Tuberculoid Patients Research Articles

Quantitative Real-Time Polymerase Chain Reaction for Detection of Mycobacterium leprae DNA in Tissue Specimens from Patients with Leprosy.

In leprosy, early diagnosis is crucial to prevent transmission and onset of disabilities of the disease. The purpose of this study was to determine usefulness of quantitative real-time polymerase chain reaction (PCR) in clinically diagnosed cases of leprosy. Thirty-two leprosy cases were included. The real-time PCR was performed using commercial kit targeting Mycobacterium leprae-specific insertion sequence element. The slit skin smear was positive in two (22.2%) borderline tuberculoid (BT) patients, five (83.3%) borderline lepromatous (BL) patients, and seven (50%) lepromatous leprosy (LL). The positivity of quantitative real-time PCR in BT, BL, LL, and pure neuritic leprosy were 77.8%, 83.3%, 100%, and 33.3%, respectively. Using histopathology as the gold standard, sensitivity of quantitative real-time PCR was 93.1%, and specificity was 100%. The DNA load was higher in LL (3,854.29/106 cells), followed by BL (140.37/106 cells), and BT (2.69/106 cells). Because of the high sensitivity and specificity of real-time PCR, our study strongly suggests the use of real-time PCR as a diagnostic tool for leprosy.

Mycobacterium leprae Induces Neutrophilic Degranulation and Low-Density Neutrophil Generation During Erythema Nodosum Leprosum.

Erythema Nodosum Leprosum (ENL) is a recurrent acute inflammatory complication of leprosy affecting up to 50% of all Borderline Lepromatous and Lepromatous Leprosy (BL/LL) patients. Although ENL is described as an immune reaction mediated by neutrophils, studies demonstrating the direct role of neutrophils in ENL are still rare. One subpopulation of low-density neutrophils (LDNs), present within the fraction of peripheral blood mononuclear cells (PBMC), has been associated with the pathogenesis and severity of diseases like sepsis, lupus, and tuberculosis. We herein analyzed LDNs and high-density neutrophils (HDNs) in terms of frequency, phenotype, and morphology. Serum levels of MMP-9 (a neutrophilic degranulation marker) were evaluated by ELISA; and LDNs were generated in vitro by stimulating healthy-donor, whole-blood cultures. PBMC layers of ENL patients presented segmented/hypersegmented cells that were morphologically compatible with neutrophils. Immunofluorescence analyses identified LDNs in ENL. Flow cytometry confirmed the elevated frequency of circulating LDNs (CD14−CD15+) in ENL patients compared to healthy donors and nonreactional Borderline Tuberculoid (BT) patients. Moreover, flow cytometry analyses revealed that ENL LDNs had a neutrophilic-activated phenotype. ENL patients under thalidomide treatment presented similar frequency of LDNs as observed before treatment but its activation status was lower. In addition, Mycobacterium leprae induced in vitro generation of LDNs in whole blood in a dose-dependent fashion; and TGF-β, an inhibitor of neutrophilic degranulation, prevented LDNs generation. MMP-9 serum levels of BL/LL patients with or without ENL correlated with LDNs frequency at the same time that ultrastructural observations of ENL LDNs showed suggestive signs of degranulation. Together, our data provide new insights into the knowledge and understanding of the pathogenesis of ENL while enriching the role of neutrophils in leprosy.

Leprosy patients with deformities at post-elimination stage: The Bangladesh experience.

