A new paradigm for leprosy diagnosis based on host gene expression.

Transcriptional profiling is a powerful tool to investigate and detect human diseases. In this study, we used bulk RNA-sequencing (RNA-Seq) to compare the transcriptomes in skin lesions of leprosy patients or controls affected by other dermal conditions such as granuloma annulare, a confounder for paucibacillary leprosy. We identified five genes capable of accurately distinguishing multibacillary and paucibacillary leprosy from other skin conditions. Indoleamine 2,3-dioxygenase 1 (IDO1) expression alone was highly discriminatory, followed by TLR10, BLK, CD38, and SLAMF7, whereas the HS3ST2 and CD40LG mRNA separated multi- and paucibacillary leprosy. Finally, from the main differentially expressed genes (DEG) and enriched pathways, we conclude that paucibacillary disease is characterized by epithelioid transformation and granuloma formation, with an exacerbated cellular immune response, while multibacillary leprosy features epithelial-mesenchymal transition with phagocytic and lipid biogenesis patterns in the skin. These findings will help catalyze the development of better diagnostic tools and potential host-based therapeutic interventions. Finally, our data may help elucidate host-pathogen interplay driving disease clinical manifestations.

A report of rifampin-resistant leprosy from northern and eastern India: identification and in silico analysis of molecular interactions.

Presence of point mutations within the drug resistance determining regions of Mycobacterium leprae (M. leprae) genome confers molecular basis of drug resistance to dapsone, rifampin and ofloxacin in leprosy. This study is focused on the identification of mutations within the rpoB gene region of M. leprae that are specific for rifampin interaction, and further in silico analysis was carried out to determine the variations in the interactions. DNA and RNA were isolated from slit skin scrapings of 60 relapsed leprosy patients. PCR targeting rpoB gene region and amplicon sequencing was performed to determine point mutations. mRNA expression levels of rpoB and high-resolution melt analysis of mutants were performed using Rotor Gene Q Realtime PCR. Molecular docking was performed using LigandFit Software. Ten cases having point mutations within the rpoB gene region were identified and were clinically confirmed to be resistant to rifampin. A new mutation at codon position Gln442His has been identified. There is a 9.44-fold upregulation in the mRNA expression of rpoB gene in mutant/resistant samples when compared with the wild/sensitive samples. In silico docking analysis of rifampin with wild-type and Gln442His mutant RpoB proteins revealed a variation in the hydrogen-bonding pattern leading to a difference in the total interaction energy and conformational change at position Asp441. These preliminary downstream functional observations revealed that the presence of point mutations within the rifampin resistance determining regions of rpoB gene plays a vital role in conferring genetic and molecular basis of resistance to rifampin in leprosy.

Genome-wide screening of mRNA expression in leprosy patients

Leprosy, an infectious disease caused by Mycobacterium leprae, affects millions of people worldwide. However, little is known regarding its molecular pathophysiological mechanisms. In this study, a comprehensive assessment of human mRNA was performed on leprosy skin lesions by using DNA chip microarrays, which included the entire spectrum of the disease along with its reactional states. Sixty-six samples from leprotic lesions (10TT, 10BT, 10BB, 10BL, 4LL, 14R1, and 10R2) and nine skin biopsies from healthy individuals were used as controls (CC) (ages ranged from 06 to 83 years, 48 were male and 29 female). The evaluation identified 1580 differentially expressed mRNAs [Fold Change (FC) ≥ 2.0, p ≤ 0.05] in diseased lesions vs. healthy controls. Some of these genes were observed in all forms of the disease (CD2, CD27, chit1, FA2H, FAM26F, GZMB, MMP9, SLAMF7, UBD) and others were exclusive to reactional forms (Type "1" reaction: GPNMB, IL1B, MICAL2, FOXQ1; Type "2" reaction: AKR1B10, FAM180B, FOXQ1, NNMT, NR1D1, PTX3, TNFRSF25). In literature, these mRNAs have been associated with numerous pathophysiological processes and signaling pathways and are present in a large number of diseases. The role of these mRNAs maybe studied in the context of developing new diagnostic markers and therapeutic targets for leprosy.

mRNA and DNA PCR tests in cutaneous tuberculosis.

