HLA characterization in adult asymptomatic cyst passers of Entamoeba histolytica/E. dispar

The present work aimed at studying the possible association of HLA antigens with Entamoeba histolytica/E. dispar asymptomatic infection in a Mexican mestizo population. A case-control design was selected for evaluation of the role of genetic markers in parasite infection. For this purpose the HLA-A, HLA-B, and HLA-DR profiles of a population of asymptomatic E. histolytica/E. dispar adult cyst passers (cases) and a corresponding nonparasitized adult group (controls) followed for 12 months were identified. Entamoeba species were identified through zymodeme patterns and/or amplification of species-specific DNA sequences. A healthy, nonparasitized group of individuals was included as a control. Our results show that apparently, no specific HLA marker is associated with the asymptomatic cyst passers' condition. These findings have to be added to previous results in which, in contrast to a demonstrated association between HLA-DR3 and amebic liver abscess in Mexican mestizo adults and infants, no significant association with amebic rectocolitis was found. Received: 18 March 1999 / Accepted: 16 April 1999     

Mucosal immunity to asymptomatic Entamoeba histolytica and Entamoeba dispar infection is associated with a peak intestinal anti-lectin immunoglobulin A antibody response

We monitored 93 subjects cured of amebic liver abscess (ALA) and 963 close associate controls in Durban, South Africa, and determined by enzyme-linked immunosorbent assay that the intestinal immunoglobulin A (IgA) antibody response to the Entamoeba histolytica galactose-inhibitable adherence lectin is most accurately represented by a complex pattern of transitory peaks. One or more intestinal anti-lectin IgA antibody peaks occurred in 85.9% of ALA subjects over 36 months compared to 41.6% of controls (P < 0.0001). ALA subjects exhibited a greater number of anti-lectin IgA antibody peaks (P < 0.0001) than controls. In addition, their peak optical density values were higher (peak numbers 1 to 3, P < 0.003), peaks were of longer duration (for peaks 1 and 2, P 相似文献   

Comparison of a stool antigen detection kit and PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar infections in asymptomatic cyst passers in Iran

The present study was conducted to compare stool antigen detection with PCR for the diagnosis of Entamoeba sp. infection in asymptomatic cyst passers from Iran. Entamoeba dispar and, in one case, E. moshkovskii were the Entamoeba spp. found in the amebic cyst passers. There was a 100% correlation between the results from the TechLab E. histolytica II stool antigen kit and those from nested PCR. We concluded that E. dispar is much more common in asymptomatic cyst passers in Iran and that antigen detection and PCR are comparable diagnostic modalities.     

Entamoeba histolytica as a commensal intestinal parasite in homosexual men

Entamoeba histolytica is considered to be an uncommon, imported organism in the United Kingdom and in many parts of North America, but recent attention has been drawn to the possibility of sexual transmission of this parasite among homosexual men. To determine the prevalence and clinical importance of enteric parasitic infections in men attending a clinic in London for the treatment of sexually transmitted diseases, we studied 354 randomly selected patients who provided a single stool sample that was examined for E. histolytica and other intestinal parasites. Forty-five of the 225 homosexual patients (20 percent) were infected with E. histolytica, but no such infections were found among the 129 heterosexual subjects (P less than 0.0001). With the use of isoenzyme electrophoresis, 34 of the 45 E. histolytica isolates were classified according to zymodeme. All were Zymodeme I or III, which are considered to be nonpathogenic. There was no correlation between the presence of E. histolytica and gastrointestinal symptoms. These findings suggest that E. histolytica is a common commensal in the homosexual population and that, in the absence of evidence of invasive disease, treatment of persons passing cysts of the organism may have little practical benefit.     

Entamoeba histolytica and Entamoeba dispar infections in cynomolgus monkeys imported into Japan for research

Three hundred and three stool samples of cynomolgus monkeys (Macaca fascicularis) imported from China and the Philippines were examined for Entamoeba histolytica/Entamoeba dispar infections. Microscopy detected E. histolytica/E. dispar cysts in 41 samples. Positive rates were higher in the monkeys from China (37.5%) than in the monkeys from the Philippines (3.7%). PCR analysis of 25 samples successfully cultured from the cysts demonstrated that 24 were E. dispar, one of the samples from China was E. histolytica. The one sample was also identified as E. histolytica by an antigen detection kit, although the monkey was asymptomatic and serology was negative. To our knowledge, this is the first report of E. histolytica isolation from cynomolgus monkeys based on the discrimination between E. histolytica and E. dispar.     

