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1.
Nature ; 593(7859): 418-423, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33727703

RESUMO

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.


Assuntos
Antivirais/farmacologia , Clofazimina/farmacologia , Coronavirus/classificação , Coronavirus/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antivirais/farmacocinética , Antivirais/uso terapêutico , Disponibilidade Biológica , Fusão Celular , Linhagem Celular , Clofazimina/farmacocinética , Clofazimina/uso terapêutico , Coronavirus/crescimento & desenvolvimento , Coronavirus/patogenicidade , Cricetinae , DNA Helicases/antagonistas & inibidores , Sinergismo Farmacológico , Feminino , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Masculino , Mesocricetus , Profilaxia Pré-Exposição , SARS-CoV-2/crescimento & desenvolvimento , Especificidade da Espécie , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/genética
2.
PLoS One ; 16(3): e0249184, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33770127

RESUMO

Mycobacterium leprae (M. leprae) is the etiological agent of leprosy, and the skin lesions of lepromatous leprosy are filled with numerous foamy or xanthomatous histiocytes that are parasitized by M. leprae. Lipids are an important nutrient for the intracellular survival of M. leprae. In this study, we attempted to determine the intracellular lipid composition and underlying mechanisms for changes in host cell lipid metabolism induced by M. leprae infection. Using high-performance thin-layer chromatography (HPTLC), we demonstrated specific induction of triacylglycerol (TAG) production in human macrophage THP-1 cells following M. leprae infection. We then used [14C] stearic acid tracing to show incorporation of this newly synthesized host cell TAG into M. leprae. In parallel with TAG accumulation, expression of host glycerol-3-phosphate acyltransferase 3 (GPAT3), a key enzyme in de novo TAG synthesis, was significantly increased in M. leprae-infected cells. CRISPR/Cas9 genome editing of GPAT3 in THP-1 cells (GPAT3 KO) dramatically reduced accumulation of TAG following M. leprae infection, intracellular mycobacterial load, and bacteria viability. These results together suggest that M. leprae induces host GPAT3 expression to facilitate TAG accumulation within macrophages to maintain a suitable environment that is crucial for intracellular survival of these bacilli.


Assuntos
Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Fator de Transcrição STAT3/genética , Triglicerídeos/biossíntese , Linhagem Celular , Expressão Gênica , Humanos , Monócitos/citologia
3.
Immunity ; 53(4): 878-894.e7, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-33053333

RESUMO

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inflamação/genética , RNA Citoplasmático Pequeno/genética , Pele/patologia , Animais , Linhagem Celular , DNA Complementar/genética , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcrição Genética/genética , Transcriptoma/genética
4.
Bioorg Med Chem Lett ; 30(20): 127469, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32768650

RESUMO

The pentacyclic triterpene 3ß,6ß,16ß-tri-hydroxilup-20(29)-ene is a natural product produced by the Brazilian medicinal plant Combretum leprosum. Its cytotoxicity has been previously reported against breast cancer cell lines. The low water solubility of this natural product, that hampers its bioavailability, motivated the investigation of a new nanoparticle formulation containing the triterpene in order to improve its bioactivity. The triterpene was encapsulated in polycaprolactone (PCL) polymer by nanoprecipitation, producing homogenic nanoparticles with nanometer sizes (122.7 ± 2.06 nm), which were characterized by FT-IR, SEM imaging and DSC. The cytotoxicity (MTT method) of the nanoparticle containing the triterpene 1, besides the free natural product and the nanoparticle control (without 1), was assayed against three human tumor cell lines [human colon carcinoma line (HCT116), prostate (PC3) and glioblastoma (SNB19)] and the normal epithelial embryo kidney human cell line (Hek293T). The nanocarrier produced a significative effect in the cytotoxicity of the natural product in the nanoformulation (IC50 0.11-0.26 µg mL-1) when compared with its free form (IC50 1.07-1.44 µg mL-1). Additionally, higher selectivity of the triterpene to the tumor cells was found when it was encapsulated (SI 1.92-4.54) than in its free form (SI 0.42-0.56). In this case, the nanoencapsulated triterpene was more selective to PC3 (SI 3.33) and SNB19 (SI 4.54) tumor cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Combretum/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Cápsulas , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Folhas de Planta/química , Relação Estrutura-Atividade
5.
J Immunol ; 205(1): 102-112, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32434940

