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1.
Genomics ; 112(1): 990-997, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31229555

RESUMO

Trypanosoma cruzi is the etiologic agent of Chagas disease, a life-threatening disease that affects different tissues. Within its mammalian host, T. cruzi develops molecular strategies for successful invasion of different cell types and adaptation to the intracellular environment. Conversely, the host cell responds to the infection by activating intracellular pathways to control parasite replication. Here, we reviewed genome-wide expression studies based on microarray and RNA-seq data from both parasite and host genes generated from animal models of infection as well as from Chagas disease patients. As expected, analyses of T. cruzi genes highlighted changes related to parasite energy metabolism and cell surface molecules, whereas host cell transcriptome emphasized the role of immune response genes. Besides allowing a better understanding of mechanisms behind the pathogenesis of Chagas disease, these studies provide essential information for the development of new therapies as well as biomarkers for diagnosis and assessment of disease progression.

2.
Front Immunol ; 10: 1637, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396209

RESUMO

MicroRNAs (miRs) are master regulators of post-transcriptional gene expression, and they are often dysregulated in individuals suffering from diabetes. We investigated the roles of miR-101-3p and miR-204-5p, both of which negatively regulate insulin secretion and cell survival and are highly expressed in pancreatic ß cells, in the context of type 1 diabetes (T1D) pathogenesis. Using quantitative real time PCR, we evaluated serum levels of miR-101-3p and miR-204-5p in four groups, including recent-onset T1D patients (T1D group; n = 50), individuals with normal glucose levels expressing one islet autoantibody (Ab) (single Ab group; n = 26) or multiple autoantibodies (multiple Ab group; n = 12), and healthy controls (control group; n = 43). An in silico analysis was performed to identify potential target genes of these miRNAs and to delineate enriched pathways. The relative expression of serum miR-101-3p was approximately three times higher in the multiple Ab and T1D groups than that in the single Ab and control groups (p < 0.0001). When considering all groups together, miR-101-3p expression was positively correlated with the level of islet autoantibodies GADA (r = 0.267; p = 0.0027) and IA-2A (r = 0.291; p = 0.001), and the expression of the miRNA was not correlated with levels of ZnT8A (r = 0.125; p = 0.183). miR-101-3p expression did not correlate with HbA1c (r = 0.178; p = 0.052) or glucose levels (r = 0.177; p = 0.051). No significant differences were observed in miR-204-5p expression among the analyzed groups. Computational analysis of the miR-101-3p target gene pathways indicated a potential activation of the HGF/c-Met, Ephrin receptor, and STAT3 signaling pathways. Our study demonstrated that the circulating levels of miR-101-3p are higher in T1D patients and in individuals with normal glucose levels, testing positive for multiple autoantibodies, indicating that miR-101-3p precedes loss of glucose homeostasis. The pathogenic role of miR-101-3p in T1D may involve multiple molecular pathways.

3.
Oncotarget ; 8(4): 6994-7002, 2017 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-28052002

RESUMO

Cardiotoxicity is associated with the chronic use of doxorubicin leading to cardiomyopathy and heart failure. Identification of cardiotoxicity-specific miRNA biomarkers could provide clinicians with a valuable prognostic tool. The aim of the study was to evaluate circulating levels of miRNAs in breast cancer patients receiving doxorubicin treatment and to correlate with cardiac function. This is an ancillary study from "Carvedilol Effect on Chemotherapy-induced Cardiotoxicity" (CECCY trial), which included 56 female patients (49.9±3.3 years of age) from the placebo arm. Enrolled patients were treated with doxorubicin followed by taxanes. cTnI, LVEF, and miRNAs were measured periodically. Circulating levels of miR-1, -133b, -146a, and -423-5p increased during the treatment whereas miR-208a and -208b were undetectable. cTnI increased from 6.6±0.3 to 46.7±5.5 pg/mL (p<0.001), while overall LVEF tended to decrease from 65.3±0.5 to 63.8±0.9 (p=0.053) over 12 months. Ten patients (17.9%) developed cardiotoxicity showing a decrease in LVEF from 67.2±1.0 to 58.8±2.7 (p=0.005). miR-1 was associated with changes in LVEF (r=-0.531, p<0.001). In a ROC curve analysis miR-1 showed an AUC greater than cTnI to discriminate between patients who did and did not develop cardiotoxicity (AUC = 0.851 and 0.544, p= 0.0016). Our data suggest that circulating miR-1 might be a potential new biomarker of doxorubicin-induced cardiotoxicity in breast cancer patients.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Cardiotoxicidade/genética , Doxorrubicina/efeitos adversos , MicroRNAs/sangue , Biomarcadores , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Carbazóis , Cardiotoxicidade/sangue , Cardiotoxicidade/fisiopatologia , Carvedilol , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Propanolaminas , Curva ROC , Volume Sistólico/efeitos dos fármacos , Troponina C/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos
4.
PLoS Pathog ; 12(4): e1005593, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-27128676