Entamoeba histolytica induces intestinal cathelicidins but is resistant to cathelicidin-mediated killing

The enteric protozoan parasite Entamoeba histolytica is the cause of potentially fatal amebic colitis and liver abscesses. E. histolytica trophozoites colonize the colon, where they induce inflammation, penetrate the mucosa, and disrupt the host immune system. The early establishment of E. histolytica in the colon occurs in the presence of antimicrobial human (LL-37) and murine (CRAMP [cathelin-related antimicrobial peptide]) cathelicidins, essential components of the mammalian innate defense system in the intestine. Studying this early step in the pathogenesis of amebic colitis, we demonstrate that E. histolytica trophozoites or their released proteinases, including cysteine proteinase 1 (EhCP1), induce intestinal cathelicidins in human intestinal epithelial cell lines and in a mouse model of amebic colitis. Despite induction, E. histolytica trophozoites were found to be resistant to killing by these antimicrobial peptides, and LL-37 and CRAMP were rapidly cleaved by released amebic cysteine proteases. The cathelicidin fragments however, did maintain their antimicrobial activity against bacteria. Degradation of intestinal cathelicidins is a novel function of E. histolytica cysteine proteinases in the evasion of the innate immune system in the bowel. Thus, early intestinal epithelial colonization of invasive trophozoites involves a complex interplay in which the ultimate outcome of infection depends in part on the balance between degradation of cathelicidins by amebic released cysteine proteinases and upregulation of proinflammatory mediators which trigger the inflammatory response.     

Purine-metabolising enzymes in Entamoeba histolytica

The enzymes that catalyse the salvage of purines in Entamoeba histolytica trophozoites have been surveyed. Adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4), guanine deaminase (EC 3.5.4.3), adenine phosphoribosyltransferase (PRTase) (EC 2.4.2.7), xanthine PRTase (EC 2.4.2.22) and hypoxanthine PRTase (EC 2.4.2.8) were all detected in cell homogenates but only at low activities, whereas AMP deaminase (EC 3.5.4.6) and guanine PRTase (EC 2.4.2.8) were not found. Phosphorylases (EC 2.4.2.1) active in both anabolic and catabolic directions were present and all nucleosides tested were phosphorylated by kinases (EC 2.7.1.15, EC 2.7.1.20, EC 2.7.1.73). 3'-Nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were found, the former being mainly particulate. Nucleotide interconversion enzymes (adenylosuccinate lyase, EC 4.3.2.2; adenylosuccinate synthetase, EC 6.3.4.4; IMP dehydrogenase, EC 1.2.1.14; GMP synthetase, EC 6.3.5.2 and GMP reductase, EC 1.6.6.8) were not detected. The results suggest that in E. histolytica the main route of nucleotide synthesis is from the individual bases through the actions of phosphorylases and kinases.     

Chromatin organization in Entamoeba histolytica

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.     

Diagnostic methods for differentiation of Entamoeba histolytica and Entamoeba dispar in carriers: performance and clinical implications in a non-endemic setting

Unpreserved faecal samples, suspected to contain Entamoeba histolytica/Entamoeba dispar cysts or trophozoites on the basis of microscopic examination, and serum samples from 416 patients were collected in a prospective study to determine whether stool antigen assays and detection of antibodies in serum are reliable methods to distinguish between carriers of E. histolytica and E. dispar in comparison to the reference test: real-time PCR. In 283 patients (68%) DNA of E. histolytica or E. dispar was amplified by real-time PCR: 6 patients with amoebic colitis (2%), 19 carriers of E. histolytica (6.7%), and 258 carriers of E. dispar (91.2%). In 133 patients (31%) no DNA of E. histolytica or E. dispar could be amplified in the stool samples. This patient group was used as control for the evaluation of diagnostic tests. Using real-time PCR as a reference test, the sensitivity and specificity of (1) the Entamoeba test for the diagnosis of E. histolytica/E. dispar carrier were 59% and 98%, (2) E. histolytica II for the diagnosis of E. histolytica carrier was 71% and 100%, and (3) serology for the diagnosis of E. histolytica infection was 83.3% and 95.2%, respectively. Applied to carriers that did not originate from an endemic country the sensitivity of serology for E. histolytica infection was 90% and specificity was 98.8%. In comparison to real-time PCR the performances of Entamoeba test and E. histolytica II lacked sensitivity for a reliable diagnosis of E. histolytica/E. dispar infection in a non-endemic setting. In carriers of E. histolytica/E. dispar from non-endemic countries the high specificity of serology can be used to establish the diagnosis of E. histolytica infection if antibodies are present.     