RESUMO

To maintain homeostasis, macrophages must be capable of assuming either an inflammatory or an anti-inflammatory phenotype. To better understand the latter, we stimulated human macrophages in vitro with TLR ligands in the presence of high-density immune complexes (IC). This combination of stimuli resulted in a broad suppression of inflammatory mediators and an upregulation of molecules involved in tissue remodeling and angiogenesis. Transcriptomic analysis of TLR stimulation in the presence of IC predicted the downstream activation of AKT and the inhibition of GSK3. Consequently, we pretreated LPS-stimulated human macrophages with small molecule inhibitors of GSK3 to partially phenocopy the regulatory effects of stimulation in the presence of IC. The upregulation of DC-STAMP and matrix metalloproteases was observed on these cells and may represent potential biomarkers for this regulatory activation state. To demonstrate the presence of these anti-inflammatory, growth-promoting macrophages in a human infectious disease, biopsies from patients with leprosy (Hanseniasis) were analyzed. The lepromatous form of this disease is characterized by hypergammaglobulinemia and defective cell-mediated immunity. Lesions in lepromatous leprosy contained macrophages with a regulatory phenotype expressing higher levels of DC-STAMP and lower levels of IL-12, relative to macrophages in tuberculoid leprosy lesions. Therefore, we propose that increased signaling by FcγR cross-linking on TLR-stimulated macrophages can paradoxically promote the resolution of inflammation and initiate processes critical to tissue growth and repair. It can also contribute to infectious disease progression.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Hanseníase Virchowiana/imunologia , Hanseníase Tuberculoide/imunologia , Macrófagos/imunologia , Biópsia , Diferenciação Celular/imunologia , Linhagem Celular , Progressão da Doença , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Hanseníase Virchowiana/patologia , Hanseníase Tuberculoide/patologia , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Neovascularização Fisiológica/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA-Seq , Receptores de IgG/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/citologia , Pele/imunologia , Pele/patologia , Receptores Toll-Like/metabolismo , Adulto Jovem
6.
Stem Cell Res ; 45: 101804, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32339904

RESUMO

Mutations in the Leucine rich repeat kinase 2 (LRRK2) gene are found in both familial and sporadic Parkinson's disease (PD), and are also associated with immune-related disorders including Crohn's disease (CD) and leprosy. We have generated two homozygous LRRK2 knockout human induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 in a well-characterized human iPSC clone. The LRRK2 knockout cell lines retained normal morphology, gene expression, and the capacity to differentiate into cell types of the three germ layers. These cell lines are valuable for elucidating the role of LRRK2 in innate immunity and PD.


Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Sistemas CRISPR-Cas/genética , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Doença de Parkinson/genética
7.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848273

RESUMO

New approaches are needed to control leprosy, but understanding of the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied 13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary host cell, the Schwann cell. We compared the results of this analysis with those of a related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme as a potential antileprosy drug target.IMPORTANCE Leprosy remains a major problem in the world today, particularly affecting the poorest and most disadvantaged sections of society in the least developed countries of the world. The long-term aim of research is to develop new treatments and vaccines, and these aims are currently hampered by our inability to grow the pathogen in axenic culture. In this study, we probed the metabolism of M. leprae while it is surviving and replicating inside its primary host cell, the Schwann cell, and compared it to a related pathogen, M. tuberculosis, replicating in macrophages. Our analysis revealed that unlike M. tuberculosis, M. leprae utilized host glucose as a carbon source and that it biosynthesized its own amino acids, rather than importing them from its host cell. We demonstrated that the enzyme phosphoenolpyruvate carboxylase plays a crucial role in glucose catabolism in M. leprae Our findings provide the first metabolic signature of M. leprae in the host Schwann cell and identify novel avenues for the development of antileprosy drugs.


Assuntos
Carbono/metabolismo , Glucose/metabolismo , Mycobacterium leprae/fisiologia , Células de Schwann/metabolismo , Células de Schwann/microbiologia , Metabolismo dos Carboidratos , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Hanseníase/metabolismo , Hanseníase/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Redes e Vias Metabólicas
8.
Food Res Int ; 126: 108616, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31732074

