Distinct Roles of Th17 and Th1 Cells in Inflammatory Responses Associated with the Presentation of Paucibacillary Leprosy and Leprosy Reactions.

It is well established that helper T cell responses influence resistance or susceptibility to Mycobacterium leprae infection, but the role of more recently described helper T cell subsets in determining severity is less clear. To investigate the involvement of Th17 cells in the pathogenesis of leprosy, we determined the immune profile with variant presentations of leprosy. Firstly, IL-17A, IFN-γ and IL-10 were evaluated in conjunction with CD4+ T cell staining by confocal microscopy of lesion biopsies from tuberculoid (TT) and lepromatous leprosy (LL) patients. Secondly, inflammatory cytokines were measured by multiplex assay of serum samples from Multibacillary (MB, n = 28) and Paucibacillary (PB, n = 23) patients and household contacts (HHC, n = 23). Patients with leprosy were also evaluated for leprosy reaction occurrence: LR+ (n = 8) and LR- (n = 20). Finally, peripheral blood mononuclear cells were analysed by flow cytometry used to determine the phenotype of cytokine-producing cells. Lesions from TT patients were found to have more CD4+ IL-17A+ cells than those from LL patients. Higher concentrations of IL-17A and IL-1ß were observed in serum from PB than MB patients. The highest serum IFN-γ concentrations were, however, detected in sera from MB patients that developed leprosy reactions (MB LR+ ). Together, these results indicate that Th1 cells were associated with both the PB presentation and also with leprosy reactions. In contrast, Th17 cells were associated with an effective inflammatory response that is present in the PB forms but were not predictive of leprosy reactions in MB patients.

Antigen-specific secretion of IFNγ and CXCL10 in whole blood assay detects Mycobacterium leprae infection but does not discriminate asymptomatic infection from symptomatic leprosy.

To advance toward a whole blood assay (WBA)-based test capable of facilitating the diagnosis of paucibacillary (PB) leprosy, we evaluated a prototype in-tube WBA using combinations of Mycobacterium leprae antigens. Blood was collected from newly diagnosed untreated PB (n=38), multibacillary (MB) (n=30), healthy household contacts (HHC) of MB (n=27), and endemic controls (n=61) residing in Goiânia and Fortaleza, Brazil. Blood was incubated with M. leprae cell sonicate, recombinant proteins (46f+LID-1; ML0276+LID-1), or controls (phosphate-buffered saline, phytohemagglutinin, M. tuberculosis purified protein derivative). Antigen-specific IFNγ production was observed in 71-84% and 55% of PB and HHC, respectively. Antigen-specific CXCL10 levels were similarly assessed to determine if, unlike IFNγ, CXCL10 could differentiate PB from HHC with repeated exposure/asymptomatic M. leprae infection. The CXCL10 levels induced in response to M. leprae antigens could not, however, differentiate PB from HHC. Despite these limitations, the WBAs reported here still represent important tools for assessing M. leprae infection rates and evaluating the impact of control measures.

CD4+ TCRγδ+ FoxP3+ cells: An unidentified population of immunosuppressive cells towards disease progression leprosy patients.

This study, for the first time, reveals the role of M. leprae-specific CD4+ TCRγδ+ FoxP3+ cells in the progression and pathogenesis of leprosy. Co-culture with CD4+ CD25- cells suggested the immunosuppressive nature of CD4+ TCRγδ+ cells in dose-dependent manner. Isolation of CD4+ TCRγδ+ cells from leprosy patients and then culture in presence of M. leprae cell wall antigens (MLCwA) along with TGF ß, IPP and IL-2 suggested that these cells are M. leprae specific. TGF-ß-mediated SMAD3 signalling was turned out to be major factor towards the expression of FoxP3 in these cells. SMAD3 silencing during induction of these cells barely showed the induction of FoxP3. High density of SMAD3 binding at TGFßRII in CD4+ TCRγδ+ FoxP3+ furthermore suggested the TGF-ß-directed SMAD3 signalling in these cells. Taken together the above data, we can conclude that CD4+ TCRγδ+ FoxP3+ cells possess the potential to track the severity of the disease in leprosy patients.

Estresse oxidativo e micronutrientes na hanseníase / Oxidative stress and micronutrients in leprosy