BACKGROUND: The microbiologic diagnosis of cutaneous tuberculosis is difficult because most lesions harbor only a small number of mycobacteria that cannot usually be detected by staining for the organism or by culture. Nucleic acid amplification tests based on the polymerase chain reaction (PCR) are potentially useful in this situation. AIMS: To evaluate the utility of mRNA PCR and DNA PCR in the diagnosis of cutaneous tuberculosis. METHODS: Biopsies from 28 cases of cutaneous tuberculosis and 19 controls with other diseases were subjected to microbiologic tests including direct smears for mycobacteria, culture and both mRNA PCR and DNA PCR. The laboratory was blinded to the clinical diagnosis. RESULTS: None of the patients or controls showed a positive reaction on mRNA PCR test. Seven of 28 cases and 5 out of 19 controls showed a positive result on DNA PCR test yielding a sensitivity of 25% and a specificity of 73.7%. CONCLUSION: The results of PCR tests in cutaneous tuberculosis should be interpreted in the light of clinical and histopathological findings.

Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases.

Mycobacterium leprae, the causative agent of leprosy, is uncultivable in defined media. Development of new diagnostic tools which do not depend on growth of bacteria is needed for the early detection of M. leprae and for monitoring the effectiveness of chemotherapy. We used a real-time PCR-based assay to quantify the copy number of bacterial DNA and hsp18 mRNA from 47 leprosy patients using paraffin-embedded biopsy samples. The assay used was specific, sensitive and reproducible. The applicability of this approach in monitoring the chemotherapy of leprosy was examined. A reduction in DNA and mRNA during chemotherapy was observed and hsp18 mRNA could not be detected in patients who underwent 2 years of multidrug therapy (MDT). However, a considerable amount of M. leprae DNA could be detected even after 2 years of MDT. A significant amount of hsp18 mRNA was found in reactional cases as well. This raises important questions regarding the role of bacterial antigens in leprosy reactions and the rationale of omitting antibiotics in the treatment of reactional cases. Results in this study show that real-time PCR could be a better tool for the careful monitoring of bacillary DNA and mRNA in lesions, which will help to improve diagnosis, disease progression and the treatment regimen.

Mycobacterium leprae inhibits dendritic cell activation and maturation.

Leprosy presents with a clinical spectrum of skin lesions that span from strong Th1-mediated cellular immunity and control of bacillary growth at one pole to poor Ag-specific T cell immunity with extensive bacillary load and Th2 cytokine-expressing lesions at the other. To understand how the immune response to Mycobacterium leprae is regulated, human dendritic cells (DC), potent inducers of adaptive immune responses, exposed to M. leprae, Mycobacterium tuberculosis (Mtb), and Mycobacterium bovis bacillus Calmette-Guerin (BCG) were studied for their ability to be activated and to prime T cell proliferation. In contrast with Mtb and BCG, M. leprae did not induce DC activation/maturation as measured by the expression of selected surface markers and proinflammatory cytokine production. In MLR, T cells did not proliferate in response to M. leprae-stimulated DC. Interestingly, M. leprae-exposed MLR cells secreted increased Th2 cytokines as well as similar Th1 cytokine levels as compared with Mtb- and BCG-exposed cells. Gene expression analysis revealed a reduction in levels of mRNA of DC activation and maturation markers following exposure to M. leprae. Our data suggest that M. leprae does not induce and probably suppresses in vitro DC maturation/activation, whereas Mtb and BCG are stimulatory.

Decreased expression of CD3zeta and nuclear transcription factor kappa B in patients with pulmonary tuberculosis: potential mechanisms and reversibility with treatment.

