Functional biomarker signatures of circulating T-cells and its association with distinct clinical status of leprosy patients and their respective household contacts.

BACKGROUND: Leprosy is a chronic infectious disease classified into two subgroups for therapeutic purposes: paucibacillary (PB) and multibacillary (MB), closely related to the host immune responses. In this context it is noteworthy looking for immunological biomarkers applicable as complementary diagnostic tools as well as a laboratorial strategy to follow-up leprosy household contacts. METHODS: The cross-sectional study enrolled 49 participants, including 19 patients and 30 healthy controls. Peripheral blood mononuclear cells (PBMC) were isolated and incubated in the presence of Mycobacterium leprae bacilli. The cells were prepared for surface (CD4+ and CD8+) and intracytoplasmic cytokine staining (IFN-γ, IL-4 and IL-10). Multiple comparisons amongst groups were carried out by ANOVA, Kruskal-Wallis, Student T or Mann-Whitney test. Comparative analysis of categorical variables was performed by Chi-square. Functional biomarker signature analysis was conducted using the global median values for each biomarker index as the cut-off edge to identify the proportion of subjects with high biomarker levels. RESULTS: The cytokine signature analysis demonstrated that leprosy patients presented a polyfunctional profile of T-cells subsets, with increased frequency of IFN-γ+ T-cell subsets along with IL-10+ and IL-4+ from CD4+ T-cells, as compared to health Controls (Venn diagram report). Moreover, statistical analysis was carried out using parametric or non-parametric variance analysis followed by pairwise multiple comparisons, according to the data normality distribution. L(PB) displayed a polyfunctional profile characterized by enhanced percentage of IFN-γ+, IL-10+ and IL-4+ produced by most T-cell subsets, as compared to L(MB) that presented a more restricted cytokine functional profile mediated by IL-10+ and IL-4+ T-cells with minor contribution of IFN-γ produced by CD4+ T-cells. Noteworthy was that HHC(MB) exhibited enhanced frequency of IFN-γ+ T-cells, contrasting with HHC(PB) that presented a cytokine profile limited to IL-10 and IL-4. CONCLUSIONS: Our data demonstrated that L(PB) displayed enhanced percentage of IFN-γ+, IL-10+ and IL-4+ as compared to L(MB) that presented functional profile mediated by IL-10+ and IL-4+ T-cells and HHC(MB) exhibited enhanced frequency of IFN-γ+ T-cells, contrasting with HHC(PB). Together, our findings provide additional immunological features associated with leprosy and household contacts. These data provide evidence that biomarkers of immune response can be useful complementary diagnostic/prognostic tools as well as insights that household contacts should be monitored to access putative subclinical infection.

Association of IL-10 Gene Polymorphism With IL-10 Secretion by CD4 and T Regulatory Cells in Human Leprosy.

Leprosy is a chronic bacterial disease caused by Mycobacterium leprae. Cytokines are known to play vital role as a peacekeeper during inflammatory and other immunocompromised conditions such as leprosy. This study has tried to bridge the gap of information on cytokine gene polymorphisms and its potential role in the pathogenesis of leprosy. Interleukin-10 (IL-10) is an immunosuppressive cytokine, found to be elevated in leprosy that accounted for the suppression of host's immune system by regulating the functions of other immune cells. T helper cells and T regulatory (Tregs) cells are the major source of IL-10 in lepromatous leprosy patients. In this study, we have documented the association of IL-10 cytokine gene polymorphism with the disease progression. A total of 132 lepromatous leprosy patients and 120 healthy controls were analyzed for IL-10 cytokine gene polymorphisms using PCR-SSP assay and flow cytometry was used to analyze IL-10 secretion by CD4 and Tregs in various genotype of leprosy patients. The frequencies of IL-10 (-819) TT and IL-10 (-1082) GG genotypes were significantly higher in leprosy patients as compared to healthy controls. This observation advocates that these genotypes were associated with the susceptibility and development of the disease. In addition, flow cytometry analysis demonstrated an increased number of IL-10 producing CD4 and Treg cells in IL-10 (819) TT genotype compared to CT and CC genotypes. These observations were further supported by immunohistochemical studies. Therefore, we can conclude that IL-10 cytokine gene polymorphisms by affecting its production can determine the predilection and progression of leprosy in the study population.

A distinct double positive IL-17A+/F+ T helper 17 cells induced inflammation leads to IL17 producing neutrophils in Type 1 reaction of leprosy patients.