Objetivo Avaliar o estresse oxidativo, perfil antioxidante e de micronutrientes em pacientes portadores de hanseníase multibacilar e paucibacilar antes do tratamento poliquimioterápico. Métodos Analisaram-se 52 amostras de soro de pacientes portadores de hanseníase - 38 multibacilares e 14 paucibacilares -, usuários do ambulatório de dermatologia de um hospital público universitário, além de 30 amostras controles. Quantificaram-se marcador de peroxidação lipídica malondialdeído pelo método de substâncias reativas ao ácido tiobarbitúrico, antioxidante glutationa reduzida pelo método baseado na quantificação de tiol solúvel em ácido, antioxidante vitamina E por cromatografia líquida de alta eficiência, minerais selênio, zinco, cobre, magnésio por espectrometria de massa com fonte plasma acoplado, e sorologia do anticorpo glicolipídio fenólico I pelo método Enzyme-Linked Immunosorbent Assay. Foi utilizado teste não paramétrico de Mann-Whitney para comparar as variáveis quantificadas neste estudo entre os diferentes grupos, e correlação de Pearson para verificar associação dessas variáveis com o anticorpo. O critério de significância adotado foi de p<0,05. Resultados Houve diferença significativa para o malondialdeído (p<0,001) e vitamina E (p<0,001) no grupo controle comparado aos grupos com hanseníase, multibacilar e paucibacilar. No entanto, essas mesmas variáveis não diferiram entre os grupos multibacilar e paucibacilar (p=0,495 e p=0,920 respectivamente). A glutationa reduzida foi superior no grupo controle em relação ao grupo com hanseníase (p=0,012) e multibacilar (p=0,001), no entanto não diferiu do grupo paucibacilar (p=0,920). Quando comparada com os multibacilares e paucibacilares, a glutationa reduzida também não diferiu (p=0,063). Quanto aos minerais, todos se apresentaram dentro da normalidade, exceto o magnésio, cujos níveis foram deficientes em todos os pacientes do estudo. Não foi possível observar correlação do ...

Objective To determine the oxidative stress and the antioxidant and micronutrient profile of patients with multibacillary and paucibacillary leprosy before polychemotherapeutic treatment. Methods Thirty control samples and fifty-two serum samples from leprosy patients who attended the dermatology outpatient clinic of a public university hospital were analyzed; 38 of them had multibacillary and 14 paucibacillarty. Malondialdehyde, a marker of lipid peroxidation, was determined using the thiobarbituric acid reacting substances assay; the antioxidant reduced glutathione was determined using a method based on the quantification of acid-soluble thiol; the antioxidant vitamin E was determined using High-Performance Liquid Chromatography; the minerals selenium, zinc, copper and magnesium were determined using coupled-mass spectrometry, and the serum phenol I glycolipid antibody was determined using Enzyme-Linked Immunosorbent Assay. The nonparametric Mann-Whitney test was used to compare the variables quantified in the present study between the different groups, and Pearson's correlation analysis was used to verify the association between these variables and the antibody. The significance level was set at p<0.05. Results There was a significant difference in the content of malondialdehyde (p<0.001) and vitamin E (p<0.001) between the groups with multibacillary and paucibacillary leprosy (p=0.495 and p=0.920, respectively) and the control groups. Reduced glutathione levels were higher in the control group compared with those of the group with leprosy (p=0.012) and multibacillary leprosy (p=0.001), but did it not differ from that of the paucibacillary group (p=0.920). Reduced glutathione levels did not differ between the multibacillary and paucibacillary groups (p=0.063) either. All minerals were within normal limits, except for magnesium; magnesium deficiency was detected in all groups studied. No correlation was observed between the ...

Menstrual irregularities, fertility status, and ovarian function in female patients with leprosy in India.

BACKGROUND: Gonadal involvement in males in lepromatous leprosy is not uncommon, but there is a paucity of literature on the involvement of gonads in female patients with leprosy. This study was undertaken to determine if there is any menstrual dysfunction, alteration in fertility status and circulating luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, and estradiol in female patients with paucibacillary (PB) and multibacillary (MB) leprosy. MATERIALS AND METHODS: In a cross-sectional study, 229 patients with leprosy (79 with PB leprosy and 150 with MB leprosy) and 100 age-matched non-leprosy controls were evaluated for menstrual function, fertility status, and circulating sex hormones. RESULTS: Twenty percent of patients with MB leprosy had menstrual irregularities post-dating the onset of leprosy in comparison to 6.3% patients with PB leprosy, and this difference was statistically significant (P < 0.001). However, the fertility profile of patients with PB leprosy was comparable to that of patients with MB leprosy (P > 0.05). A significantly higher number of patients with MB leprosy (9.3%) had elevation of circulating FSH, LH, and prolactin vis-à-vis patients with PB leprosy (1.3%), and this difference was statistically significant (P < 0.05). Similarly, the mean levels of LH, FSH, and prolactin were significantly elevated in patients with MB leprosy vis-à-vis patients with PB leprosy and controls (P < 0.05). CONCLUSIONS: Multibacillary leprosy may be associated with menstrual irregularities and elevation of gonadotropin hormones, indicating an ovarian dysfunction.

Synergistic antigen combinations for the development of interferon gamma release assays for paucibacillary leprosy.

