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.

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.

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.

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.

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.

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.

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

Use of protein microarrays to define the humoral immune response in leprosy patients and identification of disease-state-specific antigenic profiles.

Although the global prevalence of leprosy has decreased over the last few decades due to an effective multidrug regimen, large numbers of new cases are still being reported, raising questions as to the ability to identify patients likely to spread disease and the effects of chemotherapy on the overall incidence of leprosy. This can partially be attributed to the lack of diagnostic markers for different clinical states of the disease and the consequent implementation of differential, optimal drug therapeutic strategies. Accordingly, comparative bioinformatics and Mycobacterium leprae protein microarrays were applied to investigate whether leprosy patients with different clinical forms of the disease can be categorized based on differential humoral immune response patterns. Evaluation of sera from 20 clinically diagnosed leprosy patients using native protein and recombinant protein microarrays revealed unique disease-specific, humoral reactivity patterns. Statistical analysis of the serological patterns yielded distinct groups that correlated with phenolic glycolipid I reactivity and clinical diagnosis, thus demonstrating that leprosy patients, including those diagnosed with the paucibacillary, tuberculoid form of disease, can be classified based on humoral reactivity to a subset of M. leprae protein antigens produced in recombinant form.

Mannan-binding lectin plasma levels in leprosy: deficiency confers protection against the lepromatous but not the tuberculoid forms.

Mannan-binding lectin (MBL) is an important component of the first-line defence against infections. Evidence has shown that MBL deficiency, reducing phagocytosis and internalization of intracellular pathogens may protect the host against intracellular infections such as leprosy. In this study, we speculated whether genetically determined low MBL serum levels confer protection against Mycobacterium leprae infection. One hundred and ninety-one patients with leprosy, presenting lepromatous (n = 118), tuberculoid (n = 31), dimorph (n = 30) and indeterminate (n = 12) clinical forms and 110 healthy controls matched with the patients according to sex, age and ethnic background were investigated. MBL concentrations were measured in a double-antibody enzyme immune assay and C-reactive protein (CRP) serum levels by nephelometry. A significant negative association of MBL low values (< 100 ng/ml) was observed with lepromatous patients when comparing with controls and tuberculoid patients [10/118, 8.47%versus 21/110, 19.09%P = 0.03 chi(2) with Yates' correction, odds ratio (OR) 0.39, confidence interval (CI) 0.18-0.88 and 8/31, 25.81%, P = 0.02, OR 0.27, CI 0.09-0.75, respectively]. There was no significant difference in the distribution of MBL levels between patients and controls or among the clinical forms. The concentration of CRP was significantly increased in the patients (P = 0.0002) and in the lepromatous form (P = 0.0001) when compared to controls. A weak positive correlation between MBL and CRP levels was observed in the patients (P = 0.010, R = 0.255). These data suggest a protective role for MBL deficiency against the development of the most severe and multi-bacillary form of leprosy.

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.

Adrenocortical hormones and interleukin patterns in leprosy.

The functional status of adrenocortical hormones and their relationship to the pattern of inflammatory cytokines in the lepromatous and tuberculoid poles of leprosy were investigated. Interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha plasma levels, C-reactive protein (CRP) concentrations and erythrocyte sedimentation rates (ESR) were significantly higher in LL/BL (lepromatous) leprosy patients than in control subjects. There was a significant positive correlation between IL-6 and TNF-alpha plasma levels and ESR and CRP concentrations. IL-1beta was positively correlated with ESR but not with CRP. Both baseline and stimulated adrenocorticotropic hormone and cortisol plasma levels were not different between patients and control subjects. In contrast, adrenal androgen dehydroepiandrosterone sulphate (DHEA-S) plasma levels were significantly lower in leprosy patients than in sex-matched control subjects. There was a significant inverse correlation between DHEA-S and IL-6, TNF-alpha, and CRP concentrations. This finding may be of pathogenetic significance in this disease and in other inflammatory states.

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 transforming growth factor-beta 1 (TGF-beta1) by blood monocytes from patients with different clinical forms of leprosy.