BackgroundDisability assessment in leprosy patients is a very important factor in the evaluation of the effectiveness of a leprosy elimination program. Little information exists on deformities in leprosy patients in Bangladesh.ObjectivesTo describe the pattern and prevalence of deformities in leprosy patients after leprosy has been declared eliminated from Bangladesh in 1998.MethodsA descriptive retrospective cross‐sectional study was carried out in Chittagong Medical College Hospital using the registered records of patients for the period 2004–2013.ResultsOut of 670 leprosy patients, 213 (31.79%) had deformities. The prevalence of deformity was for grade 1: 92 (43.20%), for grade 2: 121 (56.80%). Among the patients with deformity, males 144 (67.60%) outnumbered females 69 (32.40%). Four age groups were considered. The calculated age‐specific cumulative detection rates showed the highest case detection in >40 years group at 81 (38.02%). The rate of children (<14 years) was less at 7 (3.29%). Of the 213 patients with deformity, the borderline tuberculoid patients were totalled 79 (37.08%), which was higher than other forms of leprosy. Among the 121 patients with limb deformity, 57 (8.50%) had claw hand followed by wrist drop 31 (4.63%), foot drop 30 (4.48%). Three (0.45%) had a nerve abscess, 27 (4.02%) had a trophic ulcer and 7 (1.05%) patients had ocular complications.ConclusionThe grade 2 deformities among newly detected leprosy patients were still high. Claw hand was the most common deformity in the upper limbs, whereas foot drop and trophic ulcer were the most common deformities in the lower limbs. Although leprosy according to the World Health Organization has been eliminated globally, the disease continues to be a significant cause of peripheral neuropathy, deformity, disability and disfigurement in some developing countries like Bangladesh.

Role of Histopathology in Predicting type 1 Lepra Reaction in Borderline Tuberculoid Leprosy.

Context:Lepra reactions if not managed promptly are an important cause of sudden onset nerve palsy and disability due to leprosy.Aim:To evaluate the usefulness of histology in predicting type 1 lepra reaction.Setting and Design:After obtaining clearance from institutional research and ethics committees, all histologically proven borderline tuberculoid patients diagnosed at our center from 1.8.2016 to 31.7.2018 were included in this retrospective cross-sectional study.Method:Clinical details were collected from patient records. The pathologist who was blinded to clinical evidence of type 1 lepra reaction at the time of biopsy re-evaluated the histopathology slides for evidence of type 1 reaction. The data of individual patient was analyzed to identify those who had a type 1 reaction at the time of the biopsy or who developed a lepra reaction during follow up.Statistical Analysis Used:Association between histological evidence of type 1 reaction and clinical manifestation of the same subsequently, was assessed using Pearson's Chi square test.Results:Study group comprised of 22 females and 18 males. Clinicohistological concordance was noted in 27 patients (67.5%). Subclinical type 1 reaction was documented in 11 patients (27.5%) based on histopathology evaluation. Five (45.5%) of these 11 patients subsequently developed clinical features of type 1 reaction. This was found to be statistically significant (P value 0.02).Limitations:Main limitation was the small sample size.Conclusions:Histology could serve as a useful tool in predicting future type 1 lepra reaction.

Can anti–PGL-1 and anti–NDO-LID-1 antibody titers be used to predict the risk of reactions in leprosy patients?

Leprosy patients may present reactional episodes classified as type I or reversal reaction and type II or erythema nodosum leprosum. Early diagnosis of these reactions is hampered by lack of diagnostic tests. This study aimed at evaluating anti–Mycobacterium leprae antibody levels in reactional and nonreactional leprosy patients at the time of diagnosis. Clinical data and serum samples of 224 patients diagnosed between 2009 and 2010 were collected in the municipality of Rondonópolis-MTBR. Quantification of anti–phenolic glycolipid-1 (PGL-1) IgM antibodies of M. leprae was obtained by the enzyme-linked immunosorbent assay method and anti–natural octyl disacharide-leprosy IDRI diagnostic (NDO-LID-1) IgM/IgG semiquantitative rapid test. We obtained low serological levels of anti–PGL-1 and anti–NDO-LID-1 for tuberculoid (T) (1.56% and 15.62%) and borderline tuberculoid (BT) patients (7.95% and 26.13%), medium levels in the borderline-borderline (BB) (47.91% and 68.75%), and high levels in lepromatous (LL) (93.33% and 100%) and borderline-lepromatous (BL) (88.0% and 100%). When comparing the reactional groups (RI and RII) with without reaction (WR) group at the time of diagnosis, we observed a statistically significant difference between the groups; patients with RII presented higher serological response: 66.66% anti–PGL-1 and 91.66% anti–NDO-LID-1. In respect to patients who developed a reaction after the initial diagnosis, they also showed significant positivity for both anti–PGL-1 and anti–NDO-LID-1 in comparison to the patients who stayed without reaction in the study period (P<0.0001). These results allow us to conclude that serological tests may contribute to an early diagnosis of RII and that the anti–NDO-LID-1 test was demonstrated to be a better indicator.