Type 1 reactions (T1R) an inflammatory condition, of local skin patches in 30-40% leprosy patients during the course of MDT. IL-17A and IL-17F play an important role in regulating skin inflammation through neutrophils. In the present study, we have analyzed 18 of each T1R and Non-reactions (NR) patients through flow cytometry and qPCR. Interestingly we found that, CD3+CD4+ gated IL-17A+IL-17F+ cells were significantly high in T1R in both MLSA stimulated PBMCs and skin lesions as compared to NR leprosy patients. Hierarchical clustering analysis of gene expression showed that CXCL6, CXCL5, CCL20, CCL7, MMP13 and IL-17RB expression were significantly associated with IL-17A and IL-17F expression (Spearman r2 = 0.77 to 0.98), neutrophils and monocyte markers respectively. In this study, the inflammation noted in lesions of T1R is a different phenotype of Th17 which produce double positive IL-17A+IL17F+ and also contributes IL-17 producing neutrophils and thus would be useful for monitoring, diagnosis and treatment response before reactions episodes.

Evaluation of monocyte subsets and markers of activation in leprosy reactions.

Understanding host immune pathways associated with tissue damage during reactions are of upmost importance to the development of immune intervention strategies. The participation of monocytes in leprosy reactions was evaluated by determining the frequency of monocyte subsets and the degree of cellular activation through the expression of MHCII and the co-stimulatory molecules CD40, CD80, CD86. Leprosy subjects with or without reactions were included in this cross-sectional study. Peripheral blood mononuclear cell were isolated and stained ex vivo to determine monocyte subsets and the degree of cellular activation by flow cytometry. Intermediate monocytes were increased in leprosy patients with reactions when compared to patients without reactions. Although no difference was detected in the frequency of monocyte subsets between type 1 and 2 reactions, the expression of CD80 was increased in monocytes from patients with type 1 reactions and CD40 was higher in paucibacillary subjects presenting type 1 reactions. Moreover, CD86 and MHC II expression were higher in intermediate monocytes when compared to the other subsets in leprosy reaction types 1 and 2. Intermediate monocyte activation with CD86 and MHCII expression is involved with both type 1 and 2 reactions, whereas CD80 and CD40 expression is related to type 1 reactions.

Autophagy Impairment Is Associated With Increased Inflammasome Activation and Reversal Reaction Development in Multibacillary Leprosy.

Leprosy reactions are responsible for incapacities in leprosy and represent the major cause of permanent neuropathy. The identification of biomarkers able to identify patients more prone to develop reaction could contribute to adequate clinical management and the prevention of disability. Reversal reaction may occur in unstable borderline patients and also in lepromatous patients. To identify biomarker signature profiles related with the reversal reaction onset, multibacillary patients were recruited and classified accordingly the occurrence or not of reversal reaction during or after multidrugtherapy. Analysis of skin lesion cells at diagnosis of multibacillary leprosy demonstrated that in the group that developed reaction (T1R) in the future there was a downregulation of autophagy associated with the overexpression of TLR2 and MLST8. The autophagy impairment in T1R group was associated with increased expression of NLRP3, caspase-1 (p10) and IL-1ß production. In addition, analysis of IL-1ß production in serum from multibacillary patients demonstrated that patients who developed reversal reaction have significantly increased concentrations of IL-1ß at diagnosis, suggesting that the pattern of innate immune responses could predict the reactional episode outcome. In vitro analysis demonstrated that the blockade of autophagy with 3-methyladenine (3-MA) in Mycobacterium leprae-stimulated human primary monocytes increased the assembly of NLRP3 specks assembly, and it was associated with an increase of IL-1ß and IL-6 production. Together, our data suggest an important role for autophagy in multibacillary leprosy patients to avoid exacerbated inflammasome activation and the onset of reversal reaction.

ISG15-Induced IL-10 Is a Novel Anti-Inflammatory Myeloid Axis Disrupted during Active Tuberculosis.

IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.

Determination of Lipoprotein Z-Specific IgA in Tuberculosis and Latent Tuberculosis Infection.

