that these antigens could be involved in immunopathological effects. Our findings showed that the antigens tested were associated with a heterogeneous cytokine production in leprosy patients. Further studies are required to establish if an individual antigen can be identified as inducing a protective immune response in leprosy.

Detection of S-100 protein and anticeramide antibodies in leprosy patients by ELISA.

The status of assay for S-100 antigen protein and anticeramide antibodies in serum in understanding nerve damage in different forms of leprosy were evaluated by the enzyme immunoassay. Based on the clinical and smear examination, patients were classified as indeterminate (Ind), tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL) and lepromatous (LL). Antibody levels against ceramide were observed in sera of leprosy patients with 37.5% of Ind, 28% of TT, 66% BT, 78% BL and 62% LL patients positive as against 8% endemic normal sera. The mean OD ranged from 0.141 to 0.275 in different groups of leprosy. In contrast, S-100 was detected in 71.4% Ind, 88.8% TT, 76.4% BT, 100% BL and 95.8% LL, while 5% of ENL samples were positive for S-100 antigen. Mean S-100 levels in these different categories of patients were significantly higher Ind--0.45 ng/ml, TT--0.32 ng/ml, BT--0.23 ng/ml, BL--0.23 ng/ml, LL--0.19 ng/ml as compared to that of normal 0.07 ng/ml. In general S-100 seems to be a more sensitive and reliable marker than anticeramide antibodies for nerve damage. Five out of 7 indeterminate cases show increased levels of S-100, showing an extent of nerve damage similar to that of TT and could be a useful marker for assessing nerve damage in indeterminate patients for better management.

Coronary heart disease risk factors in Hansen's disease sufferers.

In the frame of an epidemiologic study of Hansen's disease (HD) sufferers, several risk factors have been investigated which might explain the high prevalence of coronary heart disease (CHD) among HD patients. The data analyzed in the present study are derived from 293 HD patients (157 men and 136 women). The patients, after having completed a WHO adopted questionnaire, were given a complete physical examination, a resting and an exercise electrocardiogram, and biochemical as well as hematological examinations. Coronary HD patients, when compared to noncoronary HD patients, showed statistically significant differences in the following parameters: (1) mean age, (2) mean concentration of the electrophoretic fraction of alpha-lipoproteins, (3) deviation from mean weight, (4) prevalence of hypertension, and (5) prevalence of the borderline lepromatous form of HD. However, the differences found when comparing other parameters, such as blood pressure, smoking, diabetes mellitus, total cholesterol, triglycerides, pre-beta and beta-lipoproteins, uric acid, erythrocyte sedimentation rate, ABO blood groups, etc., did not reach the level of significance. These findings suggest that HD sufferers are a special population subgroup with reference to CHD risk factors, differing in many ways from the general population.

Glucose tolerance test in leprosy.

Glucose tolerance test was carried out in 43 cases of leprosy. They included cases of tuberculoid, borderline, lepromatous leprosy and those with lepra reaction. It was observed that normal curve was common in tuberculoid leprosy. Flat glucose tolerance curve was observed in borderline and lepromatous leprosy. However, the diabetic curve was common in Lepra reaction. Fasting blood sugar was low in lepromatous leprosy and it tended to be marginally high in lepra reaction. Normal GTT response was observed in those with duration of disease between 0-6 months, flat curves in those with duration of disease between 7-12 months while diabetic curve was more common in those with disease duration of more than 2 years.

Hormone profile in leprosy.

Hormone profile were carried out in 35 male cases of leprosy. They were divided into tuberculoid leprosy, borderline leprosy, lepromatous leprosy and lepra reaction. Serum testosterone, follicle-stimulating hormone, Luteinizing hormone, and Oestradiol level were measured in these cases of leprosy. It was observed that serum testosterone were significantly low in lepromatous leprosy (P less than 0.001) and lepra reaction (P less than 0.01). The serum levels of follicle-stimulating hormone and luteinizing hormone were significantly high in lepromatous leprosy (P less than 0.02) and lepra reaction (P less than 0.05). Serum Oestradiol was raised in approx. 60% cases in borderline leprosy, lepromatous leprosy and lepra reaction.

Correlation of serum lipids and lipoproteins in leprosy.