Characterization of MicroRNA Expression Profiles and Identification of Potential Biomarkers in Leprosy.

Leprosy is an important cause of disability in the developing world. Early diagnosis is essential to allow for cure and to interrupt transmission of this infection. MicroRNAs (miRNAs) are important factors for host-pathogen interaction and they have been identified as biomarkers for various infectious diseases. The expression profile of 377 microRNAs were analyzed by TaqMan low-density array (TLDA) in skin lesions of tuberculoid and lepromatous leprosy patients as well as skin specimens from healthy controls. In a second step, 16 microRNAs were selected for validation experiments with reverse transcription-quantitative PCR (qRT-PCR) in skin samples from new individuals. Principal-component analysis followed by logistic regression model and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic potential of selected miRNAs. Four patterns of differential expression were identified in the TLDA experiment, suggesting a diagnostic potential of miRNAs in leprosy. After validation experiments, a combination of four miRNAs (miR-101, miR-196b, miR-27b, and miR-29c) was revealed as able to discriminate between healthy control and leprosy patients with 80% sensitivity and 91% specificity. This set of miRNAs was also able to discriminate between lepromatous and tuberculoid patients with a sensitivity of 83% and 80% specificity. In this work, it was possible to identify a set of miRNAs with good diagnostic potential for leprosy.

Increased expression of interleukin-17A in the lesional skin indicates increase of serum antibody anti-phenolic glycolipid-I in leprosy patients

Background: In lepromatous type of leprosy, the serum titer of the antibody against phenolic glycolipid (PGL)-I-a-specific antigen in leprosy that is produced by B cell is high. Whereas, the interleukin (IL)-17A expressed by T-helper 17 cells could induce B-cell differentiation. The role of IL-17A in leprosy is still unknown. Hence, this study aimed to assess a correlation between IL-17A expression in the lesional skin and anti-PGL-I immunoglobulin (Ig) M serum levels in leprosy patients. Methods: This study was performed in Leprosy Clinic, Dr. Hasan Sadikin Hospital Bandung, Indonesia, using a cross-sectional analytical study. A punch biopsy obtained from 49 leprosy patients was included through consecutive sampling for measurement of IL-17A expression in the skin by immunohistochemistry scoring (histoscore). Furthermore, the anti-PGL-I IgM level in serum is evaluated by enzyme-linked immunosorbent assay. Results: The IL-17A expressions in the skin biopsies of tuberculoid (TT), borderline tuberculoid (BT), mid-borderline (BB), borderline lepromatous (BL), and lepromatous leprosy (LL) patients using histoscore were 1.00, 2.31, 4.63, 5.06, and 10.14, respectively, and they showed significant differences ( P = 0.0001). The titer of anti-PGL-I IgM levels was 89 pg/ml, 555 pg/ml, 1.244 pg/ml, 1.920 pg/ml, and 23.591 pg/ml in TT, BT, BB, BL, and LL patients, respectively, and they showed significant differences ( P = 0.005). The results of Rank–Spearman correlation analysis between IL-17A expression in the skin and anti-PGL-I IgM serum levels were as follows: r = 0.767 and P = 0.0001. Conclusion: These results suggested that the increase of IL-17A expression in the lesional skin indicates the increase of anti-PGL-I IgM serum levels in leprosy.