Tuberculosis (TB) remains one of the most severe infectious diseases. It is still of paramount importance to establish more accurate, rapid, and efficient diagnostic methods. Since infection with Mycobacterium tuberculosis (M. tb) is largely mediated through the respiratory tract, IgA responses against mycobacterial proteins are worthy of investigation for their potential clinical utility. In this study, the IgA response targeting lipoprotein Z (LppZ) was determined by using a homemade ELISA with plasma of TB patients (N = 125), LTBI individuals (N = 92), healthy controls (HCs) (N = 165), as well as TB patients undergoing anti-TB treatment (N = 9). In parallel the antigen-specific IFN-γ release from PBMCs triggered by LppZ and M. tb-specific ESAT-6 or CFP-10 was detected by using an ELISPOT assay. It was found that the LppZ-specific IgA level was dramatically higher in TB patients than in HCs (p < 0.0001). Compared to that before anti-TB treatment, the LppZ-specific IgA level decreased substantially after 2 months of anti-TB treatment (p = 0.0297) and remained at low levels until the end of the treatment. What is more, pulmonary TB patients exhibited significantly higher LppZ-specific IgA-values than extra-pulmonary TB patients (p = 0.0296). Interestingly, the LppZ-specific IgA-values were negatively correlated to the amounts of IFN-γ released in response to LppZ with statistical significance (r = -0.5806, p = 0.0002). LppZ-specific IgA level was also higher in LTBI individuals than in HCs (p < 0.0001). Additionally there were some PPD+ HC individuals with high LppZ-specific IgA levels but the potential of this assay for identifying leaky LTBI in PPD+ HCs needs to be further investigated through follow-up studies. The sensitivity of detecting TB solely with ESAT-6 or CFP-10-specific IFN-γ release was increased by including the LppZ-specific IgA results, respectively, from 86.11 to 100% and 88.89 to 100%; the sensitivity of screening for LTBI was increased from 80.36 to 83.93% and 57.14 to 69.64%, respectively. The higher LppZ-specific IgA responses in TB and LTBI populations than in controls indicated high immunoreactivity to LppZ upon M. tb infection. Although the assay was not efficient enough for independent application in sero-diagnosis, LppZ-specific IgA might become a complementary biomarker for the improvement of TB and LTBI screening.

Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients.

Previously we showed that 65-kDa Mycobacterium leprae heat shock protein (Hsp65) is a target for the development of a tuberculosis vaccine. Here we evaluated peripheral blood mononuclear cells (PBMC) from healthy individuals or tuberculosis patients stimulated with two forms of Hsp65 antigen, recombinant DNA that encodes Hsp65 (DNA-HSP65) or recombinant Hsp65 protein (rHsp65) in attempting to mimic a prophylactic or therapeutic study in vitro, respectively. Proliferation and cytokine-producing CD4+ or CD8+ cell were assessed by flow cytometry. The CD4+ cell proliferation from healthy individuals was stimulated by DNA-HSP65 and rHsp65, while CD8+ cell proliferation from healthy individuals or tuberculosis patients was stimulated by rHSP65. DNA-HSP65 did not improve the frequency of IFN-gamma+ cells from healthy individuals or tuberculosis patients. Furthermore, we found an increase in the frequency of IL-10-producing cells in both groups. These findings show that Hsp65 antigen activates human lymphocytes and plays an immune regulatory role that should be addressed as an additional antigen for the development of antigen-combined therapies.

DNA Sensing via TLR-9 Constitutes a Major Innate Immunity Pathway Activated during Erythema Nodosum Leprosum.

The chronic course of lepromatous leprosy may be interrupted by acute inflammatory episodes known as erythema nodosum leprosum (ENL). Despite its being a major cause of peripheral nerve damage in leprosy patients, the immunopathogenesis of ENL remains ill-defined. Recognized by distinct families of germline-encoded pattern recognition receptors, endogenous and pathogen-derived nucleic acids are highly immunostimulatory molecules that play a major role in the host defense against infections, autoimmunity, and autoinflammation. The aim of this work was to investigate whether DNA sensing via TLR-9 constitutes a major inflammatory pathway during ENL. Flow cytometry and immunohistochemistry analysis showed significantly higher TLR-9 expression in ENL when compared with nonreactional lepromatous patients, both locally in the skin lesions and in circulating mononuclear cells. The levels of endogenous and pathogen-derived TLR-9 ligands in the circulation of ENL patients were also higher. Furthermore, PBMCs isolated from the ENL patients secreted higher levels of TNF, IL-6, and IL-1ß in response to a TLR-9 agonist than those of the nonreactional patients and healthy individuals. Finally, E6446, a TLR-9 synthetic antagonist, was able to significantly inhibit the secretion of proinflammatory cytokines by ENL PBMCs in response to Mycobacterium leprae lysate. Our data strongly indicate that DNA sensing via TLR-9 constitutes a major innate immunity pathway involved in the pathogenesis and evolution of ENL. Thus, the use of TLR-9 antagonists emerges as a potential alternative to more effectively treat ENL aiming to prevent the development of nerve injuries and deformities in leprosy.

Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal.

BACKGROUND: Acute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available. Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes. METHODS: To identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients. RESULTS: At all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead. CONCLUSIONS: This study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage.

Transcriptional changes that characterize the immune reactions of leprosy.

BACKGROUND: Leprosy morbidity is increased by 2 pathologic immune reactions, reversal reaction (RR) and erythema nodosum leprosum (ENL). METHODS: To discover host factors related to immune reactions, global transcriptional profiles of peripheral blood mononuclear cells were compared between 11 RR, 11 ENL, and 19 matched control patients, with confirmation by quantitative polymerase chain reaction. Encoded proteins were investigated in skin biopsy specimens by means of immunohistochemistry. RESULTS: There were 275 genes differentially expressed in RR and 517 differentially expressed in ENL on the microarray. Pathway analysis showed immunity-related pathways represented in RR and ENL transcriptional profiles, with the "complement and coagulation" pathway common to both. Interferon γ was identified as a significant upstream regulator of the expression changes for RR and ENL. Immunohistochemical staining of skin lesions showed increased C1q in both RR and ENL. CONCLUSIONS: These data suggest a previously underrecognized role for complement in the pathogenesis of both RR and ENL, and we propose new hypotheses for reaction pathogenesis.

T-cell regulation in lepromatous leprosy.

Regulatory T (Treg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25+ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25+ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25+ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25+ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25+ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25+ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25+ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3+ CD8+CD25+ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68+CD163+ as well as FoxP3+ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL.

Autophagy gene polymorphism is associated with susceptibility to leprosy by affecting inflammatory cytokines.

Autophagy and inflammation closely interact with each other, and together, they play critical roles in bacterial infection. Leprosy is caused by the infection of Mycobacterium leprae (M. leprae). The objective of the study was to investigate the association between polymorphisms in IRGM, an autophagy gene, and susceptibility to leprosy, and identify possible functions of the polymorphism in the infection of M. leprae. Two polymorphisms in IRGM, rs4958842 and rs13361189, were tested in 412 leprosy cases and 432 healthy controls. Levels of inflammatory cytokines including interleukin 1 beta, IL-4, IL-6, and interferon gamma (INF-γ) were measured after the infection of M. leprae in the peripheral blood mononuclear cell (PBMC) of subjects with different genotypes of rs13361189. Data showed that prevalence of rs13361189TC and CC genotypes were significantly higher in leprosy patients than in healthy controls (odds ratio (OR) = 1.49, 95 % confidence interval (CI) 1.09-2.04, P = 0.012; OR = 2.58, 95 % CI 1.65-4.05, P < 0.001; respectively). Furthermore, the frequency of rs13361189CC genotype was increased in patients with complications than those without complications (P = 0.011). When analyzing the effect of rs13361189 polymorphism on M. leprae infection, we identified that M. leprae-infected PBMC with rs13361189CC genotype expressed significantly elevated levels of INF-γ and IL-4 than those with TT genotype. Our results suggested autophagy gene polymorphism was associated with the increased risk of leprosy by affecting inflammatory cytokines.

An in vitro model of Mycobacterium leprae induced granuloma formation.

BACKGROUND: Leprosy is a contagious and chronic systemic granulomatous disease caused by Mycobacterium leprae. In the pathogenesis of leprosy, granulomas play a key role, however, the mechanisms of the formation and maintenance of M. leprae granulomas are still not clearly understood. METHODS: To better understand the molecular physiology of M. leprae granulomas and the interaction between the bacilli and human host cells, we developed an in vitro model of human granulomas, which mimicked the in vivo granulomas of leprosy. Macrophages were differentiated from human monocytes, and infected with M. leprae, and then cultured with autologous human peripheral blood mononuclear cells (PBMCs). RESULTS: Robust granuloma-like aggregates were obtained only when the M. leprae infected macrophages were co-cultured with PBMCs. Histological examination showed M. leprae within the cytoplasmic center of the multinucleated giant cells, and these bacilli were metabolically active. Macrophages of both M1 and M2 types co-existed in the granuloma like aggregates. There was a strong relationship between the formation of granulomas and changes in the expression levels of cell surface antigens on macrophages, cytokine production and the macrophage polarization. The viability of M. leprae isolated from granulomas indicated that the formation of host cell aggregates benefited the host, but the bacilli also remained metabolically active. CONCLUSIONS: A simple in vitro model of human M. leprae granulomas was established using human monocyte-derived macrophages and PBMCs. This system may be useful to unravel the mechanisms of disease progression, and subsequently develop methods to control leprosy.

