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1.  iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo 
Cancer immunology research  2013;2(1):59-69.
Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we find that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially-induced by iNKT cell agonists of varying TCR affinities, such as OCH, α-galactosyl ceramide and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of T cell receptor (TCR) signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell-deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T-lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T-lymphoma.
PMCID: PMC3927984  PMID: 24563871
2.  Long-term Survival and Virulence of Mycobacterium leprae in Amoebal Cysts 
Leprosy is a curable neglected disease of humans caused by Mycobacterium leprae that affects the skin and peripheral nerves and manifests clinically in various forms ranging from self-resolving, tuberculoid leprosy to lepromatous leprosy having significant pathology with ensuing disfiguration disability and social stigma. Despite the global success of multi-drug therapy (MDT), incidences of clinical leprosy have been observed in individuals with no apparent exposure to other cases, suggestive of possible non-human sources of the bacteria. In this study we show that common free-living amoebae (FLA) can phagocytose M. leprae, and allow the bacillus to remain viable for up to 8 months within amoebic cysts. Viable bacilli were extracted from separate encysted cocultures comprising three common Acanthamoeba spp.: A. lenticulata, A. castellanii, and A. polyphaga and two strains of Hartmannella vermiformis. Trophozoites of these common FLA take up M. leprae by phagocytosis. M. leprae from infected trophozoites induced to encyst for long-term storage of the bacilli emerged viable by assessment of membrane integrity. The majority (80%) of mice that were injected with bacilli extracted from 35 day cocultures of encysted/excysted A. castellanii and A. polyphaga showed lesion development that was similar to mice challenged with fresh M. leprae from passage mice albeit at a slower initial rate. Mice challenged with coculture-extracted bacilli showed evidence of acid-fast bacteria and positive PCR signal for M. leprae. These data support the conclusion that M. leprae can remain viable long-term in environmentally ubiquitous FLA and retain virulence as assessed in the nu/nu mouse model. Additionally, this work supports the idea that M. leprae might be sustained in the environment between hosts in FLA and such residence in FLA may provide a macrophage-like niche contributing to the higher-than-expected rate of leprosy transmission despite a significant decrease in human reservoirs due to MDT.
Author Summary
Leprosy is a progressive disease of the skin and nervous system caused by the bacillus, Mycobacterium leprae. Implementation of multiple drug therapy (MDT) for leprosy has significantly reduced the global cases of leprosy. Currently, only a few endemic countries remain where relatively high number of cases persists. Despite global reduction of leprosy and the concomitant decrease in human reservoirs, leprosy transmission and incidence have not declined as expected, suggesting a possible extra-human or environmental source of the bacilli. In the current study, we demonstrate that M. leprae can survive long-term within cysts of common environmental free-living amoebae. M. leprae residing in amoebal cysts for over 30 days remain fully capable of transferring disease to mouse footpads and retain viability phenotypes after several months residence within amoebal cysts. It is hypothesized that these protozoa provide an intracellular refuge for M. leprae in environments for which they would otherwise seem ill suited. Traits allowing bacilli to survive in macrophages may likely be acquired via an evolutionary response against predation by amoebae. The results from this work suggest alternative non-human reservoirs for M. leprae exist fostering further study to determine the role of amoebae in the transmission of this Mycobacterium to humans.
PMCID: PMC4270725  PMID: 25521850
3.  Regulation of Gene Expression in Autoimmune Disease Loci and the Genetic Basis of Proliferation in CD4+ Effector Memory T Cells 
PLoS Genetics  2014;10(6):e1004404.
