Myeloid derived suppressor cells and the release of micro-metastases from dormancy.

Metastasis is the primary cause of cancer mortality and an improved understanding of its pathology is critical to the development of novel therapeutic approaches. Mechanism-based therapeutic strategies require insight into the timing of tumor cell dissemination, seeding of distant organs, formation of occult lesions and critically, their release from dormancy. Due to imaging limitations, primary tumors can only be detected when they reach a relatively large size (e.g. > 1 cm3), which, based on our understanding of tumor evolution, occurs approximately 10 years and about 30 doubling times following tumor initiation. Genomic profiling of paired primary tumors and metastases has suggested that tumor seeding at secondary sites occurs early during tumor progression and frequently, years prior to clinical diagnosis. Following seeding, tumor cells may enter into and remain in a dormant state, and if they survive and are released from dormancy, they can proliferate into an overt lesion. The timeline of tumor initiation and metastatic dormancy is regulated by tumor interactions with its microenvironment, angiogenesis, and tumor-specific cytotoxic T-lymphocyte (CTL) responses. Therefore, a better understanding of the cellular interactions responsible for immune evasion and/or tumor cell release from dormancy would facilitate the development of therapeutics targeted against this critical part of tumor progression. The immunosuppressive mechanisms mediated by myeloid-derived suppressor cells (MDSCs) contribute to tumor progression and, we posit, promote tumor cell escape from CTL-associated dormancy. Thus, while clinical and translational research has demonstrated a role for MDSCs in facilitating tumor progression and metastasis through tumor escape from adoptive and innate immune responses (T-, natural killer and B-cell responses), few studies have considered the role of MDSCs in tumor release from dormancy. In this review, we discuss MDSC expansion, driven by tumor burden associated growth factor secretion and their role in tumor cell escape from dormancy, resulting in manifest metastases. Thus, the therapeutic strategies to inhibit MDSC expansion and function may provide an approach to delay metastatic relapse and prolong the survival of patients with advanced malignancies.

Fatty Acid Mediators in the Tumor Microenvironment.

Patients with cancer frequently overexpress inflammatory cytokines with an associated neutrophilia both of which may be downregulated by diets with high omega-3 polyunsaturated fatty acids (ω-3 PUFA). The anti-inflammatory activity of dietary ω-3 PUFA has been suggested to have anticancer properties and to improve survival of cancer patients. Currently, the majority of dietary research efforts do not differentiate between obesity and dietary fatty acid consumption as mediators of inflammatory cell expansion and tumor microenvironmental infiltration, initiation, and progression. In this chapter, we discuss the relationships between dietary lipids, inflammation, neoplasia and strategies to regulate these relationships. We posit that dietary composition, notably the ratio of ω-3 vs. ω-6 PUFA, regulates tumor initiation and progression and the frequency and sites of metastasis that, together, impact overall survival (OS). We focus on three broad topics: first, the role of dietary lipids in chronic inflammation and tumor initiation, progression, and regression; second, lipid mediators linking inflammation and cancer; and third, dietary lipid regulation of murine and human tumor initiation, progression, and metastasis.

Whole blood RNA signatures in leprosy patients identify reversal reactions before clinical onset: a prospective, multicenter study.