180: Elucidation and characterization of CD4+ TCRγδ+ FoxP3+ type of immune-suppressive cells with the progression of leprosy

Leprosy is one of the dreaded infectious diseases haunting the mankind for older ages. Owing to tremendous scientific advancements we now gradually understand the dysregulation of immunological networks in driving the pathogenesis of leprosy. As we move from borderline, tuberculoid to lepromatous patients we found a gradual increase in the proportion of Mycobacterium leprae specific CD4+ TCRγδ+ cells. Interestingly, CD4+ TCRγδ+ cells isolated from lepromatous patients significantly reduced the proliferation of CD4+ CD25− cells in co-culture experiments draw out attention towards the immune- suppressive nature of these cells. Furthermore, we have also evaluated TGF-β mediated SMAD3 signaling and induction of FoxP3 inside these cells. We observed comparatively increased nuclear ingress of SMAD3 in lepromatous patients as compared to SMAD7 in tuberculoid patients. Thus, the induction of FoxP3 in CD4 + TCRγδ+ was found to associate with the severity of disease. To establish this molecular event further, we silenced SMAD3 in these cells in the culture. A remarkable reduction of FoxP3 and a concomitant increase in the proliferation of CD4+ CD25− cells in co-culture proved the operation of TGF-β mediated SMAD3 signaling towards the induction of FoxP3 in these cells. This study for the first time reveals the role of CD4+ TCRγδ+ cells in the progression of disease.This study added a new dimension of immune suppression with the progression of leprosy. Also, for the first time we have explored the Th3 dependent TGF-β induced SMAD3 signaling inside CD4+ TCRγδ+ cells with the progression of leprosy.

A rapid ELISA for the diagnosis of MB leprosy based on complementary detection of antibodies against a novel protein-glycolipid conjugate

Despite the widespread use of multidrug therapy for treatment, delays in clinical recognition and under-reporting of leprosy indicate that Mycobacterium leprae transmission is continuing. Thus, leprosy is likely to persist as a significant burden on health systems in many regions. In this study, we combined 2 previously characterized leprosy antigens, leprosy IDRI diagnostic-1 (LID-1) and ND-O, into the single fusion complex (ND-O–LID) and determined the serum antibody responses of leprosy patients from Colombia and the Philippines. Following confirmation that antibodies recognized each component within the conjugate, we assessed the performance of a rapid enzyme-linked immunosorbent assay (ELISA) system (Leprosy DetectTM fast ELISA; InBios International, Inc., Seattle, WA, USA) based on ND-O–LID capable of generating results within 1.5hours of sample addition. We found ELISA results correlated with the bacteriological index and Ridley–Jopling categorization, with lepromatous leprosy patients having the highest responses, while those of borderline tuberculoid patients were lower. Multibacillary (MB) leprosy patients were distinguished with a high degree of sensitivity (95.7%) and specificity (93.2%), suggesting that this ELISA could potentially replace invasive and insensitive skin slit smear procedures that require expert microscopic examinations. Due to the speed and robustness of this assay, we believe this is an excellent tool for detecting MB leprosy patients in a simple and highly-quantitative manner.

Metabonomics Reveals Drastic Changes in Anti-Inflammatory/Pro-Resolving Polyunsaturated Fatty Acids-Derived Lipid Mediators in Leprosy Disease

Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases.

MicroRNAs induced during Mycobacterium leprae infection and their effects on immune response (P3019)

Abstract MicroRNAs have been shown to be crucial in regulation of immune cell development and cancer progression. Recently, it has become increasingly clear that their involvement in regulation of immune responses to infection is also important. Here we use Mycobacterium leprae, the causative agent of leprosy, as a model to determine the potential roles of microRNAs in infectious disease. The immune response to M. leprae has been well-characterized as presenting as a spectrum, ranging from a contained infection characterized by a Th1 response in tuberculoid (T-lep) patients to a disseminated infection reflecting a Th2 response in lepromatous (L-lep) patients. We performed mRNA and microRNA microarray analysis on 6 T-lep and 4 L-lep lesions and subsequently integrated this data using Ingenuity. Preliminary gene network analysis of differentially expressed microRNAs in L-lep versus T-lep yielded multiple networks potentially targeted, including antigen presentation. A supervised analysis of Th1 and Th2 gene targeting indicates that microRNAs more highly expressed in L-lep lesions, such as miR-21 and miR-146a, may preferentially target proinflammatory genes required for a Th1 immune response. Further analysis to determine the functional effects of these microRNAs and their involvement in skewing towards a Th2 immune response to infection could explain the bias in disease outcome.

