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1.  Bioassay-Guided Fractionation of Extracts from Codiaeum variegatum against Entamoeba histolytica Discovers Compounds That Modify Expression of Ceramide Biosynthesis Related Genes 
Leaves of Codiaeum variegatum (“garden croton”) are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica.
Author Summary
Amoebiasis is a disease caused by a protozoan parasite, Entamoeba histolytica, with or without clinical symptoms. Humans are the only relevant host of this parasite, which mainly targets the large intestine and the liver. The current drug, metronidazole, has been successfully used against this parasite for several years. However, some reports have shown either parasite resistance or adverse effects due to its long term usage. Our study thereby pointed to alternative treatment of this infection by investigating the rational use of Codiaeum variegatum also referred as “garden croton” which is a medicinal plant used in Cameroon against bloody diarrhoea. We moved into the identification of the most efficient fraction of the aqueous extract of this plant, and initiated the characterization of the mechanism of action of this fraction. Upon treatment with the active fraction, parasite death occurs within two days through morphological changes such as cell membrane disorganization and cell destruction. More deeply, we found that components of the active fraction modify expression of genes involved in ceramide biosynthesis, a pathway responsible for cell death and growth inhibition. Our study therefore suggests a novel finding which could be further explored for screening of anti-amoebic drugs.
PMCID: PMC3887121  PMID: 24416462
Ancient Science of Life  1993;12(3-4):358-362.
In ancient days, Physicians having the comprehensive knowledge of Bhaishajya Kalpana, used to prepare the drugs themselves to treat their patients. So there was no doubt in obtaining genuine drug with desired therapeutic effect. But in recent years, the growing population and their life style, industrialization etc have forced physicians to depend on market preparations. As such we find the necessity of standardization of these preparations. The quality assessments of a drug, which is a chemical irrespective of the system is possible by ‘Thin Layer Chromatographic technique’ using known Chemical constituents as reference standards. A herbal preparation ‘Kutajarishta’, has been standardized by using this technique and the significance of the findings is discussed.
PMCID: PMC3336554  PMID: 22556614
3.  Melatonin reduces the severity of experimental amoebiasis 
Parasites & Vectors  2011;4:62.
Melatonin has immunomodulatory effects but very little is known about its influence in protozoan infections, such as Entamoeba histolytica, which causes amoebiasis, a disease with significant morbidity and mortality. In this study, we evaluated the effects of exogenous melatonin interference in experimental amoebiasis and on interactions between human blood cells and E. histolytica trophozoites.
The effect of melatonin was investigated in models of experimental amoebiasis in hamsters and rats by evaluating the area of necrosis induced by E. histolytica. The activity of melatonin on the interactions between leukocytes and amoebae was determined by examining leukophagocytosis. For in vitro tests, polymorphonuclear and mononuclear human blood leucocytes were incubated with E. histolytica trophozoites.
The areas of amoebic necrosis were significantly reduced in animals treated with melatonin. Melatonin treatment increased leukophagocytosis but was associated with a greater number of dead amoebae.
These results suggest that melatonin may play a beneficial role in the control of amoebic lesions, raising the possibility that this drug may be used as an adjuvant in anti-amoebic therapy.
PMCID: PMC3097001  PMID: 21501501
4.  Evaluation of Amoebicidal Potential of Paneth Cell Cryptdin-2 against Entamoeba histolytica 
Amoebiasis is a major public health problem in tropical and subtropical countries. Currently, metronidazole is the gold choice medication for the treatment of this disease. However, reports have indicated towards the possibility of development of metronidazole-resistance in Entamoeba strains in near future. In view of the emergence of this possibility, in addition to the associated side effects and mutagenic ability of the currently available anti-amoebic drugs, there is a need to explore newer therapeutics against this disease. In this context, the present study evaluated the amoebicidal potential of cryptdin-2 against E. histolytica.
Methods/Principal Findings
In the present study, cryptdin-2 exhibited potent in-vitro amoebicidal activity against E. histolytica in a concentration dependent manner at a minimum amoebicidal concentration (MAC) of 4 mg/L. Scanning electron microscopy as well as phase contrast microscopic investigations of cryptdin-2 treated trophozoites revealed that the peptide was able to induce significant morphological alterations in terms of membrane wrinkling, leakage of the cytoplasmic contents and damaged plasma membrane suggesting a possible membrane dependent amoebicidal activity. N-phenyl napthylamine (NPN) uptake assay in presence of sulethal, lethal as well as twice the lethal concentrations further confirmed the membrane-dependent mode of action of cryptdin-2 and suggested that the peptide could permeabilize the plasma membrane of E. histolytica. It was also found that cryptdin-2 interfered with DNA, RNA as well as protein synthesis of E. histolytica exerting the highest effect against DNA synthesis. Thus, the macromolecular synthesis studies correlated well with the observations of membrane permeabilization studies.
