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1.
J Leukoc Biol ; 110(1): 167-176, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33040382

RESUMO

The enzyme IDO-1 is involved in the first stage of tryptophan catabolism and has been described in both microbicidal and tolerogenic microenvironments. Previous data from our group have shown that IDO-1 is differentially regulated in the distinctive clinical forms of leprosy. The present study aims to investigate the mechanisms associated with IDO-1 expression and activity in human monocyte-derived dendritic cells (mDCs) after stimulation with irradiated Mycobacterium leprae and its fractions. M. leprae and its fractions induced the expression and activity of IDO-1 in human mDCs. Among the stimuli studied, irradiated M. leprae and its membrane fraction (MLMA) induced the production of proinflammatory cytokines TNF and IL-6 whereas irradiated M. leprae and its cytosol fraction (MLSA) induced an increase in IL-10. We investigated if TLR2 activation was necessary for IDO-1 induction in mDCs. We observed that in cultures treated with a neutralizing anti-TLR2 antibody, there was a decrease in IDO-1 activity and expression induced by M. leprae and MLMA. The same effect was observed when we used a MyD88 inhibitor. Our data demonstrate that coculture of mDCs with autologous lymphocytes induced an increase in regulatory T (Treg) cell frequency in MLSA-stimulated cultures, showing that M. leprae constituents may play opposite roles that may possibly be related to the dubious effect of IDO-1 in the different clinical forms of disease. Our data show that M. leprae and its fractions are able to differentially modulate the activity and functionality of IDO-1 in mDCs by a pathway that involves TLR2, suggesting that this enzyme may play an important role in leprosy immunopathogenesis.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Tolerância Imunológica , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Hanseníase/etiologia , Hanseníase/metabolismo , Mycobacterium leprae/imunologia , Receptor 2 Toll-Like/metabolismo , Biomarcadores , Citometria de Fluxo , Humanos , Hanseníase/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
Cytokine ; 126: 154873, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31629113

RESUMO

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


Assuntos
Citocinas/metabolismo , Interleucina-17/metabolismo , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Neutrófilos/metabolismo , Células Th17/metabolismo , Adulto , Complexo CD3/metabolismo , Antígenos CD4/metabolismo , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Quimiocina CCL7/genética , Quimiocina CCL7/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Quimiocina CXCL6/genética , Quimiocina CXCL6/metabolismo , Citocinas/genética , Quimioterapia Combinada , Feminino , Citometria de Fluxo , Humanos , Inflamação/genética , Inflamação/metabolismo , Hanseníase/patologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Pessoa de Meia-Idade , Família Multigênica , Reação em Cadeia da Polimerase em Tempo Real
3.
J Infect Public Health ; 12(5): 656-659, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30904499

RESUMO

BACKGROUND AND OBJECTIVE: Leprosy is a chronic slowly progressive infection caused by Mycobacterium leprae that primarily affects the skin and peripheral nerves. Lepromatous leprosy is characterized by absence of T-cell responses to M. leprae and advanced clinical disease. It is frequently associated with the presence of autoantibodies, which might be related to CD19+CD5+ and CD19+CD5- B lymphocyte percentages. Therefore, the aim of this study was to evaluate the percentages of CD19+CD5+ and CD19+CD5- B cell subsets as well as the total B cells in lepromatous leprosy patients. MATERIALS AND METHODS: Twenty lepromatous leprosy patients and ten healthy subjects served as control were included in this study. Venous blood samples were analyzed by flow cytometry to determine the B cell subsets and total B cell percentages. RESULTS: Compared to healthy controls, the percentages of CD19+CD5+ B cell subset and total B cells were found to be significantly higher in the patient group. While, the percentage of CD19+CD5- B cell subset was found to be higher in the patient group than the control without any significantly difference. Regarding the eye affection, the percentage of total B cells was observed to be significantly higher in affected patients compared to the non-affected group. CONCLUSION: The observed significant increases in CD19+CD5+ and total B cell percentages in patients with lepromatous leprosy suggests a possible role of these cells in the disorganized protective immune response as well as the development of eye complications in these patients.