Differential expression of the costimulatory molecules CD86, CD28, CD152 and PD-1 correlates with the host-parasite outcome in leprosy

Leprosy is a spectral disease exhibiting two polar sides, namely, lepromatous leprosy (LL) characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. We studied the influence of costimulatory molecules on the immune responses of subjects along the leprosy spectrum. The expression of the costimulatory molecules was evaluated in in vitro-stimulated peripheral blood mononuclear cells of lepromatous and tuberculoid patients and healthy exposed individuals (contacts). We show that LL patients have defective monocyte CD86 expression, which likely contributes to the impairment of the antigen presentation process and to patients anergy. Accordingly, CD86 but not CD80 blockade inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the LL anergy, there was reduced expression of the positive signalling costimulatory molecules CD28 and CD86 on the T-cells in these patients. In contrast, tuberculoid leprosy patients displayed increased expression of the negative signalling molecules CD152 and programmed death-1 (PD-1), which represents a probable means of modulating an exacerbated immune response and avoiding immunopathology. Notably, the contacts exhibited proper CD86 and CD28 expression but not exacerbated CD152 or PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signalling.

Increased expression of regulatory T cells and down-regulatory molecules in lepromatous leprosy.

T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-γ (IFN-γ) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-β (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-β. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.

Role of microRNAs in immune response to infection (112.18)

Abstract Regulation of microRNAs has been shown to be critical in immune cell development as well as the progression of certain cancers. However, their role in immune response to microbial infection has remained elusive. We chose to use Mycobacterium leprae, the causative agent of leprosy, as a model to determine the potential roles of microRNA in pathogenesis. The immune response to M. leprae has been well-characterized as presenting as a spectrum, ranging from a more contained Th1 response in tuberculoid patients to a more disseminated Th2 response in lepromatous patients, providing a good system for examining the correlation of microRNAs and type of immune response. We performed mRNA and microRNA microarray analysis on 6 T-lep and 6 L-lep lesions to determine whether microRNA disregulation and subsequent downregulation of key immune genes contributes to the outcome and morbidity of infection. We discovered 7 microRNAs in T-lep and 28 microRNAs in L-lep lesions and cross-referenced them in MiRNA and Genes Integrated Analysis (MAGIA) web tool with mRNA gene transcripts that were significantly downregulated in the same lesions. Preliminary analysis of Th1 and Th2 gene targeting shows microRNAs more highly expressed in L-lep lesions may preferentially target proinflammatory genes, which could explain the bias in immune response to infection.

Lsr2 Peptides of Mycobacterium leprae Show Hierarchical Responses in Lymphoproliferative Assays, with Selective Recognition by Patients with Anergic Lepromatous Leprosy

Lsr2 protein of Mycobacterium leprae was shown earlier to elicit B and T cell responses in leprosy patients (20, 28). Lymphoproliferation to M. leprae and Lsr2 antigens was observed in >70% of tuberculoid (T) patients and in 16 and 34% of lepromatous (L) patients, respectively. We focused on the M. leprae nonresponders in the lepromatous group using 22 synthetic Lsr2 peptides (end-to-end peptides A to F and overlapping peptides p1 to p16) in in vitro T cell responses. A total of 125 leprosy and 13 tuberculosis patients and 19 healthy controls from the area of endemicity (here, healthy controls, or HC) were investigated. The highest responses were observed (67 to 100%) in HC for all peptides except p1 to p3, and the lowest was observed in tuberculosis patients. Significant differences in lymphoproliferation were observed in T, L, and HC groups (analysis of variance [ANOVA], P = 0.000 to 0.015) for all end-to-end peptides except B and for p5 and p7 to p10. Hierarchical recognition between lepromatous and tuberculoid leprosy was noted for p8 (P < 0.05) and between the HC and L groups for p7 to p10, p15, and p16 (P < 0.005 to P < 0.02). Significant lymphoproliferation was observed to peptides A to F and p1 to p9, p11, p12, p15, p16 (P = 0.000 to 0.001) with 40% responding to peptides C and p16 in L patients. Lepromatous patients also showed significantly higher levels of a gamma interferon (IFN-γ) response to peptide C than to other peptides (P < 0.05). Major histocompatibility complex (MHC) class II bias for peptide recognition was not observed. These studies indicate that Lsr2 has multiple T cell epitopes that induce in vitro T cell responses in the highly infective lepromatous leprosy patients.