RESUMO

The ß1i, ß2i and ß5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-γ (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected ß1i, ß2i and ß5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-γ+/TNF+) or single-positive (IFN-γ+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Vacinas Protozoárias/imunologia , Adolescente , Adulto , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi , Adulto Jovem
5.
J Immunol ; 181(11): 7917-24, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19017982

RESUMO

Innate immune recognition of intracellular pathogens involves both extracellular and cytosolic surveillance mechanisms. The intracellular protozoan parasite Trypanosoma cruzi triggers a robust type I IFN response in both immune and nonimmune cell types. In this study, we report that signaling through TBK1 and IFN regulatory factor 3 is required for T. cruzi-mediated expression of IFN-beta. The TLR adaptors MyD88 and TRIF, as well as TLR4 and TLR3, were found to be dispensable, demonstrating that T. cruzi induces IFN-beta expression in a TLR-independent manner. The potential role for cytosolic dsRNA sensing pathways acting through RIG-I and MDA5 was ruled out because T. cruzi was shown to trigger robust expression of IFN-beta in macrophages lacking the MAVS/IPS1/VISA/CARDif adaptor protein. The failure of T. cruzi to activate HEK293-IFN-beta-luciferase cells, which are highly sensitive to cytosolic triggers of IFN-beta expression including Listeria, Sendai virus, and transfected dsRNA and dsDNA, further indicates that the parasite does not engage currently recognized cytosolic surveillance pathways. Together, these findings identify the existence of a novel TLR-independent pathogen-sensing mechanism in immune and nonimmune cells that converges on TBK1 and IFN regulatory factor 3 for activation of IFN-beta gene expression.


Assuntos
Doença de Chagas/imunologia , Regulação da Expressão Gênica/imunologia , Fator Regulador 3 de Interferon/imunologia , Interferon beta/imunologia , Receptores Toll-Like/imunologia , Trypanosoma cruzi/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Doença de Chagas/genética , Doença de Chagas/metabolismo , DNA/imunologia , Regulação da Expressão Gênica/genética , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Listeria/imunologia , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , RNA de Cadeia Dupla/imunologia , Vírus Sendai/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
6.
An Acad Bras Cienc ; 80(1): 157-66, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18345384

RESUMO

The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast -- an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.


Assuntos
Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Imagem Tridimensional , Microscopia Eletrônica , RNA Polimerase II , Transcrição Genética , Trypanosoma cruzi/citologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/ultraestrutura
7.
An. acad. bras. ciênc ; 80(1): 157-166, Mar. 2008. ilus, graf
Artigo em Inglês | LILACS | ID: lil-477423

RESUMO

The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.