Genome-wide association studies (GWAS) and subsequent dense-genotyping of associated loci identified over a hundred single-nucleotide polymorphism (SNP) variants associated with the risk of rheumatoid arthritis (RA), type 1 diabetes (T1D), and celiac disease (CeD). Immunological and genetic studies suggest a role for CD4-positive effector memory T (CD+ TEM) cells in the pathogenesis of these diseases. To elucidate mechanisms of autoimmune disease alleles, we investigated molecular phenotypes in CD4+ effector memory T cells potentially affected by these variants. In a cohort of genotyped healthy individuals, we isolated high purity CD4+ TEM cells from peripheral blood, then assayed relative abundance, proliferation upon T cell receptor (TCR) stimulation, and the transcription of 215 genes within disease loci before and after stimulation. We identified 46 genes regulated by cis-acting expression quantitative trait loci (eQTL), the majority of which we detected in stimulated cells. Eleven of the 46 genes with eQTLs were previously undetected in peripheral blood mononuclear cells. Of 96 risk alleles of RA, T1D, and/or CeD in densely genotyped loci, eleven overlapped cis-eQTLs, of which five alleles completely explained the respective signals. A non-coding variant, rs389862A, increased proliferative response (p = 4.75×10−8). In addition, baseline expression of seventeen genes in resting cells reliably predicted proliferative response after TCR stimulation. Strikingly, however, there was no evidence that risk alleles modulated CD4+ TEM abundance or proliferation. Our study underscores the power of examining molecular phenotypes in relevant cells and conditions for understanding pathogenic mechanisms of disease variants.
Author Summary
Genome-wide association studies have identified hundreds of genetic variants associated to autoimmune diseases. To understand the mechanisms and pathways affected by these variants, follow-up studies of molecular phenotypes and functions are required. Given the diversity of cell types and specialization of functions within the immune system, it is crucial that such studies focus on specific and relevant cell types. Here, we studied genetic and cellular traits of CD4-positive effector memory T (CD4+ TEM) cells, which are particularly important in the onset of rheumatoid arthritis, celiac disease, and type 1 diabetes. In a cohort of healthy individuals, we purified CD4+ TEM cells, assayed genome-wide single nucleotide polymorphisms (SNPs), abundance of CD4+ TEM cells in blood, proliferation upon T cell receptor stimulation, and 215 gene transcripts in resting and stimulated states. We found that expression levels of 46 genes were regulated by nearby SNPs, including disease-associated SNPs. Many of these expression quantitative trait loci were not previously seen in studies of more heterogeneous peripheral blood cells. We demonstrated that relative abundance and proliferative response of CD4+ TEM cells varied in the population, however disease alleles are unlikely to confer risk by modulating these traits in this cell type.
PMCID: PMC4072514  PMID: 24968232
4.  The Challenge of Producing Skin Test Antigens with Minimal Resources Suitable for Human Application against a Neglected Tropical Disease; Leprosy 
True incidence of leprosy and its impact on transmission will not be understood until a tool is available to measure pre-symptomatic infection. Diagnosis of leprosy disease is currently based on clinical symptoms, which on average take 3–10 years to manifest. The fact that incidence, as defined by new case detection, equates with prevalence, i.e., registered cases, suggests that the cycle of transmission has not been fully intercepted by implementation of multiple drug therapy. This is supported by a high incidence of childhood leprosy. Epidemiological screening for pre-symptomatic leprosy in large endemic populations is required to facilitate targeted chemoprophylactic interventions. Such a test must be sensitive, specific, simple to administer, cost-effective, and easy to interpret. The intradermal skin test method that measures cell-mediated immunity was explored as the best option. Prior knowledge on skin testing of healthy subjects and leprosy patients with whole or partially fractionated Mycobacterium leprae bacilli, such as Lepromin or the Rees' or Convit' antigens, has established an acceptable safety and potency profile of these antigens. These data, along with immunoreactivity data, laid the foundation for two new leprosy skin test antigens, MLSA-LAM (M. leprae soluble antigen devoid of mycobacterial lipoglycans, primarily lipoarabinomannan) and MLCwA (M. leprae cell wall antigens). In the absence of commercial interest, the challenge was to develop these antigens under current good manufacturing practices in an acceptable local pilot facility and submit an Investigational New Drug to the Food and Drug Administration to allow a first-in-human phase I clinical trial.