The amoebicidal efficacy of cryptdin-2 suggests that it may be exploited as a promising option to combat amoebiasis or, at least, may act as an adjunct to metronidazole and/or other available anti-amoebic drugs.
Author Summary
Intestinal amoebiasis, caused by Enatmoeba histolytica continues to be a major public health problem in tropical and subtropical countries and is considered to be the third principal parasitic disease responsible for mortality in the world. In addition to the mutagenic ability and known toxicity of conventional anti-amoebic drugs, there are reports indicating the emergence of treatment failures to these drugs. Therefore, there has been a considerable interest in exploring the potential of various antimicrobial peptides having higher efficacy and lower toxicity to combat such parasitic infections. Herein, we present the amoebicidal efficacy of cryptdin-2, a Paneth cell alpha-defensin against E. histolytica. Cryptdin-2 was found to decrease the number of trophozoites of E. histolytica in a concentration dependent manner. By and large, cryptdin-2 could retain its amoebicidal activity in the presence of cations, bile salts and at various pH values. Microscopic analysis and N-phenyl napthylamine (NPN) uptake assay revealed membrane dependent amoebicidal action of the peptide. It was also demonstrated that cryptdin-2 has the potential to target the intracellular macromolecular synthesis machinery of Entamoeba. Based on these results, cryptdin-2 seems to be a promising agent for the development of novel therapeutics against amoebiasis or at least may act as an adjunct to conventional antibiotics against E. histolytica.
PMCID: PMC3243701  PMID: 22206022
5.  The Interplay between Entamoeba and Enteropathogenic Bacteria Modulates Epithelial Cell Damage 
Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined.
Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified.
Principal Findings
E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica.
Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage.
The in vitro system presented here provides evidence that the Entamoeba/enteropathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon.
Author Summary
In amoebiasis, a human disease that is a serious health problem in many developing countries, efforts have been made to identify responsible factors for the tissue damage inflicted by the parasite Entamoeba histolytica. This amoeba lives in the lumen of the colon without causing damage to the intestinal mucosa, but under unknown circumstances becomes invasive, destroying the intestinal tissue. Bacteria in the intestinal flora have been proposed as inducers of higher amoebic virulence, but the causes or mechanisms responsible for the induction are still undetermined. Mixed intestinal infections with Entamoeba histolytica and enteropathogenic bacteria, showing exacerbated manifestations of disease, are common in endemic countries. We implemented an experimental system to study amoebic virulence in the presence of pathogenic bacteria and its consequences on epithelial cells. Results showed that amoebae that ingested enteropathogenic bacteria became more virulent, causing more damage to epithelial cells. Bacteria induced release of inflammatory proteins by the epithelial cells that attracted amoebae, facilitating amoebic contact to the epithelial cells and higher damage. Our results, although a first approach to this complex problem, provide insights into amoebic infections, as interplay with other pathogens apparently influences the intestinal environment, the behavior of cells involved and the manifestations of the disease.
PMCID: PMC2447883  PMID: 18648517
6.  A Proteomic and Cellular Analysis of Uropods in the Pathogen Entamoeba histolytica 
Exposure of Entamoeba histolytica to specific ligands induces cell polarization via the activation of signalling pathways and cytoskeletal elements. The process leads to formation of a protruding pseudopod at the front of the cell and a retracting uropod at the rear. In the present study, we show that the uropod forms during the exposure of trophozoites to serum isolated from humans suffering of amoebiasis. To investigate uropod assembly, we used LC-MS/MS technology to identify protein components in isolated uropod fractions. The galactose/N-acetylgalactosamine lectin, the immunodominant antigen M17 (which is specifically recognized by serum from amoeba-infected persons) and a few other cells adhesion-related molecules were primarily involved. Actin-rich cytoskeleton components, GTPases from the Rac and Rab families, filamin, α-actinin and a newly identified ezrin-moesin-radixin protein were the main factors found to potentially interact with capped receptors. A set of specific cysteine proteases and a serine protease were enriched in isolated uropod fractions. However, biological assays indicated that cysteine proteases are not involved in uropod formation in E. histolytica, a fact in contrast to the situation in human motile immune cells. The surface proteins identified here are testable biomarkers which may be either recognized by the immune system and/or released into the circulation during amoebiasis.
