RESUMO
The in vitro cytopathic effect of four strains of Trichomonas vaginalis on cultured epithelial monolayers was analyzed through electrophysiology and electron microscopy. Interaction of trichomonads of two virulent strains (GT-10 and GT-13) with cultured MDCK cell monolayers mounted in Ussing chambers produced a rapid decrease in transepithelial resistance to less than 30% of control values after only 15 min. By 30 min the electrical resistance was practically abolished by the virulent parasites. In contrast, of two attenuated strains of trichomonads (GT-3 and GT-7) analyzed under similar conditions, GT-3 trophozoites required 180 min to reduce transepithelial resistance to 9% of control values, while monolayers in contact with GT-7 parasites still showed 28% of control values at this time of incubation. Sequential scanning electron microscopy confirmed the much faster and widespread cytopathic effect of virulent parasites. In contrast, the slow lytic process produced by attenuated trophozoites was reduced to focal areas of direct contact with epithelial cells. Another difference was found by measurement of the surface charge of the four strains of T. vaginalis by means of cell microelectrophoresis. While the two virulent strains showed a negative surface charge, the two attenuated strains had no detectable surface charge at neutral pH. When parasites were incubated with cationized ferritin and studied with transmission electron microscopy the surface of virulent trichomonads appeared heavily labeled, whereas the surface of attenuated parasites had only sparse and irregular ferritin binding.
Assuntos
Extensões da Superfície Celular/ultraestrutura , Propriedades de Superfície , Trichomonas vaginalis/patogenicidade , Trichomonas vaginalis/ultraestrutura , Fatores de Virulência , Animais , Linhagem Celular , Impedância Elétrica , Eletroforese , Eletrofisiologia , Feminino , Humanos , Camundongos , Microscopia Eletrônica de Varredura , Especificidade da Espécie , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/classificaçãoRESUMO
In the life cycle of Entamoeba species, the cyst and all the processes associated to it have been poorly studied. Entamoeba invadens, a serpent's parasite, has been commonly accepted as a model for the study of encystation and excystation. Here we analyzed through scanning and transmission electron microscopy the in vitro morphological differentiation of both processes. During encystation, the formation of an irregular net of fibrillar material on the surface of precysts was observed. In thin sections of cryofixed and cryosubstituted specimens, abundant vacuoles containing a microfibrillar material of similar appearance to the structural components of the cyst wall were found in the cytoplasm. Assays with a calcofluor probe on cryosections of encysting trophozoites and precysts showed the presence of fluorescent circular cytoplasmic structures. In the cyst stage, the fluorescence was located on the surface. During excystation, the detachment of the metacyst from the cyst wall was observed through scanning electron microscopy. Metacysts endocyting amorphous material which may correspond to cyst wall residues were commonly found. By transmission electron microscopy the formation of a crescent-shaped space between the plasma membrane and the cyst wall was observed. Abundant small electrondense bodies were found in the cytoplasm. Many of them were in close apposition to the plasma membrane and frequently some of them were seen projecting towards this newly formed space. Our results suggest that the microfibrillar content of the vacuoles corresponds to the cyst wall material, that the electrondense bodies may be involved in the excystation process, and that part of the cyst wall residues may be endocyted by the parasite.
Assuntos
Entamoeba/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Animais , Entamoeba/fisiologiaRESUMO
The mode of appearance and assembly of cyst wall filaments on the surface of Giardia duodenalis trophozoites committed to encyst was analysed by scanning and transmission electron microscopy (SEM and TEM) and by fluorescence microscopy (FM). SEM showed a progressive appearance of fibril patches, predominantly on the anterior area of ventral and dorsal surfaces, which then spread and coalesced. By TEM, ruthenium red (RR) displayed staining in encysting cells as rodlike spots of variable diameter (3-25 nm), possibly microfibril tips with polyanionic moieties, that displayed tangential associations and random orientations over the cell membrane. In FM assays, the 1,10-phenanthroline derivative of ruthenium red (RR/oPHE) was a specific ligand for these assembling fibrils and this staining was significantly blocked by N-acetylgalactosamine (GalNac) and galactosamine (GalN). Interestingly, RR staining was lost when the cyst wall was completely assembled and thickened as observed by TEM and FM. Kinetic FM assays, in which a mAb specific for a 26 kDa Giardia cyst wall polypeptide was used concomitantly with RR/oPHE staining, showed a differential pattern for the appearance and reactivity of polypeptide and assembling GalN/GalNac-rich moieties of Giardia cyst wall.