Author Summary
Despite reaching the global elimination target for leprosy, the need for a diagnostic tool to detect pre-symptomatic disease remains. Transmission has not been completely intercepted despite over 30 years of extensive curative treatment. With limited resources, two new leprosy skin test antigens, MLSA-LAM and MLCwA, suitable for human application were developed and manufactured in a local pilot plant. Requirements for manufacturing and clinical testing were met and an Investigational New Drug was established with the Food and Drug Administration to test both antigens in a phase I clinical trial for safety in a non-endemic region for leprosy and a phase II clinical trial for safety and efficacy in an endemic region for leprosy.
PMCID: PMC4038479  PMID: 24874086
5.  Safety and Efficacy Assessment of Two New Leprosy Skin Test Antigens: Randomized Double Blind Clinical Study 
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.
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.
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.
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 NCT01920750 (Phase I), NCT00128193 (Phase II)
Author Summary
Clinically useful skin test reagents should be safe and sufficiently sensitive to detect infection prior to physical manifestations of leprosy disease. While in these small scale human studies, leprosy reagents were safe for use in humans, they failed in respect of sensitivity at a rate of 20–25% in the key indicator group, BT/TT leprosy patients. Specificity in terms of leprosy vs. tuberculosis at a rate of 95–100% was surprisingly high in light of the extensive presence of cross-reactive antigens in the complex native M. leprae preparations. These results could justify a further trial at lower dosages.
PMCID: PMC4038488  PMID: 24874401
6.  Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens 
Chemistry & biology  2009;16(12):1230-1239.
Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.
PMCID: PMC4020808  PMID: 20064433
7.  Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis 
Future microbiology  2013;8(7):10.2217/fmb.13.52.
Most of the newly discovered compounds showing promise for the treatment of TB, notably multidrug-resistant TB, inhibit aspects of Mycobacterium tuberculosis cell envelope metabolism. This review reflects on the evolution of the knowledge that many of the front-line and emerging products inhibit aspects of cell envelope metabolism and in the process are bactericidal not only against actively replicating M. tuberculosis, but contrary to earlier impressions, are effective against latent forms of the disease. While mycolic acid and arabinogalactan synthesis are still primary targets of existing and new drugs, peptidoglycan synthesis, transport mechanisms and the synthesis of the decaprenyl-phosphate carrier lipid all show considerable promise as targets for new products, older drugs and new combinations. The advantages of whole cell- versus target-based screening in the perpetual search for new targets and products to counter multidrug-resistant TB are discussed.
PMCID: PMC3867987  PMID: 23841633
antibiotic; arabinogalactan; cell envelope; Mycobacterium; mycolic acids; peptidoglycan; tuberculosis
8.  Differences in CD1d protein structure determine species-selective antigenicity of isoglobotrihexosylceramide (iGb3) to invariant natural killer T (iNKT)Cells 
European journal of immunology  2013;43(3):815-825.
Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d-presented self-antigen for mouse iNKT cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3-dependent human iNKT-cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human-mouse cross-species analysis of iNKT-cell activation by iGb3-CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3-hCD1d was unable to support cognate interactions with the iNKT-cell TCR. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine-to-tryptophan modification within the alpha2-helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3-hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT-cell biology.
PMCID: PMC3961145  PMID: 23280365
antigen presentation; CD1d; iNKT; isogloboside 3; species differences
9.  Mycobacterium leprae in Colombia described by SNP7614 in gyrA, two minisatellites and geography 
New cases of leprosy are still being detected in Colombia after the country declared achievement of the WHO defined ‘elimination’ status. To study the ecology of leprosy in endemic regions, a combination of geographic and molecular tools were applied for a group of 201 multibacillary patients including six multi-case families from eleven departments. The location (latitude and longitude) of patient residences were mapped. Slit skin smears and/or skin biopsies were collected and DNA was extracted. Standard agarose gel electrophoresis following a multiplex PCR-was developed for rapid and inexpensive strain typing of M. leprae based on copy numbers of two VNTR minisatellite loci 27-5 and 12-5. A SNP (C/T) in gyrA (SNP7614) was mapped by introducing a novel PCR-RFLP into an ongoing drug resistance surveillance effort. Multiple genotypes were detected combining the three molecular markers. The two frequent genotypes in Colombia were SNP7614(C)/27-5(5)/12-5(4) [C54] predominantly distributed in the Atlantic departments and SNP7614 (T)/27-5(4)/12-5(5) [T45] associated with the Andean departments. A novel genotype SNP7614 (C)/27-5(6)/12-5(4) [C64] was detected in cities along the Magdalena river which separates the Andean from Atlantic departments; a subset was further characterized showing association with a rare allele of minisatellite 23-3 and the SNP type 1 of M. leprae. The genotypes within intra-family cases were conserved. Overall, this is the first large scale study that utilized simple and rapid assay formats for identification of major strain types and their distribution in Colombia. It provides the framework for further strain type discrimination and geographic information systems as tools for tracing transmission of leprosy.