Assuntos
Doenças Autoimunes/imunologia , Subpopulações de Linfócitos B/imunologia , Antígenos CD5/imunologia , Hanseníase Virchowiana/imunologia , Doenças Autoimunes/microbiologia , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Masculino , Mycobacterium leprae/imunologia , Fatores de Risco , Pele/imunologia , Pele/microbiologia
4.
Immunol Lett ; 207: 6-16, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30629982

RESUMO

The present study evaluates role of Notch1 signaling in the regulation of T cell immunity in leprosy. Peripheral blood mononuclear cells from leprosy patients and healthy controls were activated with Mycobacterium leprae antigens along with activation of Notch1 signaling pathway and then lymphoproliferation was analyzed by lymphocytes transformation test and the expression of Notch1 and its ligands DLL1, Jagged1 and Jagged 2, T cell activation marker and Th1-Th2 cytokines on Th cells in PBMCs of study subjects were analyzed by flow cytometry. Further, these parameters were also analyzed after inhibition of Notch1 signaling pathway. Higher percentage of Notch1expressing Th cells were noted in TT/BT cases and higher percentage of DLL1 expressing Th cells in TT/BT and BL/LL cases. M. leprae antigens were found to induce the expression of Jagged1 on Th cells. Interestingly activation of Notch1 signaling pathway induced lymphoproliferation in BL/LL cases in response of PGL-1. Activation of Notch1 signaling was also found to induce the expression of T cell activation markers CD25, CD69 and Th1 cytokine IFN-γ in response to M. leprae antigens. Immunomodulation through Notch1 signaling seen in our study could be helpful in augmenting Th1 response in leprosy.


Assuntos
Antígenos de Bactérias/imunologia , Glicolipídeos/imunologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Receptor Notch1/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Progressão da Doença , Citometria de Fluxo , Humanos , Imunomodulação , Interferon gama/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transdução de Sinais , Equilíbrio Th1-Th2
5.
Microbes Infect ; 21(2): 94-98, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30389463

RESUMO

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


Assuntos
Antígeno B7-1/análise , Antígeno B7-2/análise , Antígenos CD40/análise , Hanseníase/imunologia , Adolescente , Adulto , Idoso , Apresentação do Antígeno , Biomarcadores/análise , Estudos Transversais , Feminino , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Adulto Jovem
6.
J Vet Sci ; 19(6): 744-749, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30304888

RESUMO

Dapsone, an antibiotic, has been used to cure leprosy. It has been reported that dapsone has anti-inflammatory activity in hosts; however, the anti-inflammatory mechanism of dapsone has not been fully elucidated. The present study investigated the anti-inflammatory effects of dapsone on bone marrow cells (BMs), especially upon exposure to lipopolysaccharide (LPS). We treated BMs with LPS and dapsone, and the treated cells underwent cellular activity assay, flow cytometry analysis, cytokine production assessment, and reactive oxygen species assay. LPS distinctly activated BMs with several characteristics including high cellular activity, granulocyte changes, and tumor necrosis factor alpha (TNF-α) production increases. Interestingly, dapsone modulated the inflammatory cells, including granulocytes in LPS-treated BMs, by inducing cell death. While the percentage of Gr-1 positive cells was 57% in control cells, LPS increased that to 75%, and LPS plus dapsone decreased it to 64%. Furthermore, dapsone decreased the mitochondrial membrane potential of LPS-treated BMs. At a low concentration (25 µg/mL), dapsone significantly decreased the production of TNF-α in LPS-treated BMs by 54%. This study confirmed that dapsone has anti-inflammatory effects on LPS-mediated inflammation via modulation of the number and function of inflammatory cells, providing new and useful information for clinicians and researchers.


Assuntos
Células da Medula Óssea/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Dapsona/farmacologia , Lipopolissacarídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células da Medula Óssea/metabolismo , Regulação para Baixo/efeitos dos fármacos , Citometria de Fluxo , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo
7.
Scand J Immunol ; 86(1): 40-49, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28426172

RESUMO

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.