Assuntos
Cistos/parasitologia , Cistos/ultraestrutura , Giardia/fisiologia , Giardia/ultraestrutura , Animais , Western Blotting , Cistos/patologia , Microfibrilas/patologia , Microfibrilas/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Peptídeos/metabolismo , Polissacarídeos/metabolismoRESUMO
The cell coat of two Trichomonas vaginalis isolates with different degree of virulence isolated in Mexico from symptomatic women was studied by cytochemical assays. The use of carbohydrate cell surface markers allowed us to visualize greater electron-dense deposits in the highly virulent T. vaginalis isolate than in the less virulent one. On the contrary, parasites treated with concanavalin A showed a heavy uniform electron-dense deposit on the cell surface that was similar in both isolates. When parasites were treated with cationized ferritin the amount of bounded particles on the cell surface was greater in the higher virulent isolate.
Assuntos
Glicocálix/ultraestrutura , Trichomonas vaginalis/ultraestrutura , Animais , Carboidratos/análise , Glicocálix/química , Histocitoquímica , Interações Hospedeiro-Parasita , Coloração e Rotulagem , Trichomonas vaginalis/química , Trichomonas vaginalis/patogenicidade , Fatores de VirulênciaRESUMO
This paper explores the interaction of two strains of Trichomonas vaginalis, of high and low virulence, with the cell types present in the microenvironment of the parasite during human infections. With the use of transmission and scanning electron microscopy the sequence of internalization by T. vaginalis of Döderlein's lactobacilli, and of vaginal epithelial cells, leukocytes, and erythrocytes was documented. Furthermore, the degradation of ingested material by colocalization of acid phosphatase activity in phagocytic vacuoles was demonstrated. Phagocytosis of all cell types analyzed was found in both strains studied, although the highly virulent strain internalized target cells more rapidly than the less virulent one. Ultrastructural evidence indicated that phagocytosis takes place through two distinct mechanisms, only one involving the formation of a phagocytic stoma, characteristic of professional phagocytes. T. vaginalis phagocytosis may be both an efficient means of obtaining nutrients for the parasite and a significant factor in the pathogenesis of trichomonal infections of the human genitourinary tract.
Assuntos
Células Epiteliais/imunologia , Eritrócitos/imunologia , Lactobacillus/imunologia , Leucócitos/imunologia , Fagocitose , Trichomonas vaginalis/imunologia , Vagina/citologia , Fosfatase Ácida/análise , Animais , Adesão Celular , Células Epiteliais/ultraestrutura , Eritrócitos/ultraestrutura , Feminino , Humanos , Lactobacillus/ultraestrutura , Leucócitos/ultraestrutura , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Vaginite por Trichomonas/etiologia , Vaginite por Trichomonas/imunologia , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/patogenicidade , Trichomonas vaginalis/ultraestrutura , Vacúolos/enzimologia , VirulênciaRESUMO
The in vitro cytopathic effect of Trichomonas vaginalis on epithelial cells was explored through the interaction of trophozoites of the virulent strain GT-10 with MDCK monolayers. The interaction was analyzed through electrophysiology, video microscopy, and transmission and scanning electron microscopy. Electrical measurements revealed that living parasites produced severe damage to the cell monolayers within 30 min, manifested as a rapid decrease in transepithelial resistance. Microscopic observations demonstrated that when placed in contact with epithelial cells, trichomonas formed clumps through interdigitations and transient plasma membrane junctions between adjacent parasites. Also, attached trophozoites adopted an ameboid shape. The in vitro cytopathic action of T. vaginalis on MDCK cells was initially evident by modifications of the plasma membrane, resulting in opening of tight junctions, membrane blebbing, and monolayer disruption. After 15 min of interaction the damage was focal, concentrating at sites where parasite clumps adhered to the monolayer. At 30 min practically all MDCK cells were dead, whether or not trichomonas were attached to them. These events were followed by detachment of lysed cells and complete disruption of the monolayer at 60 min. Electron microscopy demonstrated a peculiar form of adhesion that appears to be specific for trichomonas, in which the basal surface of T. vaginalis formed slender channels through which microvilli and cytoplasmic fragments of epithelial cells were internalized. The same sequence of lytic events was found with the less virulent GT-3 strain. However, the time course of cytolysis with GT-3 parasites was much slower, and lysis was limited to areas of attachment of T. vaginalis.