BACKGROUND: The protective immune response against Mycobacterium tuberculosis relies both on antigen-presenting cells and on T lymphocytes. In patients with different forms of tuberculosis, varying degrees of T cell function--ranging from positive delayed-type hypersensitivity, in asymptomatic infected healthy individuals, to the absence of the response, in patients with miliary or pulmonary tuberculosis (PTB)--have been reported. The decreased expression of CD3zeta reported in T cells from patients with either cancer or leprosy has provided possible explanations for the altered immune response observed in these diseases. METHODS: The present study aimed to compare the expression of CD3zeta , nuclear transcription factor- kappa B (NF- kappa B), arginase activity, and cytokine production in 20 patients with PTB, in 20 tuberculin-positive asymptomatic subjects, and in 14 tuberculin-negative control subjects. RESULTS: Compared with those in tuberculin (purified protein derivative)-negative control subjects, peripheral-blood T lymphocytes from patients with active PTB had significantly (P < .001) decreased expression of CD3zeta and absence of the p65/p50 heterodimer of NF- kappa B. These alterations were reversed only in patients who responded to treatment. Also reported here for the first time is that the presence of arginase activity in peripheral-blood mononuclear-cell lysates of patients with PTB parallels high production of interleukin-10. CONCLUSIONS: The presence of arginase could, in part, explain the decreased expression of CD3zeta . These findings provide a novel mechanism that may explain the T cell dysfunction observed in patients with PTB.

Adipocytokine expression in placentas from pre-eclamptic and chronic hypertensive patients.

OBJECTIVE: Growing evidence suggests that concentration of the adipocytokines leptin and adiponectin may be affected by risk of hypertension during pregnancy. Leptin and leptin receptor gene expression has been studied in placentas obtained from pre-eclamptic patients, but not in those with chronic high blood pressure (CHBP). Adiponectin receptors remain unstudied in placentas obtained from hypertensive patients. METHODS: Therefore, we investigated relative mRNA expression of selected adipocytokine genes (leptin, leptin receptors (LEPRA, LEPRB, LEPRC, LEPRD) and adiponectin receptors (ADIPOR1, ADIPOR2)) in placental tissues from women with pre-eclampsia (n = 6) or CHBP (n = 8). Placentas from 28 normotensive patients were analyzed as controls. mRNA extracted from biopsies taken from the maternal and fetal sides of the placenta was investigated using real-time polymerase chain reaction. RESULTS: Compared with controls, significant increases in leptin mRNA expression were seen in placentas from pre-eclamptic patients on the maternal (p = 0.01) and fetal (p = 0.02) sides, and in placentas from CHBP mothers on the fetal side (p = 0.001). Maternal-side tissue from CHBP patients was not significantly different from that of controls (p = 0.08), but this might be due to the small sample size. No significant differences were seen in mRNA expression for most of the adipocytokine receptors tested for hypertensive cases compared with controls. However, there was a decrease in LEPRC (pre-eclamptic, maternal side, p = 0.03) and LEPRD (pre-eclamptic, maternal side, p = 0.01; CHBP, fetal side, p = 0.009) in case-control analysis. CONCLUSIONS: This pilot study shows that increases seen in leptin expression in placentas from hypertensive mothers might be a consequence of defects in placentation associated with this disease, and motivates further region-specific adipocytokine gene expression analysis across this organ.

On cell signalling mechanism of Mycobacterium leprae soluble antigen (MLSA) in Jurkat T cells.

We investigated the role of Mycobaterium leprae soluble antigen (MLSA) in the modulation of calcium signalling, phosphorylation of mitogen-activated protein (MAP) kinases and IL-2 mRNA expression in human Jurkat T cells. We observed that MLSA induced an increase in free intracellular calcium concentrations, [Ca2+]i, via opening CRAC (Ca2+-release activated- Ca2+) channels. Furthermore, MLSA failed to potentiate both thapsigargin- and anti-CD3 antibodies-induced capacitative calcium influx in Jurkat T cells. We observed that MLSA failed to affect the degree of phosphorylation of two MAP kinases, i.e., ERK1/ERK2, stimulated by anti-CD3 antibodies alone or phorbol 12-myristate 13-acetate (PMA) alone. In order to mimic co-stimulation of T cells, we stimulated them by both PMA and anti-CD3 antibodies. MLSA significantly curtailed the phosphorylation of ERK1/ERK2, stimulated by both PMA and anti-CD3 antibodies in Jurkat T cells. Also MLSA was found to reduce the transcription of IL-2 gene in PMA plus anti-CD3 antibodies-activated Jurkat T cells. Our finding demonstrates that Ca2+ influx via CRAC channels, inhibition of ERK1/ERK2 phosphorylation and IL-2 gene transcription may be implicated in immunosuppressive effects of MLSA antigen.