Assuntos
Mediadores da Inflamação/imunologia , Hanseníase Paucibacilar/imunologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Células Th1/imunologia , Células Th17/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Criança , Busca de Comunicante , Feminino , Citometria de Fluxo , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Interferon gama/sangue , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/sangue , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/sangue , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-1beta/sangue , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Hanseníase/sangue , Hanseníase/microbiologia , Hanseníase Multibacilar/sangue , Hanseníase Multibacilar/imunologia , Hanseníase Multibacilar/microbiologia , Hanseníase Paucibacilar/sangue , Hanseníase Paucibacilar/microbiologia , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Mycobacterium leprae/fisiologia , Células Th1/metabolismo , Células Th17/metabolismo , Adulto Jovem
8.
Mol Immunol ; 83: 72-81, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28110210

RESUMO

Leprosy is a bacterial disease caused by M. leprae. Its clinical spectrum reflects the host's immune response to the M. leprae and provide an ideal model to investigate the host pathogen interaction and immunological dysregulation. Tregs are high in leprosy patients and responsible for immune suppression of the host by producing IL-10 and TGF-ß cytokines. In leprosy, plasticity of Tregs remain unstudied. This is the first study describing the conversion of Tregs into Th1-like and Th17-like cells using in vitro cytokine therapy in leprosy patients. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA), rIL-12 and rIL-23 for 48h. Expression of FoxP3 in CD4+CD25+ Tregs, intracellular cytokines IFN-γ, TGF-ß, IL-10 and IL-17 in Tregs cells were evaluated by flow cytometry (FACS) after stimulation. rIL-12 treatment increases the levels of pStat4 in Tregs and IFN-γ production. In the presence of rIL-23, pStat3+ and IL-17A+ cells increase. rIL-12 and r-IL-23 treatment downregulated the FoxP3 expression, IL-10 and TGF-ß production by Tregs and enhances the expression of co-stimulatory molecules (CD80, CD86). In conclusion rIL-12 converts Tregs into IFN-γ producing cells through STAT-4 signaling while rIL-23 converts Tregs into IL-17 producing cells through STAT-3 signaling in leprosy patients. This study may helpful to provide a new avenue to overcome the immunosuprression in leprosy patients using in vitro cytokine.


Assuntos
Diferenciação Celular/imunologia , Interleucina-12/imunologia , Interleucina-23/imunologia , Hanseníase/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Western Blotting , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
9.
J Immunol ; 197(5): 1905-13, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27474073

RESUMO

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


Assuntos
DNA/metabolismo , Eritema Nodoso/imunologia , Imunidade Inata , Hanseníase Virchowiana/imunologia , Transdução de Sinais , Receptor Toll-Like 9/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Eritema Nodoso/microbiologia , Feminino , Citometria de Fluxo , Humanos , Hanseníase Virchowiana/microbiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/química , Mycobacterium leprae/imunologia , Receptor Toll-Like 9/imunologia , Adulto Jovem
10.
PLoS Pathog ; 12(6): e1005705, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27355424

RESUMO

Triggering antimicrobial mechanisms in macrophages infected with intracellular pathogens, such as mycobacteria, is critical to host defense against the infection. To uncover the unique and shared antimicrobial networks induced by the innate and adaptive immune systems, gene expression profiles generated by RNA sequencing (RNAseq) from human monocyte-derived macrophages (MDMs) activated with TLR2/1 ligand (TLR2/1L) or IFN-γ were analyzed. Weighed gene correlation network analysis identified modules of genes strongly correlated with TLR2/1L or IFN-γ that were linked by the "defense response" gene ontology term. The common TLR2/1L and IFN-γ inducible human macrophage host defense network contained 16 antimicrobial response genes, including S100A12, which was one of the most highly induced genes by TLR2/1L. There is limited information on the role of S100A12 in infectious disease, leading us to test the hypothesis that S100A12 contributes to host defense against mycobacterial infection in humans. We show that S100A12 is sufficient to directly kill Mycobacterium tuberculosis and Mycobacterium leprae. We also demonstrate that S100A12 is required for TLR2/1L and IFN-γ induced antimicrobial activity against M. leprae in infected macrophages. At the site of disease in leprosy, we found that S100A12 was more strongly expressed in skin lesions from tuberculoid leprosy (T-lep), the self-limiting form of the disease, compared to lepromatous leprosy (L-lep), the progressive form of the disease. These data suggest that S100A12 is part of an innate and adaptive inducible antimicrobial network that contributes to host defense against mycobacteria in infected macrophages.