Class-I human leukocyte alleles in leprosy patients from Southern Brazil

The present study was designed to investigate a possible role of HLA (histocompatibility leucocyte antigen) class-I alleles (HLA-A, -B, and -C) in leprosy patients from Southern Brazil. Two hundred and twenty-five patients with leprosy and 450 individuals for the control group were involved in this research. HLA genotyping was performed through PCR-SSO protocols (One Lambda, USA); the frequency of these alleles was calculated in each group by direct counting, and the frequencies were then compared. There was an association between HLA-A*11 (6.9% vs 4.1%, p=0.0345, OR=1.72, 95% CI=1.05-2.81), HLA-B*38 (2.7% vs. 1.1%, p=0.0402, OR=2.44, 95% CI=1.05-5.69), HLA-C*12 (9.4% vs. 5.4%, p=0.01, OR=1.82, 95% CI=1.17-2.82), and HLA-C*16 (3.1% vs. 6.5%, p=0.0124, OR=0.47, 95% CI=0.26-0.85) and leprosy per se. In addition, HLA-B*35, HLA-C*04, and HLA-C*07 frequencies were different between lepromatous (LL) and tuberculoid (TT) patients. However, after adjusting for the number of alleles compared, Pc values became nonsignificant. Although our results do not support the previous findings that HLA class-I alleles play a role in leprosy pathogenesis, we suggest new studies because of the importance of the association between the HLA and KIR in the innate immune response to leprosy.

The role of indoleamine 2, 3-dioxygenase in lepromatous leprosy immunosuppression

To elucidate further the possible role of the tryptophan, rate-limiting enzyme indoleamine 2, 3-dioxygenase (IDO) in leprosy, the distribution of IDO-positive cells and IDO activity in the skin biopsies and sera of these patients representing the entire spectrum of the disease were studied. An increased number of macrophages/dendritic cells (DC-lineage IDO(+) cells were found in lepromatous (LL) compared to tuberculoid (BT) and reversal reaction (RR) patients. IDO-positive cells showing CD68 and CD86 surface markers predominated in LL lesions, while higher levels of IDO activity were observed in the sera of LL versus BT patients. Tests revealed an increased IDO message in Mycobacterium leprae-stimulated peripheral blood mononuclear cells (PBMC) by real-time polymerase chain reaction (PCR) and increased IDO expression in M. leprae-stimulated CD14(+) cells of both healthy controls (HC) and LL patients, as evaluated via flow cytometry. Increased M. leprae-induced IDO-protein synthesis was also confirmed by Western blot. Based on our in vitro studies, it was confirmed that M. leprae up-regulated IDO expression and activity in HC and LL monocytes. Interferon (IFN)-γ synergized with M. leprae in promoting IDO expression and activity in monocytes. IDO expression induced by both IFN-γ and M. leprae was abrogated by 1-methyltryptophan (1-MT). Our data suggest that M. leprae chronic infection activates the suppressive molecule IDO which, in turn, contributes to the specific immunosuppression observed in LL leprosy.