RESUMO

A novel valorization strategy is proposed in this work for the sustainable utilization of a major mango processing waste (i.e. mango seed kernel, MSK), integrating green pressurized-liquid extraction (PLE), bioactive assays and comprehensive HRMS-based phytochemical characterization to obtain bioactive-rich fractions with high antioxidant capacity and antiproliferative activity against human colon cancer cells. Thus, a two steps PLE procedure was proposed to recover first the non-polar fraction (fatty acids and lipids) and second the polar fraction (polyphenols). Efficient selection of the most suitable solvent for the second PLE step (ethanol/ethyl acetate mixture) was based on the Hansen solubility parameters (HSP) approach. A comprehensive GC- and LC-Q-TOF-MS/MS profiling analysis allowed the complete characterization of the lipidic and phenolic fractions obtained under optimal condition (100% EtOH at 150 °C), demonstrating the abundance of oleic and stearic acids, as well as bioactive xanthones, phenolic acids, flavonoids, gallate derivatives and gallotannins. The obtained MSK-extract exhibited higher antiproliferative activity against human colon adenocarcinoma cell line HT-29 compared to traditional extraction procedures described in literature for MSK utilization (e.g. Soxhlet), demonstrating the great potential of the proposed valorization strategy as a valuable opportunity for mango processing industry to deliver a value-added product to the market with health promoting properties.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Resíduos Industriais/análise , Mangifera/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Sementes/química , Antioxidantes/análise , Linhagem Celular , Proliferação de Células , Neoplasias do Colo/patologia , Manipulação de Alimentos/métodos , Frutas , Células HT29 , Humanos , Fenóis/análise , Compostos Fitoquímicos/análise , Espectrometria de Massas em Tandem , Xantonas/análise
9.
Sci Rep ; 9(1): 11413, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31388035

RESUMO

Thalidomide is widely used for several diseases; however, it causes malformations in embryos exposed during pregnancy. The complete understanding of the mechanisms by which thalidomide affects the embryo development has not yet been obtained. The phenotypic similarity makes TE a phenocopy of syndromes caused by mutations in ESCO2, SALL4 and TBX5 genes. Recently, SALL4 and TBX5 were demonstrated to be thalidomide targets. To understand if these genes act in the TE development, we sequenced them in 27 individuals with TE; we verified how thalidomide affect them in human pluripotent stem cells (hPSCs) through a differential gene expression (DGE) analysis from GSE63935; and we evaluated how these genes are functionally related through an interaction network analysis. We identified 8 variants in ESCO2, 15 in SALL4 and 15 in TBX5. We compared allelic frequencies with data from ExAC, 1000 Genomes and ABraOM databases; eight variants were significantly different (p < 0.05). Eleven variants in SALL4 and TBX5 were previously associated with cardiac diseases or malformations; however, in TE sample there was no association. Variant effect prediction tools showed 97% of the variants with potential to influence in these genes regulation. DGE analysis showed a significant reduction of ESCO2 in hPSCs after thalidomide exposure.


Assuntos
Acetiltransferases/genética , Proteínas Cromossômicas não Histona/genética , Predisposição Genética para Doença , Proteínas com Domínio T/genética , Teratogênese/genética , Talidomida/efeitos adversos , Fatores de Transcrição/genética , Anormalidades Múltiplas/induzido quimicamente , Anormalidades Múltiplas/genética , Brasil , Linhagem Celular , Anormalidades Craniofaciais/induzido quimicamente , Anormalidades Craniofaciais/genética , Conjuntos de Dados como Assunto , Síndrome da Retração Ocular/induzido quimicamente , Síndrome da Retração Ocular/genética , Ectromelia/induzido quimicamente , Ectromelia/genética , Feminino , Perfilação da Expressão Gênica , Frequência do Gene , Cardiopatias Congênitas/induzido quimicamente , Cardiopatias Congênitas/genética , Comunicação Interatrial/induzido quimicamente , Comunicação Interatrial/genética , Humanos , Hipertelorismo/induzido quimicamente , Hipertelorismo/genética , Hanseníase/tratamento farmacológico , Deformidades Congênitas das Extremidades Inferiores/induzido quimicamente , Deformidades Congênitas das Extremidades Inferiores/genética , Masculino , Mutação , Células-Tronco Pluripotentes , Polimorfismo de Nucleotídeo Único , Gravidez , Complicações na Gravidez/tratamento farmacológico , Mapas de Interação de Proteínas/genética , Teratogênese/efeitos dos fármacos , Deformidades Congênitas das Extremidades Superiores/induzido quimicamente , Deformidades Congênitas das Extremidades Superiores/genética
11.
J Dermatol ; 46(10): 853-858, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432529