Assuntos
Hanseníase/imunologia , Macrófagos/imunologia , Proteína S100A12/imunologia , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Macrófagos/microbiologia , Infecções por Mycobacterium/imunologia , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma
11.
PLoS Negl Trop Dis ; 10(4): e0004592, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27035913

RESUMO

BACKGROUND: 50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4+ subsets of Th17 cells and CD25+FOXP3+ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions. METHODOLOGY AND PRINCIPLE FINDINGS: Quantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3+ Treg cells as well as reduction in TGF-ß and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients. CONCLUSIONS: Patients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-ß and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.


Assuntos
Interleucina-6/metabolismo , Hanseníase/patologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Fator de Crescimento Transformador beta/metabolismo , Adulto , Animais , Biópsia , Sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Masculino , Camundongos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto Jovem
12.
Am J Trop Med Hyg ; 94(4): 721-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26903606

RESUMO

Leprosy is frequently complicated by the appearance of reactions that are difficult to treat and are the main cause of sequelae. We speculated that disturbances in regulatory T-cells (Tregs) could play a role in leprosy reactions. We determined the frequency of circulating Tregs in patients with type 1 reaction (T1R) and type 2 reaction (T2R). The in situ frequency of Tregs and interleukin (IL)-17, IL-6, and transforming growth factor beta (TGF)-ß-expressing cells was also determined. T2R patients showed markedly lower number of circulating and in situ Tregs than T1R patients and controls. This decrease was paralleled by increased in situ IL-17 expression but decreased TGF-ß expression. Biopsies from T1R and T2R patients before the reaction episodes showed similar number of forkhead box protein P3+ (FoxP3+) and IL-17+ cells. However, in biopsies taken during the reaction, T2R patients showed a decrease in Tregs and increase in IL-17+ cells, whereas T1R patients showed the opposite: Tregs increased but IL-17+ cells decreased. We also found decreased expansion of Tregs upon in vitro stimulation with Mycobacterium leprae and a trend for lower expression of FoxP3 and the immunosuppressive molecule cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in T2R Tregs. Our results provide some evidence to the hypothesis that, in T2R, downmodulation of Tregs may favor the development of T-helper-17 responses that characterize this reaction.


Assuntos
Hanseníase/imunologia , Linfócitos T Reguladores/fisiologia , Adulto , Feminino , Citometria de Fluxo , Humanos , Imunidade Celular , Interleucina-17/sangue , Interleucina-6/sangue , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/sangue
13.
Braz. j. med. biol. res ; 48(12): 1095-1100, Dec. 2015. graf
Artigo em Inglês | LILACS | ID: lil-762920

RESUMO

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.


Assuntos
Animais , Masculino , Camundongos , Linfócitos B/imunologia , Proteínas de Choque Térmico/imunologia , Imunomodulação/genética , /genética , RNA Mensageiro/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos B/metabolismo , Citometria de Fluxo , Expressão Gênica/genética , Proteínas de Choque Térmico/uso terapêutico , Memória Imunológica/fisiologia , Imunofenotipagem/classificação , Mediadores da Inflamação/análise , Interferon gama/análise , /imunologia , /análise , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/genética , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/classificação , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico
14.
Artigo em Inglês | MEDLINE | ID: mdl-26577191

RESUMO

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0(+)CD69(+)CD4(+) memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Citometria de Fluxo/métodos , Testes de Liberação de Interferon-gama/métodos , Interferon gama/sangue , Tuberculose Bovina/diagnóstico , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Bovinos , Memória Imunológica , Lectinas Tipo C/imunologia , Antígenos Comuns de Leucócito/imunologia , Linfocinas , Mycobacterium bovis/imunologia , Peptídeos/imunologia , Sensibilidade e Especificidade , Teste Tuberculínico
15.
J Transl Med ; 13: 296, 2015 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-26362198