Assuntos
Morte Celular , Trichomonas vaginalis/patogenicidade , Trichomonas vaginalis/ultraestrutura , Adulto , Animais , Adesão Celular , Linhagem Celular , Membrana Celular/parasitologia , Cães , Impedância Elétrica , Células Epiteliais , Epitélio/parasitologia , Epitélio/ultraestrutura , Feminino , Humanos , Rim , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Vaginite por Trichomonas/parasitologia , Trichomonas vaginalis/fisiologia , VirulênciaRESUMO
An in vitro study of the adhesion and invasion of Shigella flexneri was implemented, by means of incubation of laminary cuts of cecal mucosa of Dunkin-Hartley guinea pigs in a suspension of Shigella flexneri, which was isolated from a patient with bacillary dysentery. The laminae were placed in plastic chambers for two hours at 37%C. After this the bacterial suspension was discarded so as to eliminate the bacilli which were not adhered. The epithelium was washed with saline and was processed for analysis with scanning electron microscope. The topology of the mucosa incubated with Shigella flexneri was similar to that of the witnesses. The bacteria which adhered to the mucosa were dispersed individually or in clumps of varied numbers. The main alteration observed upon the epithelial surface were depressions due to a lateral separation of the microvilli which may have originated the endocytic stomas containing bacterias. The results of this study allow the proposition of the use of explants, so as to study the interaction between Shigella flexneri and the intestinal epithelium, with the possibility of modifying different experimental variables.
Assuntos
Aderência Bacteriana , Mucosa Intestinal/microbiologia , Shigella flexneri/fisiologia , Animais , Ceco , Disenteria Bacilar/microbiologia , Epitélio/microbiologia , Feminino , Cobaias , Humanos , Masculino , Microvilosidades/microbiologia , Técnicas de Cultura de Órgãos , Shigella flexneri/isolamento & purificaçãoRESUMO
The effect of antimicrobial drugs on epimural bacteria of guinea pig cecum was assessed by scanning electron microscopy. Metronidazole (500 hg/ml), neomycin (50 hg/ml) or both combined were given orally in drinking water ad libitum for five consecutive days, and representative fragments of cecum were processed for analysis. In untreated animals large areas of mucosa were lined by spiral-shaped bacteria were eliminated by treatment with neomycin, only remaining fusiform bacteria at openings of crypts. metronidazole was effective for eliminating all bacterial populations; the same effect was achieved with a combination of neomycin and metronidazole, rendering the cecum free of epimural bacteria, the health of guinea pig was unaffected by these treatments. The cecal epithelium of antimicrobial-treated animals can be used for experimental studies without interference of epimural bacteria.
Assuntos
Bactérias/efeitos dos fármacos , Ceco/microbiologia , Metronidazol/farmacologia , Neomicina/farmacologia , Administração Oral , Animais , Bactérias/isolamento & purificação , Ceco/ultraestrutura , Combinação de Medicamentos , Sinergismo Farmacológico , Epitélio/microbiologia , Epitélio/ultraestrutura , Cobaias , Mucosa Intestinal/microbiologia , Mucosa Intestinal/ultraestrutura , Masculino , Metronidazol/administração & dosagem , Microscopia Eletrônica de Varredura , Microvilosidades/microbiologia , Microvilosidades/ultraestrutura , Neomicina/administração & dosagemRESUMO
The presence of IgA anti-Entamoeba histolytica antibodies has been demonstrates in intestinal secretions and serum of human amebiasis. We investigated which are the cellular components of trophozoites that react with IgA anti-ameba antibodies from immune serum, colostrum and human milk. The cellular localization of such antigens was accomplished by an indirect immunoperoxidase technique using anti-human IgA (alpha chain specific) labeled with peroxidase, both for light and transmission electron microscopy. Intracellular antigens were localized after permeating the parasites with cold acetone (-10 degrees C) for 3 min. and cryosections of 1 micron thick. The antigens that react with IgA antibodies from immune serum, colostrum and human milk were located in the plasma membrane and the internal portion of some cytoplasmic vesicles. So far, it is unknown what is the biological function of IgA in human amebiasis but in other systems it protects against certain parasites.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/análise , Entamoeba histolytica/imunologia , Imunoglobulina A/imunologia , Animais , Anticorpos Antiprotozoários/isolamento & purificação , Colostro/imunologia , Entamoeba histolytica/ultraestrutura , Entamebíase/sangue , Entamebíase/imunologia , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina A/isolamento & purificação , Microscopia Imunoeletrônica , Leite Humano/imunologia , Frações Subcelulares/imunologiaAssuntos
Adesão Celular , Entamoeba histolytica/fisiologia , Lectinas , Glicoproteínas de Membrana/análise , Proteínas de Protozoários/análise , Animais , Membrana Celular/química , Entamoeba histolytica/química , Entamoeba histolytica/patogenicidade , Eritrócitos , Imunofluorescência , Humanos , Fagocitose , Frações Subcelulares/química , Vacúolos/química , VirulênciaRESUMO
The body wall structure of muscle and newborn larvae of Trichinella spiralis was studied using transmission and scanning electron microscopy. Differences were found in the structure of the cuticle of the two developmental stages. In the case of the cuticle of the muscle larvae only transverse striae were present whereas the newborn larvae cuticle showed both transverse and longitudinal striations. In the two parasite stages the outer surface of the cuticle appears as a three-layered structure. The hypodermis presents well defined cellular components similar in fine structure in both stages. The plasma membrane of the hypodermal cells in the muscle larvae shows abundant short finger-like projections, that are not present in newborn larvae.