Serum lipids and lipoproteins were assessed in sixty leprosy and forty age and sex matched healthy controls. The study subjects included cases of LL with reactions, LL without reactions, BL with reactions, BL without reactions, BT and TT types of leprosy. The levels of serum phospholipids, triglycerides, total cholesterol, LDL and VLDL fractions were significantly decreased in leprosy patients compared to control subjects. The levels of serum HDL cholesterol and HDL fraction were significantly elevated in leprosy patients. Maximum elevation in serum HDL cholesterol level and HDL fraction and maximum reduction in the levels of serum phospholipids, triglycerides, total cholesterol and LDL and VLDL fractions were observed in lepromatous leprosy (LL) patients with reactions.

Evaluation of circulating immune complexes in leprosy.

Circulating immune complexes (CIC) were evaluated in leprosy by 4 methods: the 125I-C1q binding assay (C1q), the platelet aggregation test (PAT), the 3.5% polyethylene glycol (PEG) precipitation test and the 2.5% PEG precipitation assay. Serum samples belonged to lepromatous leprosy bacilloscopy positive (LL+), lepromatous leprosy bacilloscopy negative (LL-), tuberculoid (TT) and first grade contact group (Co). Studies performed by the 3 first methods showed higher CIC levels in LL+ group (p less than 0.01) and lower values in the 3 others, all of them when compared to normals. On the contrary, the 2.5% PEG precipitation test gave less discriminative results giving only p less than 0.01 in LL+. CIC values obtained in the contact group showed significant results compared to normals but similar to LL- and TT groups. The C1q binding assay and the PAT were the most discriminative methods giving r = 0.90; C1q versus 3.5% PEG, r = 0.36; C1q vs 2.5% PEG, r = 0.14. The PAT compared to 3.5% PEG, r = 0.48 and PAT vs. 2.5% PEG, r = 0.24. Therefore it may be concluded as follows: a) The radioiodinated C1q binding assay and the PAT are recommended for the study of CIC in leprosy; b) The 2.5% PEG precipitation assay offers less sensitivity since it gave similar value in LL-, TT, Co and controls; c) CIC levels observed in LL+ patients may be induced by the antigenic overload demonstrated by the positive bacilloscopy; d) The contacts have CIC levels significantly different from the normal population possibly caused by a subclinical infection.

Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients.

Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5) with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5) of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively) compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively). The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1]) of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]). The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.

Seropositivity to anti-phenolic glycolipid-I in leprosy cases, contacts and no known contacts of leprosy in an endemic and a non-endemic area in northeast Brazil.

The seroprevalence rates of IgM anti-phenolic glycolipid-I (PGL-I) antibodies in four study groups with differing exposure to Mycobacterium leprae in Ceará, Brazil were investigated between March 2005 and August 2006. The first three groups in a high prevalence area included 144 cases of leprosy, their 380 contacts and 317 participants with no known leprosy contact. The fourth group in a low prevalence area consisted of 87 participants with no known leprosy contact living in an area in which no cases of leprosy had been reported in the previous 6 months. Seropositivity and levels of IgM antibodies to PGL-I were investigated using ELISA. The seropositivity levels of anti-PGL-I among the different clinical forms of leprosy cases were 61% for lepromatous, 25% for tuberculoid and 27% indeterminate. The levels of anti-PGL-I antibodies in the endemic area differentiated leprosy cases from non-cases. However, the seropositivity was similar among contact cases (15.8%) and no known leprosy contact cases from high (15.1%) and low (13.8%) prevalence areas. The seropositivity of both contacts and no known contacts was much higher than previously reported among no known contacts in other endemic areas. The study indicates that anti-PGL-I antibodies are not useful as immunological markers of household leprosy contacts and no known leprosy contacts in endemic areas.

The functional state of the complement system in leprosy.

Ninety-one patients with different clinical forms of leprosy, 36 lepromatous (LL), 33 tuberculoid (TL), and 22 dimorphic (DL), and 31 healthy volunteer donors were included in this study. Total complement system (CS) activity was assessed by hemolytic methods, whereas individual components were quantified by the enzyme-linked immunosorbent assay. Under conditions allowing initiation of cascade by the classic pathway (CP) but not alternative pathway (AP) activation, significant CS consumption was detected only in sera from patients with LL. In this group of patients, C4 but not factor B (fB) or C3 was significantly reduced, whereas mannose-binding lectin (MBL) serum levels were significantly higher. These results indicate that the CP is involved in CS activation in patients infected with Mycobacterium leprae manifesting LL clinical form of leprosy. An association is likely between circulating immune complexes and MBL high serum levels for initiation of CS activation in patients with LL form of leprosy.

Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients

Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5) with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5) of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively) compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively). The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1]) of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]). The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.