RESUMO
Highly dynamic ribosomes, glycogen granules, thinly fibrillar material, and multiple membrane-bound vesicles are embedded in the matrix-rich cytoplasm of Entamoeba spp. trophozoites. The absence of a Golgi apparatus in these amoebae has been commonly accepted. Here we challenge this observation by incubating Entamoeba histolytica and Entamoeba dispar with monensin, an ionophore that produces swelling of the Golgi apparatus. We observe changes in the trophozoites through standard transmission electron microscopy, cryofixation and cryosubstitution, and analyze the label and expression of known resident proteins of the cis-GM130 and trans-TGN38 Golgi network through confocal microscopy and Western blot assays. Cryosubstitution and standard methods using the treatment, preserved membranous lamellae resembling Golgi components. GM130 and TGN38 Golgi antigens were found by immunoelectron, immunoblot, and co-localization by confocal microscopy using the reagent NBD C6-ceramide. Our results indicate that previously undetected Golgi apparatus components are present in the cytoplasm of E. histolytica and E. dispar.
Assuntos
Entamoeba histolytica , Entamoeba , Complexo de Golgi , Microscopia Confocal , Monensin/farmacologiaRESUMO
An association between varicella zoster virus (VZV) and multiple sclerosis (MS) has been reported in Mexican populations. The aim of this study was to compare the response of T cells from MS patients, during relapse and remission, to in vitro stimulation with VZV, adenovirus (AV) and Epstein-Barr virus (EBV). Proliferation and cytokine secretion of T cells from 29 relapsing-remitting MS patients and 38 healthy controls (HC) were analyzed by flow cytometry after stimulating with VZV, AV or EBV. IgG and IgM levels against VZV and EBV were quantified using Enzyme-Linked Immunosorbent Assay. Relapsing MS patients showed a higher percentage of responding CD4+ and CD8+ T cells against VZV compared to AV. In HC and remitting MS patients, proliferation of CD4+ T cells was higher when stimulated with VZV as compared to EBV. Moreover, T cells isolated from remitting patients secreted predominantly Th1 cytokines when cell cultures were stimulated with VZV. Finally, high concentration of anti-VZV IgG was found in sera from patients and controls. The results support previous studies of an VZV-MS association in the particular population studied and provide additional information about the possible role of this virus in the pathogenesis of MS.
Assuntos
Herpesvirus Humano 3/fisiologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/virologia , Linfócitos T/imunologia , Adenoviridae/fisiologia , Adulto , Anticorpos Antivirais/imunologia , Citocinas/metabolismo , Feminino , Herpesvirus Humano 3/imunologia , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/fisiologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/virologia , Recidiva , Indução de RemissãoRESUMO
Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.
Assuntos
Acanthamoeba castellanii/ultraestrutura , Balamuthia mandrillaris/ultraestrutura , Peroxinas/genética , Peroxissomos/ultraestrutura , Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Balamuthia mandrillaris/enzimologia , Balamuthia mandrillaris/genética , Catalase/metabolismo , Microscopia Eletrônica de Transmissão , Peroxinas/metabolismo , Peroxissomos/enzimologia , Peroxissomos/genética , FilogeniaRESUMO
Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.
Assuntos
Entamoeba/fisiologia , Fibronectinas/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas dos Microfilamentos/metabolismo , Entamoeba/efeitos dos fármacos , Entamoeba/ultraestrutura , Entamoeba histolytica/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Microscopia Confocal , Proteínas de Protozoários/metabolismoRESUMO
The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.
Assuntos
Giardia lamblia/química , Peroxissomos/química , Proteínas de Protozoários/isolamento & purificação , 3,3'-Diaminobenzidina/química , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Western Blotting , Cério/química , Coenzima A Ligases/imunologia , Coenzima A Ligases/metabolismo , Biologia Computacional , Imunofluorescência , Giardia lamblia/enzimologia , Giardia lamblia/imunologia , Giardia lamblia/ultraestrutura , Histocitoquímica , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Oxirredutases/metabolismo , Peroxinas/análise , Peroxinas/imunologia , Peroxissomos/enzimologia , Proteínas de Protozoários/análise , Coelhos , Coloração e RotulagemRESUMO
The virulence of various amoebic parasites has been correlated with the presence of electron-dense granules (EDGs) in the cytoplasm of trophozoites. Here, we report the finding by transmission electron microscopy of a large number of EDGs in a recent culture of Acanthamoeba culbertsoni, isolated from a severe case of human keratitis. When this isolate was maintained in culture for 6 mo, the granules almost disappeared. However, after induction of mice brain lesions with the long-term cultured isolate, recovered amoebas had abundant EDGs. Trophozoites of the original isolate, or those recovered from experimental lesions, secreted EDGs into the medium when incubated with MDCK cells. To analyze a possible cytotoxic effect the conditioned medium was incubated with MDCK monolayers. After 5 h, the media containing EDGs produced opening of the tight junctions; at 24 h, cell viability was compromised, and at 48 h most of the cells were detached from the monolayer. In contrast, trophozoites in long-term cultures did not release EDGs to the medium during incubation with MDCK cells, and the corresponding conditioned medium did not have any effect on MDCK monolayers. Our observations further support the hypothesis that EDGs play a role in the cytopathogenic mechanisms of A. culbertsoni.