Early diagnosis of leprosy is challenging, particularly its inflammatory reactions, the major cause of irreversible neuropathy in leprosy. Current diagnostics cannot identify which patients are at risk of developing reactions. This study assessed blood RNA expression levels as potential biomarkers for leprosy. Prospective cohorts of newly diagnosed leprosy patients, including reactions, and healthy controls were recruited in Bangladesh, Brazil, Ethiopia and Nepal. RNA expression in 1,090 whole blood samples was determined for 103 target genes for innate and adaptive immune profiling by dual color Reverse-Transcription Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) followed by cluster analysis. We identified transcriptomic biomarkers associated with leprosy disease, different leprosy phenotypes as well as high exposure to Mycobacterium leprae which respectively allow improved diagnosis and classification of leprosy patients and detection of infection. Importantly, a transcriptomic signature of risk for reversal reactions consisting of five genes (CCL2, CD8A, IL2, IL15 and MARCO) was identified based on cross-sectional comparison of RNA expression. In addition, intra-individual longitudinal analyses of leprosy patients before, during and after treatment of reversal reactions, indicated that several IFN-induced genes increased significantly at onset of reaction whereas IL15 decreased. This multi-site study, situated in four leprosy endemic areas, demonstrates the potential of host transcriptomic biomarkers as correlates of risk for leprosy. Importantly, a prospective five-gene signature for reversal reactions could predict reversal reactions at least 2 weeks before onset. Thus, transcriptomic biomarkers provide promise for early detection of these acute inflammatory episodes and thereby help prevent permanent neuropathy and disability in leprosy patients.

Immune regulation and anti-cancer activity by lipid inflammatory mediators.

Rodent and clinical studies have documented that myeloid cell infiltration of tumors is associated with poor outcomes, neutrophilia and lymphocytopenia. This contrasts with increased lymphocyte infiltration of tumors, which is correlated with improved outcomes. Lifestyle parameters, such as obesity and diets with high levels of saturated fat and/or omega (ω)-6 polyunsaturated fatty acids (PUFAs), can influence these inflammatory parameters, including an increase in extramedullary myelopoiesis (EMM). While tumor secretion of growth factors (GFs) and chemokines regulate tumor-immune-cell crosstalk, lifestyle choices also contribute to inflammation, abnormal pathology and leukocyte infiltration of tumors. A relationship between obesity and high-fat diets (notably saturated fats in Western diets) and inflammation, tumor incidence, metastasis and poor outcomes is generally accepted. However, the mechanisms of dietary promotion of an inflammatory microenvironment and targeted drugs to inhibit the clinical sequelae are poorly understood. Thus, modifications of obesity and dietary fat may provide preventative or therapeutic approaches to control tumor-associated inflammation and disease progression. Currently, the majority of basic and clinical research does not differentiate between obesity and fatty acid consumption as mediators of inflammatory and neoplastic processes. In this review, we discuss the relationships between dietary PUFAs, inflammation and neoplasia and experimental strategies to improve our understanding of these relationships. We conclude that dietary composition, notably the ratio of ω-3 vs ω-6 PUFA regulates tumor growth and the frequency and sites of metastasis that together, impact overall survival (OS) in mice.

Long-chain omega-3 polyunsaturated fatty acids decrease mammary tumor growth, multiorgan metastasis and enhance survival.

Epidemiological studies show a reduced risk of breast cancer (BC) in women consuming high levels of long-chain (LC) omega-3 (ω-3) fatty acids (FAs) compared with women who consumed low levels. However, the regulatory and mechanistic roles of dietary ω-6 and LC-ω-3 FAs on tumor progression, metastasis and survival are poorly understood. Female BALB/c mice (10-week old) were pair-fed with a diet containing ω-3 or an isocaloric, isolipidic ω-6 diet for 16 weeks prior to the orthotopic implantation of 4T1 mammary tumor cells. Major outcomes studied included: mammary tumor growth, survival analysis, and metastases analyses in multiple organs including pulmonary, hepatic, bone, cardiac, renal, ovarian, and contralateral MG (CMG). The dietary regulation of the tumor microenvironment was evaluated in mice autopsied on day-35 post tumor injection. In mice fed the ω-3 containing diet, there was a significant delay in tumor initiation and prolonged survival relative to the ω-6 diet-fed group. The tumor size on day 35 post tumor injection in the ω-3 group was 50% smaller and the frequencies of pulmonary and bone metastases were significantly lower relative to the ω-6 group. Similarly, the incidence/frequencies and/or size of cardiac, renal, ovarian metastases were significantly lower in mice fed the ω-3 diet. The analyses of the tumor microenvironment showed that tumors in the ω-3 group had significantly lower numbers of proliferating tumor cells (Ki67+)/high power field (HPF), and higher numbers of apoptotic tumor cells (TUNEL+)/HPF, lower neo-vascularization (CD31+ vessels/HPF), infiltration by neutrophil elastase+ cells, and macrophages (F4/80+) relative to the tumors from the ω-6 group. Further, in tumors from the ω-3 diet-fed mice, T-cell infiltration was 102% higher resulting in a neutrophil to T-lymphocyte ratio (NLR) that was 76% lower (p < 0.05). Direct correlations were observed between NLR with tumor size and T-cell infiltration with the number of apoptotic tumor cells. qRT-PCR analysis revealed that tumor IL10 mRNA levels were significantly higher (six-fold) in the tumors from mice fed the ω-3 diet and inversely correlated with the tumor size. Our data suggest that dietary LC-ω-3FAs modulates the mammary tumor microenvironment slowing tumor growth, and reducing metastases to both common and less preferential organs resulting in prolonged survival. The surrogate analyses undertaken support a mechanism of action by dietary LC-ω-3FAs that includes, but is not limited to decreased infiltration by myeloid cells (neutrophils and macrophages), an increase in CD3+ lymphocyte infiltration and IL10 associated anti-inflammatory activity.