RESUMO

In patients with lepromatous leprosy, Mycobacterium leprae is often observed inside the human microvascular endothelial cells (HMVEC) surrounding Schwann cells (SC) at the site of lesions in the peripheral nerves. Based on this observation, it is considered that the nasal mucous may be the invasion pathway for M. leprae and HMVEC serve as an important reservoir for the bacteria before they invade SC. In light of previous research which revealed that Mce1A protein mediates bacterial invasion into nasal epithelial cells and HMVEC, we conducted a study to determine whether the invasion of M. leprae into HMVEC can be suppressed by blocking the Mce1A protein. In this study, we analyzed bacterial invasive activity by adding recombinant Escherichia coli, which express the active region (InvX:72 a.a.) of Mce1A protein on their external membrane, into cultured HMVEC, using the adhesin involved in the diffuse adherence mechanism. The number of bacteria that invaded into the cells was then measured by a colony counting method. The active region of Mce1A was divided into four sections, and hyperimmune antisera was prepared for each section for analyzing the inhibitory effect against invasion. The invasive activity was suppressed by antibodies against InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. This suggests that the InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. of Mce1A protein play an important role in the invasion of M. leprae into HMVEC and that it may be possible to suppress entry of M. leprae in HMVEC with antibodies against these regions.


Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/imunologia , Células Endoteliais/microbiologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Animais , Anticorpos Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Linhagem Celular , Contagem de Colônia Microbiana , Humanos , Soros Imunes/imunologia , Soros Imunes/isolamento & purificação , Hanseníase/microbiologia , Hanseníase/prevenção & controle , Mycobacterium leprae/patogenicidade , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
12.
PLoS Negl Trop Dis ; 13(3): e0006704, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30835734

RESUMO

Leprosy is a chronic infection where the skin and peripheral nervous system is invaded by Mycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet for M. leprae. Mce1A protein (442 aa) is coded by mce1A (1326 bp) of M. leprae. The Mce1A homolog in Mycobacterium tuberculosis is known to be associated with M. tuberculosis epithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that Mce1A of M. leprae is also associated with epithelial cell entry. This study is aimed at identifying particular sequences within Mce1A associated with M. leprae epithelial cell entry. Recombinant proteins having N-terminus and C-terminus truncations of the Mce1A region of M. leprae were created in Escherichia coli. Entry activity of latex beads, coated with these truncated proteins (r-lep37 kDa and r-lep27 kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316-921 bp region was divided into three sub-regions: 316-531 bp (InvX), 532-753 bp (InvY), and 754-921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface of E. coli. Entry of these E. coli into monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. Only E. coli harboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa-InvXd, containing sequences 1-24 aa, 25-46 aa, 47-57 aa, and 58-72 aa, respectively. Recombinant E. coli, expressing each of InvXa-InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of Mce1A are important for M. leprae invasion into nasal epithelial cells.


Assuntos
Proteínas de Bactérias/metabolismo , Hanseníase/microbiologia , Mycobacterium leprae/patogenicidade , Septo Nasal/microbiologia , Proteínas de Bactérias/genética , Linhagem Celular , Contagem de Colônia Microbiana , DNA Bacteriano/genética , Escherichia coli/genética , Vetores Genéticos/genética , Células HeLa , Humanos , Microesferas , Mycobacterium leprae/genética , Mycobacterium leprae/crescimento & desenvolvimento , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
PLoS Negl Trop Dis ; 12(12): e0007001, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30566440

RESUMO

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.


Assuntos
Vetores Aracnídeos/fisiologia , Ixodes/microbiologia , Ixodidae/microbiologia , Hanseníase/transmissão , Mycobacterium leprae/fisiologia , Animais , Vetores Aracnídeos/microbiologia , Linhagem Celular , Feminino , Humanos , Ixodes/fisiologia , Ixodidae/fisiologia , Hanseníase/microbiologia , Masculino , Mycobacterium leprae/genética , Coelhos
14.
mBio ; 9(5)2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30301850