Effective treatment of erythema nodosum leprosum with thalidomide is associated with immune stimulation.

The immunomodulatory drug thalidomide is the treatment of choice for erythema nodosum leprosum (ENL), an inflammatory cutaneous and systemic complication of multibacillary leprosy. To elucidate the mechanism of action of thalidomide in this syndrome, we prospectively investigated 20 patients with ENL who were treated with thalidomide for 21 days. All patients responded to treatment, with the majority of them having complete resolution of cutaneous lesions within 7 days. This response was associated with a marked but transient increase in ex vivo mitogen-induced expression of interleukin (IL)-2 and interferon- gamma by CD4(+) and CD8(+) T cells that was observed on treatment day 7, but these returned to pretreatment levels by day 21. Plasma tumor necrosis factor- alpha levels were not high at baseline, and they increased modestly during treatment. Plasma levels of IL-12 increased steadily during thalidomide treatment. Hence, the therapeutic effect of thalidomide in ENL appears to be associated with transient immune stimulation, which suggests that the drug may promote an active immunoregulatory response.

Effects of prednisolone treatment on cytokine expression in patients with leprosy type 1 reactions.

Leprosy type 1 reactions (T1R) are due to increased cell-mediated immunity and result in localized tissue damage. The anti-inflammatory drug prednisolone is used for treatment, but there is little good in vivo data on the molecular actions of prednisolone. We investigated the effect of prednisolone treatment on tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-10, and transforming growth factor beta1 (TGF-beta1) mRNA and protein expression in blood and skin biopsies from 30 patients with T1R in India. After 1 month of prednisolone treatment the sizes of the skin granulomas were reduced, as were the grades of cells positive for TNF-alpha and IL-10 in skin lesions. Increased production of TGF-beta1 was seen in skin lesions after 6 months of prednisolone treatment. Expression of mRNA for TNF-alpha, IL-1beta, and TGF-beta1 was reduced, whereas no change in IL-10 mRNA expression was detected during treatment. The circulating cytokine profiles were similar in patients with and without T1R, and prednisolone treatment had no detectable effects on cytokine expression in the blood. The data emphasize the compartmentalization of pathology in T1R and the importance of the immune response in the skin. Clinical improvement and cytokine expression were compared. Surprisingly, patients with improved skin and nerve function and patients with nonimproved skin and nerve function had similar cytokine profiles, suggesting that clinical improvement is not directly mediated by the cytokines studied here. This in vivo well-controlled study of the immunosuppressive effects of prednisolone showed that the drug does not switch off cytokine responses effectively.

Susceptibility to leprosy is associated with PARK2 and PACRG.

Leprosy is caused by Mycobacterium leprae and affects about 700,000 individuals each year. It has long been thought that leprosy has a strong genetic component, and recently we mapped a leprosy susceptibility locus to chromosome 6 region q25-q26 (ref. 3). Here we investigate this region further by using a systematic association scan of the chromosomal interval most likely to harbour this leprosy susceptibility locus. In 197 Vietnamese families we found a significant association between leprosy and 17 markers located in a block of approx. 80 kilobases overlapping the 5' regulatory region shared by the Parkinson's disease gene PARK2 and the co-regulated gene PACRG. Possession of as few as two of the 17 risk alleles was highly predictive of leprosy. This was confirmed in a sample of 975 unrelated leprosy cases and controls from Brazil in whom the same alleles were strongly associated with leprosy. Variants in the regulatory region shared by PARK2 and PACRG therefore act as common risk factors for leprosy.

Expression of CC and CXC chemokines and chemokine receptors in human leprosy skin lesions.