Characterization of Entamoeba histolytica alpha-actinin

We have cloned, expressed and characterized a alpha-actinin-like protein of Entamoeba histolytica. Analysis of the primary structure reveals that the essential domains of the alpha-actinin protein family are conserved: an N-terminus actin-binding domain, a C-terminus calcium-binding domain and a central helical rod domain. However, the rod domain of this Entamoeba protein is considerably shorter than the rod domain in alpha-actinins of higher organisms. The cloned Entamoeba 63 kDa protein is recognized by conventional alpha-actinin antibodies as well as binds and cross-links filamentous actin and calcium ions in the same manner as alpha-actinins. Despite the shorter rod domain this protein has conserved the most important functions of alpha-actinins. Therefore, it is suggested that this 63 kDa protein is an atypical and ancestral alpha-actinin.     

Entamoeba histolytica interactions with polarized human intestinal Caco-2 epithelial cells.

To model the initial pathogenic effects of Entamoeba histolytica trophozoites on intestinal epithelial cells, the interactions of E. histolytica HM1-IMSS trophozoites with polarized human intestinal Caco-2 cell monolayers grown on permeabilized filters were examined. Trophozoites, when incubated with the apical surface of the monolayers at 37 degrees C, induced a rapid decrease in transepithelial resistance over 15 to 60 min. The transmonolayer resistance response was not associated with changes in short-circuit current but was associated with an increase in mannitol flux, suggesting that the drop in resistance reflected a nonselective increase in epithelial permeability rather than stimulation of electrogenic ion transport. This response preceded the earliest detection of morphologic disruption of monolayer integrity by light or electron microscopy. Apical injury to the monolayer was detected by ultrastructural studies which revealed a loss of brush border in regions of contact between epithelial cells and amebas and by chromium release assays where a small increase in the apical release of 51Cr from the monolayer (6% over background) was observed. The transmonolayer resistance response was inhibited when the temperature was reduced to 4 degrees C and by addition of cytochalasin D (1 microgram/ml) to the medium at concentrations that did not directly affect transmonolayer resistance. Application of amebic lysates or medium conditioned by coincubation of amebas with Caco-2 monolayers failed to lower transmonolayer resistance, suggesting that this effect was not mediated by soluble amebic cytotoxins. Polarized Caco-2 monolayers grown on permeable filters provide a useful model for studying the initial interactions of E. histolytica trophozoites with intestinal epithelial cells.     

Lectin Activity in Entamoeba histolytica trophozoites

A lectin (carbohydrate-binding protein) has been found in extracts of a number of axenically grown trophozoites of Entamoeba histolytica strains. The strains grown in TYI-S-33 medium (Diamond et al., Trans. R. Soc. Trop. Med. Hyg. 72: 431-432, 1978) were HK-9, 200:NIH, and HM-1:IMSS. Strain HU-1:MUSC (HSC) was grown monoxenically in the same medium. The amoebic lectin agglutinated glutaraldehyde-fixed erythrocytes. This activity was pH dependent and heat and oxidation sensitive, and was destroyed by proteolysis upon autoincubation. The relative agglutinating potency of the different strains of amoebae was investigated. Strain HSC had the highest specific activity (210 U/mg of protein), and strain HM-1 had the lowest (14 U/mg). One unit of hemagglutinating activity is defined as the amount of lectin present in 1 ml of extract which will agglutinate 1 ml of 4% erythrocytes. Upon subcellular fractionation of the lectin present in extracts of strain HK-9, two-thirds of the activity was detected in the soluble, nonsedimentable (100,000 × g, 60 min) fraction. Partial hydrolysate of chitin was found to inhibit the hemagglutinating activity. Among the oligosaccharides of N-acetylglucosamine, the trimer and tetramer were the most potent inhibitors. The lectin was purified approximately 300-fold by a one-step affinity chromatography on a chitin column. The loading and elution from the column were based on the pH dependence of the lectin activity.     

The endocytic pathway in Entamoeba histolytica

We studied the endocytic pathway of Entamoeba histolytica using (a) confocal laser scanning microscopy to observe living cells labeled with fluorescent probes and (b) transmission electron microscopy of cells incubated in the presence of gold-labeled proteins or in a cytochemical medium designed for the localization of acid phosphatase activity. Images of acridine-orange-labeled cells showed that most of the intracellular vacuoles were acidic and were also labeled with Lucifer yellow, a fluorescent dye widely used for labeling of compartments of the endocytic pathway. A similar labeling pattern was observed when the cells were incubated in the present of fluorescein-labeled bovine albumin. However, no labeling was observed when fluorescein-labeled transferrin was used. Gold-labeled proteins (albumin, transferrin, horseradish peroxidase, and lactoferrin) were used for further characterization of the endocytic pathway. With the exception of transferrin, all the proteins bound to the protozoan surface and were subsequently internalized, appearing in small peripheral vesicles and some tubular structures. The ingested molecules accumulated in large vesicles located in the more central portion of the cell, which also presented acid phosphatase activity. Received: 16 February 2000 / Accepted: 30 May 2000