In the present study, the concentration of TGF-beta1 secreted by adherent cells isolated from human peripheral blood mononuclear cells (PBMC) and either stimulated with PGL-1 or lipopolysaccharide (LPS) or left unstimulated was determined by ELISA. The cells were isolated from untreated patients with different clinical forms of leprosy and healthy individuals. The adherent cells exhibited spontaneous release of TGF-beta1 in all clinical forms of leprosy and in healthy individuals; however, lepromatous leprosy/borderline leprosy (LL/BL) patients presenting erythema nodosum leprosum (ENL) displayed significantly higher concentrations of TGF-beta1 than either the other patients studied or the controls. These high TGF-beta1 levels were consistently observed when LL/BL ENL cells were stimulated with phenolic glycolipid (PGL-1) or LPS, and even in the absence of a stimulus (P < 0.01). The most significant differences in TGF-beta1 levels were observed when comparing the results in the presence of PGL-1 from ENL with, in order of significance: tuberculoid leprosy (TT) patients (P < 0.001), LL/BL patients without ENL (P < 0.01), healthy individuals (P < 0.01) and borderline-borderline/borderline-tuberculoid (BB/BT) patients with reversal reaction (RR) (P < 0.01). The BB/BT patients produced equivalent levels of TGF-beta1 compared with LL/BL patients without ENL, for all types of stimuli (P > 0.05). In contrast, TT patients produced the lowest levels of TGF-beta1 among all the subjects studied (both patients and healthy controls), especially following PGL-1 stimulation (P < 0.001, and P < 0.05, respectively). In conjunction with our previous data regarding TGF-beta1 expression in dermal lesions, it appears that TGF-beta1 probably plays different roles in leprosy: (i) to mediate a suppressive action locally, associated with the presence of PGL-1, and (ii) to induce proinflammatory effects when secreted systemically by monocytes, thereby acting as a modulatory cytokine in the acute inflammatory reactions of ENL and associated with the Th2 immune response in multibacillary forms of leprosy.

IL-18 induction of IgE: dependence on CD4+ T cells, IL-4 and STAT6.

Overproduction of immunoglobulin E (IgE) and T helper cell type 2 (TH2) cytokines, including interleukin 4 (IL-4), IL-5 and IL-13, can result in allergic disorders. Although it is known that IL-4 is critical to the polarization of naïve CD4+ T cells to a TH2 phenotype, both in vitro and in many in vivo systems, other factors that regulate in vivo IL-4 production and TH2 commitment are poorly understood. IL-18, an IL-1-like cytokine that requires cleavage with caspase-1 to become active, was found to increase IgE production in a CD4+ T cells-, IL-4- and STAT6-dependent fashion. IL-18 and T cell receptor-mediated stimulation could induce naïve CD4+ T cells to develop into IL-4-producing cells in vitro. Thus, caspase-1 and IL-18 may be critical in regulation of IgE production in vivo, providing a potential therapeutic target for allergic disorders.

A study on the methods for early serological diagnosis of leprosy and their potential use.

This is a serial study. In this series we have established 12 methods for the early serological diagnosis of leprosy, including the FLA-ABS test, ELISAs with artificial products (ND-O-, ND-P-, NT-O-, NT-P-BSA; PGL-I, whole M. leprae and M. smegmatis), monoclonal antibody specific binding assay (McAb/SBA), latex agglutination test (LAT), and MLPA. These methods were compared with each other on a large scale in leprosy patients and in the field. The results indicate that 1) Excellent results were obtained when ELISAs were conducted with skim milk or egg albumin as the blocking agent and by using blood from earlobes instead of from venipuncture. 2) According to the four "S" standard (sensitivity, specificity, simplicity and speed), among the 12 methods the ND-O-BSA-ELISA (ND-ELISA) is the best and the MLPA is more suitable for use in the field because it is simple and rapid. 3) In the ND-ELISA, the increase or decrease of the OD value has a positive correlation with the BI, and the order of positive rates was a) in various types of leprosy: LL > BL > BB > BT > TT; b) in household contacts (HC), random population (RP), normal controls in endemic areas (ENC) and normal controls in nonendemic areas (NNC): HC > RP > ENC > NNC. 4) In a population with subclinical M. leprae infection, the highest risk group was between the ages of 15 and 25 and had an increase or a persistence of high OD values prior to onset of disease. 5) OD values gradually decreased over time following treatment and these declines paralleled declines in the BI. 6) In cases cured with dapsone therapy, there was an increase or a persistence of high OD values in ND-ELISA prior to the onset of a leprosy relapse. In conclusion, we have compared and evaluated 12 immuno-assays and have shown that the ND-ELISA is the most practical one for use in investigating sero-immunological epidemiology, subclinical infection with M. leprae, early detection of disease, monitoring of antimicrobial therapy, and even for the prediction of leprosy relapse.