A diferenciação de formas epimastigotas (proliferativas) do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas), pode ser reproduzida em laboratório incubando-se as células em um meio quimicamente definido que imita a urina do inseto vetor deste parasita. Os epimastigotas têm um núcleo esférico, o flagelo se projeta da metade do corpo do protozoário e o cinetoplasto (organela que possui o DNA mitocondrial) possui formato de disco. Os tripomastigotas metacíclicos têm um núcleo alongado com o flagelo emergindo da extremidade posterior da célula associado ao cinetoplasto esférico. Neste trabalho descrevemos as mudanças morfológicas que ocorrem durante essa transformação e caracterizamos uma nova forma intermediária do parasita usando reconstrução tridimensional de cortes seriados, visualizados por microscopia eletrônica de transmissão. Essa nova forma intermediária é caracterizada pela compressão do cinetoplasto contra o núcleo alongado, indicando que a metaciclogênese envolve movimentos ativos do cinetoplasto associado à estrutura flagelar em relação ao corpo celular. Como tripomastigotas metacíclicos transcrevem menos que as formas epimastigotas proliferativas, verificamos a presença da RNA polimerase II e medimos a atividade transcricional durante o processo de diferenciação. A presença da enzima e a atividade transcricional permanecem inalteradas durante todas as etapas da metaciclogênese, desaparecendo apenas quando as formas metacíclicas são formadas. Sugerimos que a diferenciação requer uma atividade transcricional, necessária para uma intensa remodelação da célula, que acontece até o cinetoplasto e o flagelo atingirem uma posição posterior do corpo do tripomastigota metacíclico.


Assuntos
Animais , Trypanosoma cruzi/crescimento & desenvolvimento , Imagem Tridimensional , Microscopia Eletrônica , RNA Polimerase II , Transcrição Genética , Trypanosoma cruzi/citologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/ultraestrutura
8.
J Leukoc Biol ; 72(6): 1215-27, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12488504

RESUMO

The ability of Trypanosoma cruzi to activate macrophages is, at least in part, attributed to the glycosylphosphatidylinositol-anchored mucin-like glycoproteins (GPI-mucins) expressed in the surface of the trypomastigote stage of the parasite. The differential display reverse transcriptase-polymerase chain reaction and the reverse Northern blot were used to study modulation of gene expression in murine macrophages exposed to GPI-mucins and in cardiac tissues from mice infected with T. cruzi. Among several cDNAs that were more abundant in lanes corresponding to macrophages stimulated with GPI-mucins as compared with resting cells, we confirmed the differential expression of A1, interleukin-18, and GPIgamma4. Some of these genes were also shown to have enhanced expression in the cardiac tissue (DAP-12, A1, and GPIgamma4) from infected animals. The expression of GPIgamma4 was also enhanced in human monocytes stimulated with GPI-mucins or bacterial lipopolysaccharides. The complete sequence of the GPIgamma4 transcript and its gene including the 5' upstream region was defined. GPIgamma4 was encoded by a novel, single copy gene present in mouse as well as human genomes and showed conserved homology to different members of the guanine nucleotide exchange factor family.


Assuntos
Macrófagos/metabolismo , Camundongos/genética , Miocardite/parasitologia , Trypanosoma cruzi , Fatores ras de Troca de Nucleotídeo Guanina/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação a DNA/genética , Componentes do Gene , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicosilfosfatidilinositóis , Humanos , Interleucina-18/genética , Macrófagos/efeitos dos fármacos , Masculino , Proteínas de Membrana , Dados de Sequência Molecular , Mucinas/farmacologia , Miocardite/metabolismo , Receptores Imunológicos/genética , Proteína de Replicação C , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores ras de Troca de Nucleotídeo Guanina/biossíntese
9.
Microbes Infect ; 4(9): 1015-25, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12106796

RESUMO

Activation of cells from the innate immune system has an important role in host resistance to early infection with the intracellular protozoan parasite, Trypanosoma cruzi. Here we review the studies that have identified and structurally characterized the glycosylphosphatidylinositol (GPI) anchors, as parasite molecules responsible for the activation of cells from the macrophage lineage. We also cover the studies that have identified the receptor, signaling pathways as well as the array of genes expressed in macrophages that are activated by these glycoconjugates. We discuss the possible implications of such response on the host resistance to T. cruzi infection and the pathogenesis of Chagas disease.


Assuntos
Proteínas de Drosophila , Glicoproteínas/imunologia , Glicosilfosfatidilinositóis/imunologia , Macrófagos/imunologia , Transdução de Sinais , Trypanosoma cruzi/imunologia , Animais , Sequência de Carboidratos , Citocinas/biossíntese , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Receptores de Superfície Celular/imunologia , Relação Estrutura-Atividade , Receptores Toll-Like , Trypanosoma cruzi/fisiologia
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