The development of immunodiagnostic tests for paucibacillary leprosy (PB) is based on Mycobacterium leprae specific-cell mediated immunity (CMI)/IFN-γ production. Recently, novel M. leprae protein antigens that stimulate CMI have been described. This study evaluated different M. leprae antigen combinations in whole blood assay (WBA). Five study groups were tested (20 per group): newly diagnosed, untreated PB patients and multibacillary leprosy patients (MB); household contacts of MB patients (HHC); healthy endemic controls (EC); pulmonary tuberculosis patients (TB). WBA (heparinized, 24 h 37 °C 5% CO2) were stimulated with: 10 µg/ml of each individual M. leprae recombinant protein (rML) and five combinations of rML (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044, ML0276 + 46f, ML2055 + LID-1)-M. leprae cell sonicate (MLCS, 10 µg/ml), PHA (1 µg/ml), and PBS alone. Human IFN-γ ELISA (QuantiFERON-TB Gold/QFT-G, Cellestis) was performed using stimulated plasma (arbitrary cut-off = 50 pg/ml). Three out of five antigen combinations (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044) were able to increase the levels of IFN-γ production in WBA in a larger number of responders among both PB leprosy and contacts. However, the magnitude of IFN-γ responses was higher among contacts. The antigen combination (46f + ML0276) stimulated IFN-γ only in symptomatic PB leprosy patients and not in asymptomatic contacts. Few controls (EC, TB) responded to combinations (0-15%), indicating the specificity of the response in an endemic area with high BCG coverage. The synergistic effect of new combinations of M. leprae proteins upon IFN-γ production in WBA indicates their potential use for the development of an interferon gamma release assay/IGRA for the diagnosis of PB leprosy.

Mycobacterium leprae DNA in peripheral blood may indicate a bacilli migration route and high-risk for leprosy onset.

Leprosy epidemiological studies have been restricted to Mycobacterium leprae DNA detection in nasal and oral mucosa samples with scarce literature on peripheral blood. We present the largest study applying quantitative real-time PCR (qPCR) for the detection of M. leprae DNA in peripheral blood samples of 200 untreated leprosy patients and 826 household contacts, with results associated with clinical and laboratory parameters. To detect M. leprae DNA a TaqMan qPCR assay targeting the M. leprae ML0024 genomic region was performed. The ML0024 qPCR in blood samples detected the presence of bacillus DNA in 22.0% (44/200) of the leprosy patients: 23.2% (16/69) in paucibacillary (PB), and 21.4% (28/131) in multibacillary (MB) patients. Overall positivity among contacts was 1.2% (10/826), with similar percentages regardless of whether the index case was PB or MB. After a follow-up period of 7 years, 26 contacts have developed leprosy. Comparing the results of healthy contacts with those that become ill, ML0024 qPCR positivity at the time of diagnosis of their index case represented an impressive 14.78-fold greater risk for leprosy onset (95% CI 3.6-60.8; p <0.0001). In brief, contacts with positive PCR in blood at diagnosis of index cases are at higher risk of later leprosy onset and this marker might be combined with other prognostic markers for management of contacts, which requires further studies.

Correlation between beta-2-glycoprotein I gene polymorphism and anti-beta-2 glycoprotein I antibodies in patients with multibacillary leprosy.

Antiphospholipid antibodies, such as anti-beta2-glycoprotein I (beta2GPI), are present in multibacillary leprosy (MB) patients; however, MB patients do not usually present with antiphospholipid antibody syndrome (APS), which is characterized by thromboembolic phenomena (TEP). Rare cases of TEP occur in leprosy patients, but the physiopathology of this condition remains unclear. In this case-control study, we examined whether single-nucleotide polymorphisms (SNPs) of the beta2GPI gene contributed to the risk of leprosy and APS co-morbidity. SNPs Ser88Asn, Leu247Val, Cys306Gly and Trp316Ser were identified in 113 Brazilian leprosy patients. Additionally, anti-beta2GPI antibodies and plasma concentrations of beta2GPI were quantified. The Ser88Asn, Cys306Gly and Trp316Ser SNPs were not risk factors for APS in leprosy. A higher frequency of Val/Val homozygosity was observed in leprosy patients compared to controls (36 vs. 5%; P < 0.001). Forty-two percent of MB and 17% of paucibacillary leprosy patients were positive for anti-beta2GPI IgM (P = 0.014). There was no correlation between SNP Ser88Asn or Cys306Gly and anti-beta2GPI antibody levels. In MB patients with positive anti-beta2GPI IgM, the frequency of Val/Val homozygosity was higher than in controls (32 vs. 15%; P = 0.042). The frequency of the mutant allele Ser316 was higher in MB patients with positive rather than negative anti-beta2GPI IgM levels (6 vs. 0%; P = 0.040) and was greater than in the control group (6 vs. 1%; P = 0.034). The studied polymorphisms did not influence the plasma concentrations of beta2GPI. These results suggest that Leu247Val and Trp316Ser SNPs may represent genetic risk factors for anti-beta2GPI antibody production in MB patients.