Assuntos
Trichinella/crescimento & desenvolvimento , Animais , Larva/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Músculos/parasitologia , Trichinella/ultraestruturaRESUMO
The morphological features of early intestinal ulcerations induced in rodents with axenic cultures of Entamoeba histolytica were studied by scanning and transmission electron microscopy. Amebas did not attach to the luminal surface of the mucosa except at interglandular regions, where parasites penetrated apparently through pseudopodial movement. Once in the lamina propria, trophozoites multiplied and destroyed mucosal components. Damage extended laterally through the mucosa, but progression to deeper layers of the intestinal wall was prevented by the muscularis mucosae, which acted as a partial barrier. This was eventually breached at focal points where amebas invaded the submucosa. Electron microscopy clearly showed the lysis of abundant polymorphonuclear neutrophil leukocytes (PMNs) by the amebas at the periphery of intestinal ulcerations as well as the lack of bacteria at these sites. This study demonstrates that recruitment and destruction of inflammatory cells following intestinal amebic invasion may take place in the absence of bacterial multiplication. The observations provide histological support to the hypothesis that lysis of PMNs by trophozoites participates in the genesis of amebic intestinal lesions.
Assuntos
Ceco/ultraestrutura , Disenteria Amebiana/patologia , Entamoeba histolytica/ultraestrutura , Mucosa Intestinal/ultraestrutura , Animais , Ceco/parasitologia , Cricetinae , Modelos Animais de Doenças , Disenteria Amebiana/parasitologia , Cobaias , Mucosa Intestinal/parasitologia , Microscopia Eletrônica , Microscopia Eletrônica de VarreduraRESUMO
The surface charge of Giardia lamblia trophozoites from axenic cultures of strains recently isolated in Mexico from human cases of symptomatic and asymptomatic giardiasis was studied by means of cellular microelectrophoresis and ultrastructural cytochemistry. It is concluded that ionogenic surface groups confer a negative surface charge on trophozoites of G. lamblia and that no significant differences exist between the surface charge of trophozoites of symptomatic and asymptomatic origin.
Assuntos
Giardia/metabolismo , Giardíase/parasitologia , Animais , Eletroforese , Giardia/ultraestrutura , Histocitoquímica , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Propriedades de SuperfícieRESUMO
Se utilizo microscopia electronica de barrido para estudiar la morfologia de trofozoitos de Entamoeba histolytica de las cepas HM1:IMSS y HK-9, incubados sobre un substrato natural, mono-cepas confluentes de celulas de la linea celular MDCK y uno artificial, cubreobjetos de plastico. Se estudiaron ademas trofozoitos separados de dichos substratos, asi como parasitos crio-fracturados. Los caracteres morfologicos que presentan los trofozoitos en ambos sistemas, estan representados entre otros, por pseudopodos y estomas de macropinocitosis, protrusiones citoplasmicas y filopodos de diversos tamanos. Los trofozoitos separados de los substratos antes mencionados, no presentan en su region basal estructuras citoplasmicas especializadas y los trofozoitos crio-fracturados muestran en su citoplasma abundantes vacuolas de varios tamanos y posiblemente diferente contenido. Los resultados obtenidos sugieren que el tipo de substrato en que se encuentra el parasito no influye en la morfologia externa del mismo