PMCID: PMC3668693  PMID: 23291420
Leprosy; Mycobacterium leprae; SNP; gyrA; SNP7614; VNTR
10.  Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells 
Nature immunology  2012;14(1):90-99.
Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled iNKT cell gene expression during ontogeny and in peripheral subsets as part of the Immunological Genome Project (ImmGen). High-resolution comparative transcriptional analyses defined developmental and subset-specific iNKT cell gene expression programs. In addition, iNKT cells were found to share an extensive transcriptional program with natural killer (NK) cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Strikingly, the NK- iNKT program also operated constitutively in γδT cells and in adaptive T cells following activation. Together, our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes.
PMCID: PMC3764492  PMID: 23202270
11.  Isolation of a distinct Mycobacterium tuberculosis mannose-capped lipoarabinomannan isoform responsible for recognition by CD1b-restricted T cells 
Glycobiology  2012;22(8):1118-1127.
Mannose-capped lipoarabinomannan (ManLAM) is a complex lipoglycan abundantly present in the Mycobacterium tuberculosis cell envelope. Many biological properties have been ascribed to ManLAM, from directly interacting with the host and participating in the intracellular survival of M. tuberculosis, to triggering innate and adaptive immune responses, including the activation of CD1b-restricted T cells. Due to its structural complexity, ManLAM is considered a heterogeneous population of molecules which may explain its different biological properties. The presence of various modifications such as fatty acids, succinates, lactates, phosphoinositides and methylthioxylose in ManLAM have proven to correlate directly with its biological activity and may potentially be involved in the interactions between CD1b and the T cell population. To further delineate the specific ManLAM epitopes involved in CD1b-restricted T cell recognition, and their potential roles in mediating immune responses in M. tuberculosis infection, we established a method to resolve ManLAM into eight different isoforms based on their different isoelectric values. Our results show that a ManLAM isoform with an isoelectric value of 5.8 was the most potent in stimulating the production of interferon-γ in different CD1b-restricted T-cell lines. Compositional analyses of these isoforms of ManLAM revealed a direct relationship between the overall charge of the ManLAM molecule and its capacity to be presented to T cells via the CD1 compartment.
PMCID: PMC3382347  PMID: 22534567
CD1b; lipoarabinomannan; lipoglycans; Mycobacterium tuberculosis; T cells
12.  Gene Expression Profile and Immunological Evaluation of Unique Hypothetical Unknown Proteins of Mycobacterium leprae by Using Quantitative Real-Time PCR 
The cell-mediated immunity (CMI)-based in vitro gamma interferon release assay (IGRA) of Mycobacterium leprae-specific antigens has potential as a promising diagnostic means to detect those individuals in the early stages of M. leprae infection. Diagnosis of leprosy is a major obstacle toward ultimate disease control and has been compromised in the past by the lack of specific markers. Comparative bioinformatic analysis among mycobacterial genomes identified potential M. leprae-specific proteins called “hypothetical unknowns.” Due to massive gene decay and the prevalence of pseudogenes, it is unclear whether any of these proteins are expressed or are immunologically relevant. In this study, we performed cDNA-based quantitative real-time PCR to investigate the expression status of 131 putative open reading frames (ORFs) encoding hypothetical unknowns. Twenty-six of the M. leprae-specific antigen candidates showed significant levels of gene expression compared to that of ESAT-6 (ML0049), which is an important T cell antigen of low abundance in M. leprae. Fifteen of 26 selected antigen candidates were expressed and purified in Escherichia coli. The seroreactivity to these proteins of pooled sera from lepromatous leprosy patients and cavitary tuberculosis patients revealed that 9 of 15 recombinant hypothetical unknowns elicited M. leprae-specific immune responses. These nine proteins may be good diagnostic reagents to improve both the sensitivity and specificity of detection of individuals with asymptomatic leprosy.