Specific IgG antibody responses may be used to monitor leprosy treatment efficacy and as recurrence prognostic markers

Although curable, leprosy requires better diagnostic and prognostic tools to accompany therapeutic strategies. We evaluated the serum samples of leprosy patients from Venezuela and Brazil for reactivity against the specific recombinant proteins, ML0405 and ML2331, and the LID-1 fusion protein that incorporates both of these antigens. Antigen-specific IgG was highest in lepromatous leprosy patients (LL) and decreased across the disease spectrum, such that only a small subset of true tuberculoid patients (TT) tested positive. The impact of multidrug therapy (MDT) on these antibody responses was also examined. Several years after treatment, the vast majority of Venezuelan patients did not possess circulating anti-LID-1, anti-ML0405, and anti-ML2331 IgG, and the seropositivity of the remaining cases could be attributed to irregular treatment. At discharge, the magnitude and proportion of positive responses of Brazilian patients against the proteins and phenolic glycolipid (PGL)-I were lower for most of the clinical forms. The monthly examination of IgG levels in LL patient sera after MDT initiation indicated that these responses are significantly reduced during treatment. Thus, responses against these antigens positively correlate with bacillary load, clinical forms, and operational classification at diagnosis. Our data indicate that these responses could be employed as an auxiliary tool for the assessment of treatment efficacy and disease relapse.

MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy (147.12)

Abstract Leprosy is a human disease in which the clinical manifestations correlate with the host immune response to the pathogen, Mycobacterium leprae. In tuberculoid (T-lep) patients the infection is localized and Th1 cytokines predominate in lesions; whereas, in lepromatous patients (L-lep) the infection is disseminated and Th2 cytokines predominate. Evidence also suggests a role for the vitamin D-dependent antimicrobial pathway in contributing to host defense in leprosy. By profiling miRNAs in leprosy lesions, we identified 13 miRNAs differentially expressed in L-lep vs. T lep lesions. Bioinformatic analysis revealed a significant enrichment of L-lep-specific miRNAs that preferentially target genes important in T-lep lesions, including twelve Th1 and antimicrobial pathway genes. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in M. leprae-infected monocytes and induced by the mycobacterial virulence factor phenolic glycolipid-I (PGL-I). Hsa-mir-21, by downregulating Toll-like receptor induced CYP27B1 and IL1B and upregulating IL-10, inhibited the vitamin D-dependent antimicrobial peptide gene expression. In contrast, knockdown of hsa-mir-21 in M. leprae-infected monocytes enhanced antimicrobial peptide gene expression. The ability of the pathogen to upregulate hsa-mir-21 which targets multiple genes associated with the immunologically localized disease form provides an effective mechanism to escape the vitamin D antimicrobial pathway.

Analysis of apoptosis and Bcl-2 expression in polar forms of leprosy

Apoptosis eliminates pathogen-infected cells. Its modulation can influence the course of infections, permitting the survival of intracellular pathogens. In leprosy, which presents several clinical manifestations related to bacillary burden and host immune status, the mechanisms responsible for the persistence of the bacillus are unknown. Few studies have focused on apoptosis over the disease spectrum and as a defense mechanism against Mycobacterium leprae. We evaluated apoptosis using terminal transferase dUTP nick end labeling and the expression of Bcl-2 by immunohistochemistry in skin lesions from 11 tuberculoid and 15 lepromatous leprosy patients. Each specimen was evaluated by determining the number of positive cells in 10 fields at × 400 magnification. We observed a higher number of apoptotic cells in tuberculoid lesions in comparison with lepromatous leprosy (42.5 cells per 10 fields vs. 11.5 cells per 10 fields, P<0.0001). Expression of Bcl-2, conversely, was larger in lepromatous than in tuberculoid samples (172.0 cells per 10 fields vs. 17.7 cells per 10 fields, P<0.0001). These observations suggest modulation of apoptosis in leprosy, primarily in lepromatous patients, for which the decrease in cell death could support M. leprae survival and contribute to the success of infection. Conversely, in tuberculoid patients, apoptosis could contribute to reducing propagation of the bacillus.