Author Summary
Uropods are membrane folds formed at the rear of moving cells, e.g. lymphocytes during immune responses and the amoebic parasite Entamoeba histolytica during amoebiasis. Previous studies showed some surface receptors of E. histolytica, e.g. the Gal/GalNAc lectin, which is involved in adhesion, undergo capping and accumulate at the uropod, and these processes are driven by the activities of the actin-rich cytoskeleton. These uropods are then discarded to the extracellular medium, suggesting the components of uropods may induce anti-amoebic responses from the host. In this study, we showed that the serum from patients infected with E. histolytica, but not serum from healthy individuals, is able to induce uropod formation. To characterize the proteome of these induced uropods, we performed a proteomic analysis of the discarded complexes. In addition to the presence of several proteases and novel cytoskeleton factors, our proteomic results highlight the presence of important surface components including the Gal/GalNAc lectin, calreticulin, several adhesion molecules and the immunodominant antigen M17. Finally, we derived two important conclusions from further cellular analyses. Firstly, cysteine proteases are not involved in uropod formation in E. histolytica. Secondly, M17 was confirmed to be recruited at the uropods induced by serum from infected patients.
PMCID: PMC3071361  PMID: 21483708
7.  An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis 
Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.
Author Summary
Entamoeba histolytica is the causative agent of amoebiasis, a human disease. Like other enteric infections, the lack of animal models enhances the difficulty of studying the development of amoebiasis. To date, no experimental model has been developed that reproduces the invasive intestinal amoebic lesions seen in human colon. We present the first study that examines, using human colon explants, the early steps of the human colonic barrier invasion by E. histolytica. With this ex vivo integrative model we have investigated both parasite behaviour and the human tissue response. Remarkably, in this model E. histolytica was able to cross and destroy the intestinal barrier evoking a tissue inflammatory response, while E. dispar, a non-pathogenic species, was unable to penetrate nor induce tissue responses. Furthermore, we have explored the role of three virulence factors during the invasive process, using gene-silenced E. histolytica trophozoites, particularly the kinetics of invasion, tissue destruction and induction of an early inflammatory responses. This is, to our knowledge, the first time that their role is highlighted in a complex human system. Our study provides new insights in the molecular mechanisms involved in the early steps of human colon invasion by E. histolytica.
PMCID: PMC2777411  PMID: 19936071
8.  Neurohumoral alterations and their role in amoebiasis 
Amoebiasis is world wide in distribution and continues to be an important Public health problem. Intestinal amoebiasis may be present as dysentery, diarrhea or may stimulate other intra abdominal conditions. Clinical symptoms may not be seen in majority of patients, while amebic cysts are passed in the stool. This single-celled parasite is transmitted to humans via contaminated water and food. Amoebic dysentery can be accompanied by amebic infection of the liver and other organs. The present study was carried out to evaluate the changes in the circulating levels of neurohumors, their metabolizing enzymes and cortisol in these patients both before and after one month of chemotherapy. In the patients of amoebiasis the circulating acetylcholine (ACh), histamine, histaminase, cortisol, 5-Hydroxy tryptamine (serotonin) levels were significnatly enhanced with no change in the Dopamine-beta-hydroxylase (DBH) activity, while the activities of erythrocyte aceytylcholinesterase (AChE) and plasma Monoamine oxidase (MAO) were found decreased in comparison to normal healthy controls. After one month of treatment all the parameters reverted towards their control values, while the level of plasma histaminase remained still significantly high. The normal DBH activity reflects that there is no alteration in the circulating catecholamine levels. while the alteration in the levels of histamine, serotonin and cortisol may be due to the nonspecific response of the body to the stress of the disease and the parasitic infestation.
PMCID: PMC3453840  PMID: 23105547
Amoebiasis; Acetylcholine; Cortisol; histamine; Histaminase
9.  Identification of the Virulence Landscape Essential for Entamoeba histolytica Invasion of the Human Colon 
PLoS Pathogens  2013;9(12):e1003824.
Entamoeba histolytica is the pathogenic amoeba responsible for amoebiasis, an infectious disease targeting human tissues. Amoebiasis arises when virulent trophozoites start to destroy the muco-epithelial barrier by first crossing the mucus, then killing host cells, triggering inflammation and subsequently causing dysentery. The main goal of this study was to analyse pathophysiology and gene expression changes related to virulent (i.e. HM1:IMSS) and non-virulent (i.e. Rahman) strains when they are in contact with the human colon. Transcriptome comparisons between the two strains, both in culture conditions and upon contact with human colon explants, provide a global view of gene expression changes that might contribute to the observed phenotypic differences. The most remarkable feature of the virulent phenotype resides in the up-regulation of genes implicated in carbohydrate metabolism and processing of glycosylated residues. Consequently, inhibition of gene expression by RNA interference of a glycoside hydrolase (β-amylase absent from humans) abolishes mucus depletion and tissue invasion by HM1:IMSS. In summary, our data suggest a potential role of carbohydrate metabolism in colon invasion by virulent E. histolytica.