PMCID: PMC3571279  PMID: 23239802
13.  Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types 
Journal of Clinical Microbiology  2012;50(3):742-753.
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.
PMCID: PMC3295127  PMID: 22170923
14.  Invariant natural killer T cells recognize lipid self-antigen induced by microbial danger signals 
Nature Immunology  2011;12(12):1202-1211.
Invariant natural killer T cells (iNKT cells) play a prominent role during infection and other inflammatory processes, and these cells can be activated through their T cell receptors by microbial lipid antigens. However, increasing evidence shows that they are also activated in situations where no foreign lipid antigens are present, suggesting a role for lipid self-antigen. We now demonstrate that an abundant endogenous lipid, β-D-glucopyranosylceramide (β-GlcCer), is a potent iNKT cell self-antigen in mouse and human, and that its activity depends on N-acyl chain composition. Furthermore, β-GlcCer accumulates during infection and in response to Toll-like receptor agonists, contributing to iNKT cell activation. Thus, we propose that recognition of β-GlcCer by the invariant TCR translates innate danger signals into iNKT cell activation.
PMCID: PMC3242449  PMID: 22037601
15.  Transmission of Dapsone-Resistant Leprosy Detected by Molecular Epidemiological Approaches▿ 
Antimicrobial Agents and Chemotherapy  2011;55(11):5384-5387.
Drug resistance surveillance identified six untreated leprosy patients in the Philippines with Mycobacterium leprae folP1 mutations which confer dapsone resistance. Five patients share a village of residence; four who carried the mutation, Thr53Val, were also linked by M. leprae variable-number tandem repeat (VNTR) strain types. In India, folP1 mutations were detected in two relapse patients with a history of dapsone treatment. Mutations were not found in the rifampin target gene rpoB. These findings indicate that dapsone resistance is being transmitted.
PMCID: PMC3195063  PMID: 21859943
16.  Pathogen-Specific Epitopes as Epidemiological Tools for Defining the Magnitude of Mycobacterium leprae Transmission in Areas Endemic for Leprosy 
During recent years, comparative genomic analysis has allowed the identification of Mycobacterium leprae-specific genes with potential application for the diagnosis of leprosy. In a previous study, 58 synthetic peptides derived from these sequences were tested for their ability to induce production of IFN-γ in PBMC from endemic controls (EC) with unknown exposure to M. leprae, household contacts of leprosy patients and patients, indicating the potential of these synthetic peptides for the diagnosis of sub- or preclinical forms of leprosy. In the present study, the patterns of IFN-γ release of the individuals exposed or non-exposed to M. leprae were compared using an Artificial Neural Network algorithm, and the most promising M. leprae peptides for the identification of exposed people were selected. This subset of M. leprae-specific peptides allowed the differentiation of groups of individuals from sites hyperendemic for leprosy versus those from areas with lower level detection rates. A progressive reduction in the IFN-γ levels in response to the peptides was seen when contacts of multibacillary (MB) patients were compared to other less exposed groups, suggesting a down modulation of IFN-γ production with an increase in bacillary load or exposure to M. leprae. The data generated indicate that an IFN-γ assay based on these peptides applied individually or as a pool can be used as a new tool for predicting the magnitude of M. leprae transmission in a given population.