We have investigated the expression of chemokines and their receptors in leprosy skin lesions using immunohistochemistry. Skin biopsies from 25 leprosy patients across the leprosy spectrum, 11 patients undergoing type I reversal reactions and four normal donors were immunostained by ABC peroxidase method using antibodies against CC and CXC chemokines and their receptors. Using an in situ hybridization technique we have also studied the expression of monocyte chemoattractant protein 1 (MCP-1), RANTES and interleukin (IL)-8 chemokines mRNA in leprosy skin lesions. Chemokines and receptor expression was detected in all leprosy skin biopsies. Expression of CC chemokines MCP-1 (P < 0.01) and RANTES (P < 0.01) were elevated significantly in borderline tuberculoid leprosy in reversal reaction compared to non-reactional borderline tuberculoid leprosy, but there was no difference in the expression of IL-8 chemokine. Surprisingly, there was no significant difference in the expression of CC (CCR2 and CCR5) and CXC (CXCR2) chemokine receptors across the leprosy spectrum. Similarly, there was no significant difference in the expression of mRNA for MCP-1, regulated upon activation normal T cell expressed and secreted (RANTES) and IL-8 chemokines. Here, the presence of a neutrophil chemoattractant IL-8 in leprosy lesions, which do not contain neutrophils, suggests strongly a role of IL-8 as a monocyte and lymphocyte recruiter in leprosy lesions. These results suggest that the chemokines and their receptors, which are known to chemoattract T lymphocytes and macrophages, are involved in assembling the cellular infiltrate found in lesions across the leprosy spectrum.

Thalidomide inhibits tumor necrosis factor-alpha production and antigen presentation by Langerhans cells.

Thalidomide is an effective treatment for several inflammatory and autoimmune disorders including erythema nodosum leprosum, Behcet's syndrome, discoid lupus erythematosus, and Crohn's disease. Thalidomide is believed to exert its anti-inflammatory effects, at least in part, by inhibiting tumor necrosis factor-alpha (TNF-alpha) production by monocytes. We studied the effects of thalidomide on epidermal Langerhans cells (LC). LCs are epidermal antigen-presenting dendritic cells that play important roles in skin immune responses. Using the murine epidermis-derived dendritic cell lines, XS106A from A/J mice and XS52 from BALB/c mice as surrogates for LC, we found that thalidomide inhibited TNF-alpha production in a concentration-dependent manner. Northern blot analysis revealed that thalidomide significantly decreased the peak-induced mRNA level of TNF-alpha in XS106A cells and XS52 cells. We then examined the effect of thalidomide on fresh LC enriched to approximately 98% using positive selection of Ia+ cells with antibodies conjugated to magnetic microspheres. TNF-alpha production was reduced by 67.7% at a thalidomide concentration of 200 microg per mL. Thalidomide also had a profound inhibitory effect on the ability of LC to present antigen to a responsive TH1 clone. Thalidomide inhibits TNF-alpha production and the antigen-presenting ability of epidermal LCs. These mechanisms may contribute to the therapeutic effects observed with this agent.

DNA-PCR and RT-PCR for the 18-kDa gene of Mycobacterium leprae to assess the efficacy of multi-drug therapy for leprosy.

DNA-PCR and reverse transcription (RT)-PCR for the 18-kDa protein of Mycobacterium leprae were used to examine the efficacy of multi-drug therapy (MDT) in leprosy. MDT was administered for 0-24 months. Fourteen (63.6%) of 22 patients showed positive PCR results after treatment for 12 months and the positive results decreased to 30% after 24 months of MDT. These results did not correlate with the bacterial index (BI) or the IgM antibody titre for the phenolic glycolipid (PGL)-1. One-dimensional densitometric analysis of agarose gels from PCR from the longitudinal study showed a gradual reduction of the 360-bp band after 12-24 months of MDT. RT-PCR for mRNA of the 18-kDa protein successfully tracked bacterial RNA changes in the biopsies and confirmed a decrease in the RNA of M. leprae in patients after MDT for 12 months. Thus, DNA- and RT-PCR for the 18-kDa protein of M. leprae are effective in assessing the efficacy of MDT for leprosy.

Lepromatous leprosy patients show T helper 1-like cytokine profile with differential expression of interleukin-10 during type 1 and 2 reactions.