Long-Chain Omega-3 Polyunsaturated Fatty Acids Modulate Mammary Gland Composition and Inflammation.

Studies in rodents have shown that dietary modifications as mammary glands (MG) develop, regulates susceptibility to mammary tumor initiation. However, the effects of dietary PUFA composition on MGs in adult life, remains poorly understood. This study investigated morphological alterations and inflammatory microenvironments in the MGs of adult mice fed isocaloric and isolipidic liquid diets with varying compositions of omega (ω)-6 and long-chain (Lc)-ω3FA that were pair-fed. Despite similar consumption levels of the diets, mice fed the ω-3 diet had significantly lower body-weight gains, and abdominal-fat and mammary fat pad (MFP) weights. Fatty acid analysis showed significantly higher levels of Lc-ω-3FAs in the MFPs of mice on the ω-3 diet, while in the MFPs from the ω-6 group, Lc-ω-3FAs were undetectable. Our study revealed that MGs from ω-3 group had a significantly lower ductal end-point density, branching density, an absence of ductal sprouts, a thinner ductal stroma, fewer proliferating epithelial cells and a lower transcription levels of estrogen receptor 1 and amphiregulin. An analysis of the MFP and abdominal-fat showed significantly smaller adipocytes in the ω-3 group, which was accompanied by lower transcription levels of leptin, IGF1, and IGF1R. Further, MFPs from the ω-3 group had significantly decreased numbers and sizes of crown-like-structures (CLS), F4/80+ macrophages and decreased expression of proinflammatory mediators including Ptgs2, IL6, CCL2, TNFα, NFκB, and IFNγ. Together, these results support dietary Lc-ω-3FA regulation of MG structure and density and adipose tissue inflammation with the potential for dietary Lc-ω-3FA to decrease the risk of mammary gland tumor formation.

Dietary omega-3 and omega-6 polyunsaturated fatty acids modulate hepatic pathology.

Recent evidence has suggested that dietary polyunsaturated fatty acids (PUFAs) modulate inflammation; however, few studies have focused on the pathobiology of PUFA using isocaloric and isolipidic diets and it is unclear if the associated pathologies are due to dietary PUFA composition, lipid metabolism or obesity, as most studies compare diets fed ad libitum. Our studies used isocaloric and isolipidic liquid diets (35% of calories from fat), with differing compositions of omega (ω)-6 or long chain (Lc) ω-3 PUFA that were pair-fed and assessed hepatic pathology, inflammation and lipid metabolism. Consistent with an isocaloric, pair-fed model we observed no significant difference in diet consumption between the groups. In contrast, the body and liver weight, total lipid level and abdominal fat deposits were significantly higher in mice fed an ω-6 diet. An analysis of the fatty acid profile in plasma and liver showed that mice on the ω-6 diet had significantly more arachidonic acid (AA) in the plasma and liver, whereas, in these mice ω-3 fatty acids such as eicosapentaenoic acid (EPA) were not detected and docosahexaenoic acid (DHA) was significantly lower. Histopathologic analyses documented that mice on the ω-6 diet had a significant increase in macrovesicular steatosis, extramedullary myelopoiesis (EMM), apoptotic hepatocytes and decreased glycogen storage in lobular hepatocytes, and hepatocyte proliferation relative to mice fed the Lc ω-3 diet. Together, these results support PUFA dietary regulation of hepatic pathology and inflammation with implications for enteral feeding regulation of steatosis and other hepatic lesions.