Author Summary
Despite the efforts to treat registered leprosy patients, the number of new cases reported globally remains stable and high (about 200,000/year). As the treatment of multibacillary leprosy patients, the major recognized source for new infections, did not allow the expected reduction in new leprosy cases, additional sources must be considered. Following exposure to M. leprae infection, the evolution to active disease is estimated to take from 2 to 10 years, and it is conceivable that some of these asymptomatic individuals could be a yet unrecognized source of infection. Previously, the use of computational tools allowed us to select M. leprae-specific genes or gene regions, and derive M. leprae-specific synthetic peptides from the M. leprae genome. Ex vivo stimulation of the blood leukocytes with a subset of these peptides induced IFN-γ production that allowed the differentiation of individuals exposed to M. leprae from unexposed ones. Individuals with no known history of exposure to M. leprae, but living in an area with high frequency of leprosy cases had high-level positive responses to the peptides. This last observation raised the possibility of using this test as a tool for evaluating the level of transmission of M. leprae infection in areas of interest.
PMCID: PMC3335884  PMID: 22545169
17.  Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism 
PLoS ONE  2012;7(1):e29970.
The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.
PMCID: PMC3253812  PMID: 22253841
18.  Synthetic UDP-furanoses as potent inhibitors of mycobacterial galactan biogenesis 
Chemistry & biology  2010;17(12):1356-1366.
UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short “dead-end” intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.
PMCID: PMC3012269  PMID: 21168771
19.  Hospital-Based Comparative Effectiveness Centers: Translating Research into Practice to Improve the Quality, Safety and Value of Patient Care 
Journal of General Internal Medicine  2010;25(12):1352-1355.
Hospital-based comparative effectiveness (CE) centers provide a model that clinical leaders can use to improve evidence-based practice locally. The model is used by integrated health systems outside the US, but is less recognized in the US. Such centers can identify and adapt national evidence-based policies for the local setting, create local evidence-based policies in the absence of national policies, and implement evidence into practice through health information technology (HIT) and quality initiatives. Given the increasing availability of CE evidence and incentives to meaningfully use HIT, the relevance of this model to US practitioners is increasing. This is especially true in the context of healthcare reform, which will likely reduce reimbursements for care deemed unnecessary by published evidence or guidelines. There are challenges to operating hospital-based CE centers, but many of these challenges can be overcome using solutions developed by those currently leading such centers. In conclusion, these centers have the potential to improve the quality, safety and value of care locally, ultimately translating into higher quality and more cost-effective care nationally. To better understand this potential, the current activity and impact of hospital-based CE centers in the US should be rigorously examined.
PMCID: PMC2988155  PMID: 20697961
comparative effectiveness; evidence-based medicine; health technology assessment; quality of health care; cost-effectiveness; health information technology; organizational decision making
20.  Serum Metabolomics Reveals Higher Levels of Polyunsaturated Fatty Acids in Lepromatous Leprosy: Potential Markers for Susceptibility and Pathogenesis 
Leprosy is a disease of the skin and peripheral nervous system caused by the obligate intracellular bacterium Mycobacterium leprae. The clinical presentations of leprosy are spectral, with the severity of disease determined by the balance between the cellular and humoral immune response of the host. The exact mechanisms that facilitate disease susceptibility, onset and progression to certain clinical phenotypes are presently unclear. Various studies have examined lipid metabolism in leprosy, but there has been limited work using whole metabolite profiles to distinguish the clinical forms of leprosy.
Methodology and Principal Findings
In this study we adopted a metabolomics approach using high mass accuracy ultrahigh pressure liquid chromatography mass spectrometry (UPLC-MS) to investigate the circulatory biomarkers in newly diagnosed untreated leprosy patients. Sera from patients having bacterial indices (BI) below 1 or above 4 were selected, subjected to UPLC-MS, and then analyzed for biomarkers which distinguish the polar presentations of leprosy. We found significant increases in the abundance of certain polyunsaturated fatty acids (PUFAs) and phospholipids in the high-BI patients, when contrasted with the levels in the low-BI patients. In particular, the median values of arachidonic acid (2-fold increase), eicosapentaenoic acid (2.6-fold increase) and docosahexaenoic acid (1.6-fold increase) were found to be greater in the high-BI patients.