RESUMO

BACKGROUND: Peripheral nerve injury and bone lesions, well known leprosy complications, lead to deformities and incapacities. The phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX) encodes a homonymous protein (PHEX) implicated in bone metabolism. PHEX/PHEX alterations may result in bone and cartilage lesions. PHEX expression is downregulated by intracellular Mycobacterium leprae (M. leprae) in cultures of human Schwann cells and osteoblasts. M. leprae in vivo effect on PHEX/PHEX is not known. METHODS: Cross-sectional observational study of 36 leprosy patients (22 lepromatous and 14 borderline-tuberculoid) and 20 healthy volunteers (HV). The following tests were performed: PHEX flow cytometric analysis on blood mononuclear cells, cytokine production in culture supernatant, 25-hydroxyvitamin D (OHvitD) serum levels and (99m)Tc-MDP three-phase bone scintigraphy, radiography of upper and lower extremities and blood and urine biochemistry. RESULTS: Significantly lower PHEX expression levels were observed in lepromatous patients than in the other groups (χ(2) = 16.554, p < 0.001 for lymphocytes and χ(2) = 13.933, p = 0.001 for monocytes). Low levels of 25-(OHvitD) were observed in HV (median = 23.0 ng/mL) and BT patients (median = 27.5 ng/mL) and normal serum levels were found in LL patients (median = 38.6 ng/mL). Inflammatory cytokines, such as TNF, a PHEX transcription repressor, were lower after stimulation with M. leprae in peripheral blood mononuclear cells from lepromatous in comparison to BT patients and HV (χ(2) = 10.820, p < 0.001). CONCLUSION: Downregulation of PHEX may constitute an important early component of bone loss and joint damage in leprosy. The present results suggest a direct effect produced by M. leprae on the osteoarticular system that may use this mechanism.


Assuntos
Regulação para Baixo , Hanseníase Dimorfa/metabolismo , Hanseníase Multibacilar/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Osso e Ossos/microbiologia , Cartilagem/microbiologia , Estudos Transversais , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Voluntários Saudáveis , Humanos , Inflamação/metabolismo , Inflamação/microbiologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Osteoblastos/microbiologia , Células de Schwann/microbiologia , Medronato de Tecnécio Tc 99m , Adulto Jovem
16.
Braz J Med Biol Res ; 48(12): 1095-100, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26397973

RESUMO

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.


Assuntos
Linfócitos B/imunologia , Proteínas de Choque Térmico/imunologia , Imunomodulação/genética , Interleucina-10/genética , RNA Mensageiro/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos B/metabolismo , Citometria de Fluxo , Expressão Gênica/genética , Proteínas de Choque Térmico/uso terapêutico , Memória Imunológica/fisiologia , Imunofenotipagem/classificação , Mediadores da Inflamação/análise , Interferon gama/análise , Interleucina-10/imunologia , Interleucina-12/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/classificação , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico
17.
Cytometry A ; 87(9): 855-67, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26109497

RESUMO

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.


Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes/análise , Macrófagos/química , Macrófagos/fisiologia , Xantenos/análise , Xenobióticos/análise , Animais , Linhagem Celular , Clofazimina/análise , Clofazimina/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Xenobióticos/farmacologia
18.
Neonatology ; 106(4): 355-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25277063

RESUMO

We evaluated a neonate with severe jaundice but a negative family history. Spherocytes were present and suspected hereditary spherocytosis was confirmed by osmotic fragility and eosin-5-maleimide erythrocyte staining. We found he was a compound heterozygote for two pathogenic mutations in the gene encoding α-spectrin: a previously reported α(LEPRA) inherited from his asymptomatic mother, and a novel α-spectrin mutation in intron 45 +1 disrupting the consensus splice site, from his asymptomatic father.


Assuntos
Anemia Hemolítica Congênita/genética , Anquirinas/deficiência , Icterícia Neonatal/genética , Icterícia Obstrutiva/genética , Mutação , Espectrina/genética , Esferocitose Hereditária/genética , Anemia Hemolítica Congênita/sangue , Anemia Hemolítica Congênita/diagnóstico , Anquirinas/sangue , Anquirinas/genética , Análise Mutacional de DNA , Amarelo de Eosina-(YS)/análogos & derivados , Citometria de Fluxo , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Humanos , Lactente , Recém-Nascido , Icterícia Neonatal/sangue , Icterícia Neonatal/diagnóstico , Icterícia Obstrutiva/sangue , Icterícia Obstrutiva/diagnóstico , Masculino , Fragilidade Osmótica , Linhagem , Fenótipo , Valor Preditivo dos Testes , Índice de Gravidade de Doença , Esferocitose Hereditária/sangue , Esferocitose Hereditária/diagnóstico
19.
Int J Food Microbiol ; 191: 1-9, 2014 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-25218463