Lipid Inflammatory Mediators in Cancer Progression and Therapy.

Rodent and clinical studies have documented that myeloid cell infiltration of tumors is associated with neutrophilia, lymphocytopenia and poor patient outcomes. This contrasts with lymphocyte infiltration of tumors, which is associated with improved outcomes. Lifestyle parameters such as high fat diet s and omega (ω)-6 polyunsaturated fatty acids (PUFA) intake may influence these inflammatory parameters including extramedullary myelopoiesis that can contribute to a metastatic "niche". While, tumor secretion of growth factors (GFs) and chemokines regulate tumor-immune-cell crosstalk, in this chapter, we also emphasize how lifestyle choices, including, obesity, high-fat and high ω-6 PUFA dietary content, contribute to inflammation and myeloid cell infiltration of tumors. A relationship between obesity and high-fat diets (notably the saturated fats in Western diets) and tumor incidence, metastasis, and poor outcomes is generally accepted. However, the mechanisms of dietary promotion of inflammatory microenvironments and targeted drugs to inhibit the clinical sequel remain an unmet challenge. One approach, modification of dietary intake may have a preventative or therapeutic approach to regulate tumor-associated inflammation and remains an attractive, but little studied intervention.

Exploratory urinary metabolomics of type 1 leprosy reactions.

BACKGROUND: Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves. Although curable with multidrug therapy, leprosy is complicated by acute inflammatory episodes called reactions, which are the major causes of irreversible neuropathy in leprosy that occur before, during, and even after treatment. Early diagnosis and prompt treatment of reactions reduces the risk of permanent disability. METHODS: This exploratory study investigated whether urinary metabolic profiles could be identified that correlate with early signs of reversal reactions (RR). A prospective cohort of leprosy patients with and without reactions and endemic controls was recruited in Nepal. Urine-derived metabolic profiles were measured longitudinally. Thus, a conventional area of biomarker identification for leprosy was extended to non-invasive urine testing. RESULTS: It was found that the urinary metabolome could be used to discriminate endemic controls from untreated patients with mycobacterial disease. Moreover, metabolic signatures in the urine of patients developing RR were clearly different before RR onset compared to those at RR diagnosis. CONCLUSIONS: This study indicates that urinary metabolic profiles are promising host biomarkers for the detection of intra-individual changes during acute inflammation in leprosy and could contribute to early treatment and prevention of tissue damage.

Genetic Variation in Toll-Interacting Protein Is Associated With Leprosy Susceptibility and Cutaneous Expression of Interleukin 1 Receptor Antagonist.

Leprosy is a chronic disease characterized by skin and peripheral nerve pathology and immune responses that fail to control Mycobacterium leprae. Toll-interacting protein (TOLLIP) regulates Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling against mycobacteria. We analyzed messenger RNA (mRNA) expression of candidate immune genes in skin biopsy specimens from 85 individuals with leprosy. TOLLIP mRNA was highly and specifically correlated with IL-1R antagonist (IL-1Ra). In a case-control gene-association study with 477 cases and 1021 controls in Nepal, TOLLIP single-nucleotide polymorphism rs3793964 TT genotype was associated with increased susceptibility to leprosy (recessive, P = 1.4 × 10(-3)) and with increased skin expression of TOLLIP and IL-1Ra. Stimulation of TOLLIP-deficient monocytes with M. leprae produced significantly less IL-1Ra (P < .001), compared with control. These data suggest that M. leprae upregulates IL-1Ra by a TOLLIP-dependent mechanism. Inhibition of TOLLIP may decrease an individual's susceptibility to leprosy and offer a novel therapeutic target for IL-1-dependent diseases.