RESUMO

New drugs are needed to control the current tuberculosis (TB) pandemic caused by infection with Mycobacterium tuberculosis We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agents in vitro All five compounds showed good activity against M. tuberculosis in vitro and in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization of M. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in the qcrB gene encoding a subunit of cytochrome bc 1 oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochrome bc 1 -aa 3 is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles of M. tuberculosis treated with AX-35 were similar to those generated by other cytochrome bc 1 oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochrome bd in respiratory adaptation. In the absence of cytochrome bd oxidase, AX-35 was bactericidal against M. tuberculosis Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCE New drugs against Mycobacterium tuberculosis are urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potent in vitro and in vivo activities against M. tuberculosis AX compounds target the QcrB subunit of the cytochrome bc 1 terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochrome bd oxidase upon QcrB inhibition. In the absence of cytochrome bd oxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Piperazinas/farmacologia , Tuberculose/tratamento farmacológico , Amidas/farmacologia , Amidas/uso terapêutico , Animais , Linhagem Celular , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Tuberculose/microbiologia
15.
s.l; s.n; 2018. 25 p. ilu, tab, graf.
Não convencional em Inglês | SES-SP, HANSEN, SESSP-ILSLPROD, SES-SP, SESSP-ILSLACERVO, SES-SP | ID: biblio-1025298

RESUMO

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.


Assuntos
Humanos , Animais , Masculino , Feminino , Coelhos , Vetores Aracnídeos/fisiologia , Vetores Aracnídeos/microbiologia , Linhagem Celular , Ixodes/fisiologia , Ixodes/microbiologia , Ixodidae/fisiologia , Ixodidae/microbiologia , Hanseníase/microbiologia , Hanseníase/transmissão , Mycobacterium leprae/fisiologia , Mycobacterium leprae/genética
16.
PLoS Negl Trop Dis ; 11(6): e0005687, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28636650

RESUMO

Leprosy is a chronic infectious disease that is caused by the obligate intracellular pathogen Mycobacterium leprae (M.leprae), which is the leading cause of all non-traumatic peripheral neuropathies worldwide. Although both myelinating and non-myelinating Schwann cells are infected by M.leprae in patients with lepromatous leprosy, M.leprae preferentially invades the non-myelinating Schwann cells. However, the effect of M.leprae infection on non-myelinating Schwann cells has not been elucidated. Lipid droplets (LDs) are found in M.leprae-infected Schwann cells in the nerve biopsies of lepromatous leprosy patients. M.leprae-induced LD formation favors intracellular M.leprae survival in primary Schwann cells and in a myelinating Schwann cell line referred to as ST88-14. In the current study, we initially characterized SW-10 cells and investigated the effects of LDs on M.leprae-infected SW-10 cells, which are non-myelinating Schwann cells. SW-10 cells express S100, a marker for cells from the neural crest, and NGFR p75, a marker for immature or non-myelinating Schwann cells. SW-10 cells, however, do not express myelin basic protein (MBP), a marker for myelinating Schwann cells, and myelin protein zero (MPZ), a marker for precursor, immature, or myelinating Schwann cells, all of which suggests that SW-10 cells are non-myelinating Schwann cells. In addition, SW-10 cells have phagocytic activity and can be infected with M. leprae. Infection with M. leprae induces the formation of LDs. Furthermore, inhibiting the formation of M. leprae-induced LD enhances the maturation of phagosomes containing live M.leprae and decreases the ATP content in the M. leprae found in SW-10 cells. These facts suggest that LD formation by M. leprae favors intracellular M. leprae survival in SW-10 cells, which leads to the logical conclusion that M.leprae-infected SW-10 cells can be a new model for investigating the interaction of M.leprae with non-myelinating Schwann cells.


Assuntos
Gotículas Lipídicas/metabolismo , Viabilidade Microbiana , Mycobacterium leprae/fisiologia , Células de Schwann/metabolismo , Células de Schwann/microbiologia , Animais , Linhagem Celular , Interações Hospedeiro-Patógeno , Camundongos , Fagocitose , Fagossomos/microbiologia
17.
PLoS Negl Trop Dis ; 11(2): e0005373, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28158186

RESUMO

Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine has a long history of use and has demonstrated a good safety profile for a disease that requires chronic dosing for a period of time ranging 3-36 months. These results, taken with clofazimine's status as an FDA-approved drug with over four decades of use for the treatment of leprosy, support the continued investigation of clofazimine both as a new chemical tool for understanding cryptosporidium biology and a potential new treatment of cryptosporidiosis.