Some leprosy patients suffer from clinical episodes associated with tissue damage which are designated as Type 1 (reversal reaction) when localized to the lesions and Type 2 (erythema nodosum leprosum, ENL) when accompanied by systemic involvement. We had reported earlier that stable, non-reaction lepromatous leprosy subjects show T helper 2 (Th2)- and Th0- but not Th1-like responses in the peripheral blood. To further understand the development of Th-like responses during disease, 32 lepromatous patients undergoing reactions were studied using cytokine-specific reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in peripheral blood and some skin biopsies. Of interest was the evidence of a Th1-like response with presence of interferon-gamma (IFN-gamma) and absence of interleukin-4 (IL-4) mRNA in the peripheral blood mononuclear cells (PBMC) of 85 and 64% of Type 1 and 2 reaction patients, respectively, and in all reaction sites. Whereas a Th0- was seen in some, a Th2-like response was absent. IL-12p40 mRNA was seen in 21/25 ENL and all Type 1 reaction subjects irrespective of the Th phenotype. IL-12p40 and IFN-gamma were detectable in unstimulated PBMC suggesting an in vivo priming during reactions. IL-10 was mainly associated with adherent cells and showed a differential expression in the two reactions. It was present in the PBMC of ENL but not in reversal reaction patients. Moreover, it was not detectable in the skin lesions of either type of reactions. A Th1-like cytokine profile was associated with immunopathology and persisted up to 6-7 months after the onset of reactions.

Susceptibility to Mycobacterium leprae of ALY (alymphoplasia) mice and IFN-gamma induction in the culture supernatant of spleen cells.

The aly/aly (alymphoplasia) mice from a mutation of a colony of the C57BL/6J mouse strain, which has a systemic absence of lymph nodes and Peyer's patches, are deficient in both T- and B-cell-mediated immune functions. We have undertaken a comparison of susceptibility to Mycobacterium leprae of ALY (aly/aly, aly/+) mice with C57BL/6J mice. The aly/aly mouse was found to have an excellent high susceptibility to M. leprae with no distinction between female and male. The aly/+ mouse also was more susceptible to M. leprae at an earlier stage than the C57BL/6J mouse. Therefore, we examined and compared the cytokine gene expression and gamma interferon (IFN-gamma) induction in the splenocytes of ALY mice. The expression of interleukin 4 (IL-4), IL-10 and IL-12 mRNA was weakly stimulated with ML-lysate in inoculated aly/aly mice but IL-2, IL-6, IGIF/IL-18 and IFN-gamma mRNA were not observed. None of the cytokine genes used appeared, except the mRNA for IL-1-alpha, when uninfected cultured spleen cells were stimulated with ML-lysate. Also, IFN-gamma production was not induced. However, the appearance of these cytokine genes was observed when stimulated with concanavalin A (ConA), and IFN-gamma production was also induced in the culture supernatant by aly/+ and even aly/aly mice stimulated with ConA. To examine the reason why IFN-gamma cannot be produced by splenocytes of ALY mice inoculated with M. leprae, we detected cytokine gene expression and IFN-gamma induction in the presence of recombinant murine IL-12 or IGIF/IL-18. IL-2 mRNA expression was detected in all of the mice tested in the presence of IL-12 but not in aly/aly mice under IGIF/IL-18, and iNOS mRNA expression was not observed in aly/aly mice under IL-12 or IGIF/IL-18. IL-4 and IL-10 mRNA were detected by aly/aly mice only by exposure to IGIF/IL-18. In culture, the supernatant with ML antigens of the aly/aly mice did not produce IFN-gamma in spite of the presence of IL-12 and IGIF/IL-18, while IFN-gamma was weakly induced in aly/+ mice stimulated with ML-lysate and in the presence of IGIF/IL-18. Nevertheless, IFN-gamma production was observed in splenocytes of the aly/aly mice stimulated with ConA and also with IGIF/IL-18 plus anti-CD3 antibody. Our results suggest that ALY mice might be showing a high susceptibility to M. leprae because of deficient priming for activation of T cells with the leprosy bacilli infection. Moreover, it is possible that the phagocytic activities of the macrophages of ALY mice are also impaired.