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.

Safety and efficacy assessment of two new leprosy skin test antigens: randomized double blind clinical study.

BACKGROUND: New tools are required for the diagnosis of pre-symptomatic leprosy towards further reduction of disease burden and its associated reactions. To address this need, two new skin test antigens were developed to assess safety and efficacy in human trials. METHODS: A Phase I safety trial was first conducted in a non-endemic region for leprosy (U.S.A.). Healthy non-exposed subjects (n = 10) received three titrated doses (2.5 µg, 1.0 µg and 0.1 µg) of MLSA-LAM (n = 5) or MLCwA (n = 5) and control antigens [Rees MLSA (1.0 µg) and saline]. A randomized double blind Phase II safety and efficacy trial followed in an endemic region for leprosy (Nepal), but involved only the 1.0 µg (high dose) and 0.1 µg (low dose) of each antigen; Tuberculin PPD served as a control antigen. This Phase II safety and efficacy trial consisted of three Stages: Stage A and B studies were an expansion of Phase I involving 10 and 90 subjects respectively, and Stage C was then conducted in two parts (high dose and low dose), each enrolling 80 participants: 20 borderline lepromatous/lepromatous (BL/LL) leprosy patients, 20 borderline tuberculoid/tuberculoid (BT/TT) leprosy patients, 20 household contacts of leprosy patients (HC), and 20 tuberculosis (TB) patients. The primary outcome measure for the skin test was delayed type hypersensitivity induration. FINDINGS: In the small Phase I safety trial, reactions were primarily against the 2.5 µg dose of both antigens and Rees control antigen, which were then excluded from subsequent studies. In the Phase II, Stage A/B ramped-up safety study, 26% of subjects (13 of 50) showed induration against the high dose of each antigen, and 4% (2 of 50) reacted to the low dose of MLSA-LAM. Phase II, Stage C safety and initial efficacy trial showed that both antigens at the low dose exhibited low sensitivity at 20% and 25% in BT/TT leprosy patients, but high specificity at 100% and 95% compared to TB patients. The high dose of both antigens showed lower specificity (70% and 60%) and sensitivity (10% and 15%). BL/LL leprosy patients were anergic to the leprosy antigens. INTERPRETATION: MLSA-LAM and MLCwA at both high (1.0 µg) and low (0.1 µg) doses were found to be safe for use in humans without known exposure to leprosy and in target populations. At a sensitivity rate of 20-25% these antigens are not suitable as a skin test for the detection of the early stages of leprosy infection; however, the degree of specificity is impressive given the presence of cross-reactive antigens in these complex native M. leprae preparations. TRIAL REGISTRATION: ClinicalTrials.gov NCT01920750 (Phase I), NCT00128193 (Phase II).

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.

Diagnosis and treatment of leprosy reactions in integrated services--the patients' perspective in Nepal.

UNLABELLED: Leprosy care has been integrated with peripheral health services, away from vertical programmes. This includes the diagnosis and management of leprosy reactions, which cause significant morbidity. We surveyed patients with leprosy reactions at two leprosy hospitals in Nepal to assess their experience of leprosy reaction management following integration to identify any gaps in service delivery. METHODS: Direct and referral patients with leprosy reactions were interviewed in two of Nepal's leprosy hospitals. We also collected quantitative and qualitative data from clinical examination and case-note review to document the patient pathway. RESULTS: Seventy-five patients were interviewed. On development of reaction symptoms 39% presented directly to specialist services, 23% to a private doctor, 17% to a district hospital, 10% to a traditional healer, 7% to a health post and 4% elsewhere. Those who presented directly to specialist services were 6.6 times more likely to start appropriate treatment than those presenting elsewhere (95% CI: 3.01 to 14.45). The average delay between symptom onset to commencing corticosteroids was 2.9 months (range 0-24 months). Obstacles to early presentation and treatment included diagnostic challenge, patients' lack of knowledge and the patients' view of health as a low priority. 40% received corticosteroids for longer than 12 weeks and 72% required an inpatient stay. Treatment follow-up was conducted at locations ranging from health posts to specialist hospitals. Inconsistency in the availability of corticosteroids peripherally was identified and 41% of patients treated for leprosy and a reaction on an outpatient basis attended multiple sites for follow-up treatment. CONCLUSION: This study demonstrates that specialist services are necessary and continue to provide significant critical support within an integrated health system approach towards the diagnosis and management of leprosy reactions.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M: leprae in Brazil, Ethiopia and Nepal