RESUMO

Various molecular approaches have been applied as culture-independent techniques to monitor wine fermentations over the last decade. Among them, those based on RNA detection have been widely used for yeast cell detection, assuming that RNA only exists in live cells. Fluorescence in situ hybridization (FISH) targeting intracellular rRNA is considered a promising technique for the investigation of wine ecology. For the present study, we applied the FISH technique in combination with epifluorescence microscopy and flow cytometry to directly quantify populations of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris during alcoholic fermentations. A new specific probe that hybridizes with eight species of Hanseniaspora genus and a second probe specific for Starm. bacillaris were designed, and the conditions for their application to pure cultures, mixed cultures, and wine samples were optimized. Single and mixed fermentations were performed with natural, concentrated must at two different temperatures, 15 °C and 25 °C. The population dynamics revealed that the Sacch. cerevisiae population increased to 10(7)-10(8)cells/ml during all fermentations, whereas H. uvarum and Starm. bacillaris tended to increase in single fermentations but remained at levels similar to their inoculations at 10(6)cells/ml in mixed fermentations. Temperature mainly affected the fermentation duration (slower at the lower temperature) but did not affect the population sizes of the different species. The use of these probes in natural wine fermentations has been validated.


Assuntos
Ascomicetos/fisiologia , Fermentação , Citometria de Fluxo/normas , Microbiologia de Alimentos/métodos , Hanseniaspora/fisiologia , Hibridização in Situ Fluorescente/normas , Saccharomyces cerevisiae/fisiologia , Ascomicetos/genética , Hanseniaspora/genética , RNA Ribossômico , Saccharomyces cerevisiae/genética , Temperatura , Vinho/microbiologia
20.
Arch Dermatol Res ; 306(9): 793-801, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25018055

RESUMO

Leprosy is not only a bacteriological disease but also an immunological disease, in which T helper17 and CD4(+) CD25(high)FoxP3(+) regulatory T cells (T-regs), among others, may play a role. We aimed to evaluate serum levels of interleukin (IL)-17, IL-22 (Th17 cytokines), IL-10 and transforming growth factor (TGF)-ß (down regulatory cytokines) in 43 untreated leprosy patients and 40 controls by enzyme-linked immunosorbent assay, and to assess circulating CD4(+) CD25(high)FoxP3(+)T-regs in patients using flow cytometry. Patients were grouped into tuberculoid, pure neural, borderline, lepromatous, type 1 reactional leprosy, and erythema nodosum leprosum. IL-10 and TGF-ß were significantly higher in patients as compared to controls (p < 0.001), while IL-17, but not IL-22, was significantly lower (p < 0.001), with no significant difference comparing patients' subgroups. Significantly higher CD4(+) CD25(high)FoxP3(+)T-regs levels was detected in tuberculoid, type 1 reaction and pure neural leprosy, while the lowest levels in erythema nodosum leprosum (p < 0.001). TregsFoxP3 expression% was significantly lower in pure neural leprosy than other patients' subgroups (p < 0.05). T-regs/T-effs was lowest in erythema nodosum leprosum (p < 0.05). TGF-ß correlated negatively with TregsFoxP3 expression% and T-effs% (p = 0.009 and 0.018 respectively). Leprosy is associated with defective IL-17 and overproduction of IL-10 and TGF-ß. Tuberculoid, type 1 reaction and pure neural leprosy express significantly higher circulating T-regs, consistent with effector immune mechanisms activation, but with lower TregsFoxP3 expression (in pure neural leprosy). Erythema nodosum leprosum is characterized by deficient T-regs and increased TregsFoxP3 expression%. The present study pinpointed a potential role of Th17, CD4(+) CD25(high)FoxP3(+)T-regs, and probably CD4(+) CD25(+)IL-10(+) T regulatory cells 1 (Tr1), and Th3 in leprosy.


Assuntos
Citocinas/sangue , Fatores de Transcrição Forkhead/sangue , Subunidade alfa de Receptor de Interleucina-2/sangue , Hanseníase/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Adolescente , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Interleucina-10/sangue , Interleucina-17/sangue , Interleucinas/sangue , Hanseníase/sangue , Hanseníase/diagnóstico , Hanseníase/microbiologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/microbiologia , Células Th17/microbiologia , Fator de Crescimento Transformador beta/sangue , Adulto Jovem
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