Eicosapentaenoic acid and docosahexaenoic acid are known to exert anti-inflammatory properties, while arachidonic acid has been reported to have both pro- and anti-inflammatory activities. The observed increase in the levels of several lipids in high-BI patients may provide novel clues regarding the biological pathways involved in the immunomodulation of leprosy. Furthermore, these results may lead to the discovery of biomarkers that can be used to investigate susceptibility to infection, facilitate early diagnosis and monitor the progression of disease.
Author Summary
Leprosy is an infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. M. leprae infects the skin and nerves, leading to disfigurement and nerve damage, with the severity of the disease varying widely. We believe there are multiple factors (genetic, bacterial, nutritional and environmental), which may explain the differences in clinical manifestations of the disease. We studied the metabolites in the serum of infected patients to search for specific molecules that may contribute to variations in the severity of disease seen in leprosy. We found that there were variations in levels of certain lipids in the patients with different bacterial loads. In particular, we found that three polyunsaturated fatty acids (PUFAs) involved in the inhibition of inflammation were more abundant in the serum of patients with higher bacterial loads. However, we do not know whether these PUFAs originated from the host or the bacteria. The variations in the metabolite profile that we observed provide a foundation for future research into the explanations of how leprosy causes disease.
PMCID: PMC3167790  PMID: 21909445
21.  Analysis of Antibody Responses to Mycobacterium leprae Phenolic Glycolipid I, Lipoarabinomannan, and Recombinant Proteins To Define Disease Subtype-Specific Antigenic Profiles in Leprosy ▿  
A simple serodiagnostic test based on the Mycobacterium leprae-specific phenolic glycolipid I(PGL-I), for individuals with leprosy is nearly universally positive in leprosy patients with high bacillary loads but cannot be used as a stand-alone diagnostic test for the entire spectrum of the disease process. For patients with early infection with no detectable acid-fast bacilli in lesions or with low or no antibody titer to PGL-I, as in those at the tuberculoid end of the disease spectrum, this diagnostic approach has limited usefulness. To identify additional M. leprae antigens that might enhance the serological detection of these individuals, we have examined the reactivity patterns of patient sera to PGL-I, lipoarabinomannan (LAM), and six recombinant M. leprae proteins (ML1877, ML0841, ML2028, ML2038, ML0380, and ML0050) by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Overall, the responses to ML2028 (Ag85B) and ML2038 (bacterioferritin) were consistently high in both multibacillary and paucibacillary groups and weak or absent in endemic controls, while responses to other antigens showed considerable variability, from strongly positive to completely negative. This analysis has given a clearer understanding of some of the differences in the antibody responses between individuals at opposite ends of the disease spectrum, as well as illustrating the heterogeneity of antibody responses toward protein, carbohydrate, and glycolipid antigens within a clinical group. Correlating these response patterns with a particular disease state could allow for a more critical assessment of the form of disease within the leprosy spectrum and could lead to better patient management.
PMCID: PMC3067349  PMID: 21177913
22.  A modified synthesis and serological evaluation of neoglycoproteins containing the natural disaccharide of PGL-I from Mycobacterium leprae 
In order to generate substantial amounts of neoglycoconjugate needed for commercialization of diagnostic kits and high-throughput detection of leprosy, we developed a facile and high-yield synthesis of the corresponding disaccharide. Herein, the non-reducing disaccharide segment of phenolic glycolipid I from Mycobacterium leprae, O-(3,6-di-O-methyl-β-D-glucopyranosyl)-(1→4)-O-2,3-di-O-methyl-α-L-rhamnopyranose was synthesized by an improved procedure. The disaccharide was efficiently conjugated to bovine/human serum albumin, via acyl-azide intermediate, to form natural disaccharide-BSA/HSA neoglycoproteins that showed a high activity in serodiagnosis of leprosy. The disaccharide incorporated into the proteins was accurately measured by MALDI-ToF mass spectrometry. The serological activities of the neoglycoproteins against pooled human lepromatous leprosy were measured by ELISA and they were detectable at picogram amount.