Toll-like receptor 1 N248S single-nucleotide polymorphism is associated with leprosy risk and regulates immune activation during mycobacterial infection.

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

Lsr2 of Mycobacterium leprae and its synthetic peptides elicit restitution of T cell responses in erythema nodosum leprosum and reversal reactions in patients with lepromatous leprosy.

The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742-752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs.

Development and pre-clinical assessment of a 73 kD chimeric fusion protein as a defined sub-unit vaccine for leprosy.

Despite the advances toward the elimination of leprosy through widespread provision of multi-drug therapy to registered patients over the last 2 decades, new case detection rates have stabilized and leprosy remains endemic in a number of localized regions. A vaccine could overcome the inherent limitations of the drug treatment program by providing protection in individuals who are not already harboring the Mycobacterium leprae bacilli at the time of administration and effectively interrupt the transmission cycle over a wider timespan. In this report we present data validating the production of 73f, a chimeric fusion protein incorporating the M. leprae antigens ML2028, ML2346 and ML2044. The 73f protein was recognized by IgG in multibacillary (MB) leprosy patient sera and stimulated IFNγ production within whole blood assays of paucibacillary (PB) leprosy patient and healthy household contacts of MB patients (HHC). When formulated with a TLR4L-containing adjuvant (GLA-SE), 73f stimulated a strong and pluripotent Th1 response that inhibited M. leprae-induced inflammation in mice. We are using these data to develop new vaccine initiatives for the continued and long-term control of leprosy.

Toll-like receptor 1 N248S single-nucleotide polymorphism is associated with leprosy risk and regulates immune activation during mycobacterial infection

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

Active tuberculosis is characterized by an antigen specific and strictly localized expansion of effector T cells at the site of infection.

Mycobacterium tuberculosis (MTB)-specific cytokine responses in the peripheral blood and at the site of infection may differ significantly within the same individual, but the under-lying T-cell subset changes are largely unknown. Here, we measured effector and memory T-cell markers on CD4⁺ T cells (CD45RO, cysteine chemokine receptor (CCR)7, and CD27) in peripheral blood and at the site of active tuberculosis (TB). Additionally, T cells were stimulated overnight with purified protein derivative (PPD) and early secretory antigenic target (ESAT)-6 to determine which T-cell subset produces MTB-specific interferon (IFN)-γ. A striking decrease in CCR7 and CD27 expression on T cells was noted at the site of active TB. Likewise, IFN-γ expressing, ESAT-6 specific CD4⁺CD45RO⁺CD27⁻ T cells were dramatically increased at the site of infection but were not detectable in peripheral blood. An antigen-specific expansion of differentiated T cells at the site of active TB infection was poorly reflected in peripheral blood. Insight in these changes in MTB-specific effector T cells in different compartments of the body could lead to new approaches for immune-based diagnosis and interventions.

A framework to identify gene expression profiles in a model of inflammation induced by lipopolysaccharide after treatment with thalidomide.

BACKGROUND: Thalidomide is an anti-inflammatory and anti-angiogenic drug currently used for the treatment of several diseases, including erythema nodosum leprosum, which occurs in patients with lepromatous leprosy. In this research, we use DNA microarray analysis to identify the impact of thalidomide on gene expression responses in human cells after lipopolysaccharide (LPS) stimulation. We employed a two-stage framework. Initially, we identified 1584 altered genes in response to LPS. Modulation of this set of genes was then analyzed in the LPS stimulated cells treated with thalidomide. RESULTS: We identified 64 genes with altered expression induced by thalidomide using the rank product method. In addition, the lists of up-regulated and down-regulated genes were investigated by means of bioinformatics functional analysis, which allowed for the identification of biological processes affected by thalidomide. Confirmatory analysis was done in five of the identified genes using real time PCR. CONCLUSIONS: The results showed some genes that can further our understanding of the biological mechanisms in the action of thalidomide. Of the five genes evaluated with real time PCR, three were down regulated and two were up regulated confirming the initial results of the microarray analysis.