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Human beta-defensin 3 is up-regulated in cutaneous leprosy type 1 reactions.

BACKGROUND: Leprosy, a chronic granulomatous disease affecting the skin and nerves, is caused by Mycobacterium leprae (M. leprae). The type of leprosy developed depends upon the host immune response. Type 1 reactions (T1Rs), that complicate borderline and lepromatous leprosy, are due to an increase in cell-mediated immunity and manifest as nerve damage and skin inflammation. Owing to the increase in inflammation in the skin of patients with T1Rs, we sought to investigate the activation of the innate immune system during reactionary events. Specifically, we investigated the expression levels of human beta-defensins (hBDs) 2 and 3 in the skin of patients with T1Rs, in keratinocytes, and in macrophages stimulated with M. leprae and corticosteroids. RESULTS: Skin biopsies from twenty-three patients with Type 1 reactions were found to have higher transcript levels of hBD3 as compared to fifteen leprosy patients without Type 1 reactions, as measured by qPCR. Moreover, we observed that keratinocytes but not macrophages up-regulated hBD2 and hBD3 in response to M. leprae stimulation in vitro. Corticosteroid treatment of patients with T1Rs caused a suppression of hBD2 and hBD3 in skin biopsies, as measured by qPCR. In vitro, corticosteroids suppressed M. leprae-dependent induction of hBD2 and hBD3 in keratinocytes. CONCLUSIONS: This study demonstrates that hBD3 is induced in leprosy Type 1 Reactions and suppressed by corticosteroids. Furthermore, our findings demonstrate that keratinocytes are responsive to M. leprae and lend support for additional studies on keratinocyte innate immunity in leprosy and T1Rs. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN31894035.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal.

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Real-time PCR and high-resolution melt analysis for rapid detection of Mycobacterium leprae drug resistance mutations and strain types.

Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR-high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.

Common polymorphisms in the NOD2 gene region are associated with leprosy and its reactive states.

BACKGROUND: Because of its wide spectrum of clinical manifestations and its well-defined immunological complications, leprosy is a useful disease for studying genetic regulation of the host response to infection. We hypothesized that polymorphisms in the nucleotide-binding oligomerization domain containing 2 (NOD2) gene, for a cytosolic receptor known to detect mycobacteria, are associated with susceptibility to leprosy and its clinical outcomes. METHODS: We used a case-control study design with 933 patients in Nepal. Our study included 240 patients with type 1 (reversal) reactions and 124 patients with type 2 (erythema nodosum leprosum) reactions. We compared the frequencies of 32 common polymorphisms in the NOD2 gene region between patients with the different clinical types of leprosy as well as between the patients and 101 control participants without leprosy. RESULTS: Four polymorphisms were associated with susceptibility to leprosy when comparing allele frequencies, and 8 were associated when comparing genotype frequencies with a dominant model. Five polymorphisms were associated with protection from reversal reaction in an allelic analysis, and 7 were associated with reversal reaction with a dominant model. Four polymorphisms were associated with increased susceptibility to erythema nodosum leprosum in an allelic analysis, whereas 7 of 32 polymorphisms were associated with a dominant model. CONCLUSION: These data suggest that NOD2 genetic variants are associated with susceptibility to leprosy and the development of leprosy reactive states.