PMCID: PMC2917636  PMID: 20462755
23.  Biogenesis of the cell wall and other glycoconjugates of Mycobacterium tuberculosis 
The reemergence of tuberculosis in its present-day manifectations – single, multiple and extensive drug resistant forms and as HIV-TB coinfections – has resulted in renewed research on fundamental questions such as the nature of the organism itself, Mycobacterium tuberculosis, the molecular basis of its pathogenesis, definition of the immunological response in animal models and humans, and development of new intervention strategies such as vaccines and drugs. Foremost among these developments has been the precise chemical definition of the complex and distinctive cell wall of M. tuberculosis, elucidation of the relevant pathways and underlying genetics responsible for the synthesis of the hallmark moities of the tubercle bacillus such as the mycolic acid-arabinogalactan-peptidoglycan complex, the phthiocerol- and trehalose-containing effector lipids, the phosphatidylinositol-containing mannosides, lipomannosides and lipoarabinomannosides, major immunomodulators, and others. In this review, the laboratory personnel that have been the focal point of some to these developments review recent progress towards a comprehensive understanding of the basic physiology and functions of the cell wall of M. tuberculosis.
PMCID: PMC3066434  PMID: 19729090
24.  Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate and kinetic studies of Mycobacterium tuberculosis IspF, a potential drug target 
Chemistry & biology  2010;17(2):117-122.
Many pathogenic bacteria utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, two major building blocks of isoprenoid compounds. The fifth enzyme in the MEP pathway, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP) synthase (IspF), catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to ME-CPP with a corresponding release of cytidine 5-monophosphate (CMP). Since there is no ortholog of IspF in human cells IspF is of interest as a potential drug target. However, study of IspF has been hindered by a lack of enantiopure CDP-ME2P. Herein, we report the first synthesis of enantiomerically pure CDP-ME2P from commercially available D-arabinose. Cloned, expressed, and purified M. tuberculosis IspF was able to utilize the synthetic CDP-ME2P as a substrate, a result confirmed by mass spectrometry. A convenient, sensitive, in vitro IspF assay was developed by coupling the CMP released during production of ME-CPP to mononucleotide kinase, which can be used for high throughput screening.
PMCID: PMC2837070  PMID: 20189102
25.  AftD, a novel essential arabinofuranosyltransferase from mycobacteria 
Glycobiology  2009;19(11):1235-1247.
Arabinogalactan (AG) and lipoarabinomannan (LAM) are the two major cell wall (lipo)polysaccharides of mycobacteria. They share arabinan chains made of linear segments of α-1,5-linked d-Araf residues with some α-1,3-branching, the biosynthesis of which offers opportunities for new chemotherapeutics. In search of the missing arabinofuranosyltransferases (AraTs) responsible for the formation of the arabinan domains of AG and LAM in Mycobacterium tuberculosis, we identified Rv0236c (AftD) as a putative membrane-associated polyprenyl-dependent glycosyltransferase. AftD is 1400 amino acid-long, making it the largest predicted glycosyltransferase of its class in the M. tuberculosis genome. Assays using cell-free extracts from recombinant Mycobacterium smegmatis and Corynebacterium glutamicum strains expressing different levels of aftD indicated that this gene encodes a functional AraT with α-1,3-branching activity on linear α-1,5-linked neoglycolipid acceptors in vitro. The disruption of aftD in M. smegmatis resulted in cell death and a decrease in its activity caused defects in cell division, reduced growth, alteration of colonial morphology, and accumulation of trehalose dimycolates in the cell envelope. Overexpression of aftD in M. smegmatis, in contrast, induced the accumulation of two arabinosylated compounds with carbohydrate backbones reminiscent of that of LAM and a degree of arabinosylation dependent on aftD expression levels. Altogether, our results thus indicate that AftD is an essential AraT involved in the synthesis of the arabinan domain of major mycobacterial cell envelope (lipo)polysaccharides.
PMCID: PMC2757576  PMID: 19654261
arabinogalactan; arabinosyltransferase; lipoarabinomannan; Mycobacterium; tuberculosis

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