Assuntos
Antiprotozoários/farmacologia , Clofazimina/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Reposicionamento de Medicamentos , Animais , Automação Laboratorial , Linhagem Celular , Criptosporidiose/parasitologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/parasitologia , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Resultado do Tratamento
18.
J Ethnopharmacol ; 195: 298-308, 2017 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-27880883

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: 3ß,6ß,16ß-Trihydroxylup-20(29)-ene (TTHL) is a triterpene isolated from the flowers of Combretum leprosum, a plant used in folk medicine in the north of Brazil for the treatment of skin disorders. AIM OF THE STUDY: In the present study, TTHL was evaluated as a potential topical anti-inflammatory and anti-proliferative agent through in vivo and in vitro models. MATERIAL AND METHODS: Anti-inflammmatory and anti-proliferative effects of TTHL were assessed using Swiss mice in acute and chronic models of skin inflammation induced by 12-O-tetradecanoylphorbol-acetate (TPA) application. Anti-proliferative activity was proved through in vitro experiments with the HaCaT human keratinocyte cell line. RESULTS: Treatment with TTHL inhibited inflammatory parameters such as oedema formation and cellular infiltration in acute and chronic models. In the chronic model, TTHL also inhibited epidermal hyperproliferation, as evidenced by reduction of epidermis thickness and proliferating cell nuclear antigen expression. The anti-proliferative effect was confirmed by the capability of TTHL in reducing the proliferation and inducing cell apoptosis of HaCaT cells. Suggesting a mechanism of action, TTHL showed activation of corticosteroid receptors, but without the induction of corticosteroid-related cutaneous side effects. CONCLUSION: Our results demonstrate consistent anti-inflammatory and anti-proliferative activity and assign TTHL as a valuable tool in the development of a new treatment for skin inflammatory and proliferative diseases, such as psoriasis.


Assuntos
Anti-Inflamatórios/farmacologia , Proliferação de Células/efeitos dos fármacos , Dermatite de Contato/prevenção & controle , Queratinócitos/efeitos dos fármacos , Psoríase/prevenção & controle , Pele/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Doença Crônica , Dermatite de Contato/etiologia , Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/prevenção & controle , Feminino , Antagonistas de Hormônios/farmacologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Mifepristona/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Psoríase/induzido quimicamente , Psoríase/metabolismo , Psoríase/patologia , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Pele/metabolismo , Pele/patologia , Acetato de Tetradecanoilforbol , Fatores de Tempo
19.
Sci Rep ; 6: 35106, 2016 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-27731364

RESUMO

Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson's disease, Crohn's disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identified. To investigate the signalling events through which LRRK2 acts to influence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase.


Assuntos
Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Inibidores de Proteínas Quinases/farmacologia
20.
J Biol Chem ; 291(41): 21375-21387, 2016 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-27555322

RESUMO

Mycobacterium leprae, the intracellular etiological agent of leprosy, infects Schwann promoting irreversible physical disabilities and deformities. These cells are responsible for myelination and maintenance of axonal energy metabolism through export of metabolites, such as lactate and pyruvate. In the present work, we observed that infected Schwann cells increase glucose uptake with a concomitant increase in glucose-6-phosphate dehydrogenase (G6PDH) activity, the key enzyme of the oxidative pentose pathway. We also observed a mitochondria shutdown in infected cells and mitochondrial swelling in pure neural leprosy nerves. The classic Warburg effect described in macrophages infected by Mycobacterium avium was not observed in our model, which presented a drastic reduction in lactate generation and release by infected Schwann cells. This effect was followed by a decrease in lactate dehydrogenase isoform M (LDH-M) activity and an increase in cellular protection against hydrogen peroxide insult in a pentose phosphate pathway and GSH-dependent manner. M. leprae infection success was also dependent of the glutathione antioxidant system and its main reducing power source, the pentose pathway, as demonstrated by a 50 and 70% drop in intracellular viability after treatment with the GSH synthesis inhibitor buthionine sulfoximine, and aminonicotinamide (6-ANAM), an inhibitor of G6PDH 6-ANAM, respectively. We concluded that M. leprae could modulate host cell glucose metabolism to increase the cellular reducing power generation, facilitating glutathione regeneration and consequently free-radical control. The impact of this regulation in leprosy neuropathy is discussed.


Assuntos
Metabolismo Energético , Glucose/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Hanseníase Tuberculoide/metabolismo , Mycobacterium leprae/metabolismo , Células de Schwann/metabolismo , Linhagem Celular , Humanos , Metionina/análogos & derivados , Metionina/farmacologia , Mitocôndrias/metabolismo , Células de Schwann/microbiologia
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