Assuntos
Acanthamoeba/patogenicidade , Acanthamoeba/ultraestrutura , Amebíase/parasitologia , Ceratite/parasitologia , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Trofozoítos/crescimento & desenvolvimento , Trofozoítos/ultraestrutura , VirulênciaRESUMO
Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection.
Assuntos
Ceratite por Acanthamoeba/diagnóstico , Acanthamoeba/classificação , Encefalite/diagnóstico , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Ceratite por Acanthamoeba/parasitologia , Adulto , Lentes de Contato/parasitologia , Córnea/parasitologia , DNA Ribossômico/genética , Encefalite/parasitologia , Feminino , Genótipo , Humanos , México , Análise de Sequência de DNA , Adulto JovemRESUMO
Three months after the World Health Organization declared the epidemic of Zika virus infections to be a Public Health Emergency of International Concern, we can look back at what we have learned and prospects for controlling the disease. Although Zika virus infections may explain many cases of brain damage in newborns, it may not be the only cause. We need a clear association between confirmed cases of Zika infections in pregnant women and microcephaly in newborns. Until we reach a firm conclusion, past experience with another virus that causes damage to newborns offers some hope. The development and almost universal use of rubella vaccine has all but eliminated the congenital rubella syndrome in the world. Rapid development of Zika virus vaccine might well do the same for this epidemic.
Assuntos
Infecção por Zika virus/prevenção & controle , Epidemias/prevenção & controle , Feminino , Humanos , Recém-Nascido , Microcefalia/etiologia , Microcefalia/prevenção & controle , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controle , Rubéola (Sarampo Alemão)/prevenção & controle , Síndrome da Rubéola Congênita/prevenção & controle , Vacina contra Rubéola/uso terapêutico , Infecção por Zika virus/epidemiologiaRESUMO
A serious accident experienced firsthand by a senior authority of the American medical establishment serves as a catalyst to comment with regards to the excellent attention provided at the intensive care facilities of "ivy league" hospitals, in contrast with the poor attention received by the patient in other institutions. On this article it is also highlighted the importance of the nurses' labor to the hospitalized patient.
Se resume la experiencia "en carne propia" de una de las grandes autoridades médicas de los Estados Unidos al sufrir un gravísimo accidente casero. Se comentan las excelencias de los cuidados intensivos en hospitales de tercer nivel, en contraste con la despersonalización y el desinterés que recibió el paciente en otras instituciones médicas. Se realza la importancia de la labor de las enfermeras en la atención al paciente hospitalizado.
Assuntos
Vértebras Cervicais/lesões , Hospitais de Ensino/normas , Médicos , Relações Profissional-Paciente , Qualidade da Assistência à Saúde , Fraturas da Coluna Vertebral/terapia , Idoso de 80 Anos ou mais , História do Século XXI , Humanos , Masculino , MassachusettsRESUMO
Entamoeba histolytica is the causative agent of human intestinal and liver amebiasis. The extraordinary phagocytic activity of E. histolytica trophozoites has been accepted as one of the virulence mechanisms responsible for their invasive capacity. The recognition of the noninvasive Entamoeba dispar as a different species has raised the question as to whether the lack of pathogenic potential of this ameba correlates with a limited phagocytic capacity. We have therefore compared the process of erythrophagocytosis in both species by means of light and video microscopy, hemoglobin measurement, and the estimation of reactive oxygen species (ROS). In the present study, we confirmed that E. dispar has lower erythrophagocytic capacity. We also observed by video microscopy a new event of erythrocyte opsonization-like in both species, being more characteristic in E. histolytica. Moreover, E. dispar showed a lower capacity to produce ROS compared with the invasive species and also showed a large population of amoebae that did not engulf any erythrocyte over time. Our results demonstrate that E. histolytica has a higher phagocytic capacity than E. dispar, including a higher rate of production of ROS in the course of ingesting red blood cells.