Author Summary
Entamoeba histolytica is an intestinal parasite which displays diverse phenotypes with respect to pathogenesis in the human colon. Trophozoites can remain as commensal, without causing evident intestinal damage, or they can destroy the colonic mucosa leading to amoebiasis. Using human colon explants and transcriptome analysis, we investigated the gene expression profile of two E. histolytica strains (virulent and non-virulent) during their contact with the intestinal mucus to gain insights into the molecular basis responsible for amoebic divergent phenotypes. Our results suggest that the virulent E. histolytica, when in contact with the intestinal barrier, specifically increases the rate of gene transcription for enzymes necessary to exploits the carbohydrate resources present in the human colon. Using RNA interference methodologies to knockdown gene expression, our data revealed the potential role of amoebic β-amylase (a glycosydase) in colon invasion and mucus depletion. Our data implies that the ability of an E. histolytica strain to exploit the carbohydrate resources might affect its ability to invasion the intestine.
PMCID: PMC3868522  PMID: 24385905
10.  Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica. 
Journal of Clinical Pathology  1994;47(3):236-239.
AIMS--To assess the reliability of the detection of erythrophagocytic amoebic trophozoites in stool samples in the diagnosis of dysentery associated with invasive Entamoeba histolytica. METHODS--Amoebic culture was carried out on single stool samples collected from patients from Mexico, Colombia, and Bangladesh. The stools had been examined by light microscopy. Amoebic dysentery was diagnosed when erythrophagocytic E histolytica trophozoites were observed in a case of bloody diarrhoea. E histolytica isolates were characterised by isoenzyme electrophoresis and results correlated with microscopical findings in stools. Statistical analysis was performed using the chi 2 test. RESULTS--Where erythrophagocytic amoebae had been observed in dysenteric stool specimens the E histolytica phenotype was invariably invasive (p < 0.0001). Observation of erythrophagocytic amoebae in dysentery is 100% specific and predictive of infection with invasive E histolytica. When amoebic culture-positive cases only are considered it is 96% sensitive. In this study E histolytica of zymodeme XIV was more commonly associated with amoebic dysentery than zymodeme II. There was no significant difference between the carriage rate of invasive and non-invasive E histolytica in non-dysenteric diarrhoea. Asymptomatic subjects carried non-invasive E histolytica more frequently than invasive E histolytica. Patients with non-amoebic dysentery, when shown to be infected with E histolytica, carried non-invasive strains (12%). CONCLUSIONS--Sensitivity and specificity of microscopical examination of a single stool specimen for diagnosing amoebic dysentery is very high; intestinal carriage of invasive E histolytica detected by culture is not necessarily an indication of active disease as patients with diarrhoea and asymptomatic subjects shed invasive and non-invasive E histolytica. There are possibly two subpopulations of invasive E histolytica with different pathogenic potential which can be differentiated by zymodeme analysis.
PMCID: PMC501902  PMID: 8163695
11.  New Assembly, Reannotation and Analysis of the Entamoeba histolytica Genome Reveal New Genomic Features and Protein Content Information 
In order to maintain genome information accurately and relevantly, original genome annotations need to be updated and evaluated regularly. Manual reannotation of genomes is important as it can significantly reduce the propagation of errors and consequently diminishes the time spent on mistaken research. For this reason, after five years from the initial submission of the Entamoeba histolytica draft genome publication, we have re-examined the original 23 Mb assembly and the annotation of the predicted genes.
Principal Findings
The evaluation of the genomic sequence led to the identification of more than one hundred artifactual tandem duplications that were eliminated by re-assembling the genome. The reannotation was done using a combination of manual and automated genome analysis. The new 20 Mb assembly contains 1,496 scaffolds and 8,201 predicted genes, of which 60% are identical to the initial annotation and the remaining 40% underwent structural changes. Functional classification of 60% of the genes was modified based on recent sequence comparisons and new experimental data. We have assigned putative function to 3,788 proteins (46% of the predicted proteome) based on the annotation of predicted gene families, and have identified 58 protein families of five or more members that share no homology with known proteins and thus could be entamoeba specific. Genome analysis also revealed new features such as the presence of segmental duplications of up to 16 kb flanked by inverted repeats, and the tight association of some gene families with transposable elements.
This new genome annotation and analysis represents a more refined and accurate blueprint of the pathogen genome, and provides an upgraded tool as reference for the study of many important aspects of E. histolytica biology, such as genome evolution and pathogenesis.
Author Summary
Entamoeba histolytica is an anaerobic parasitic protozoan that causes amoebic dysentery. The parasites colonize the large intestine, but under some circumstances may invade the intestinal mucosa, enter the bloodstream and lead to the formation of abscesses such amoebic liver abscesses. The draft genome of E. histolytica, published in 2005, provided the scientific community with the first comprehensive view of the gene set for this parasite and important tools for elucidating the genetic basis of Entamoeba pathogenicity. Because complete genetic knowledge is critical for drug discovery and potential vaccine development for amoebiases, we have re-examined the original draft genome for E. histolytica. We have corrected the sequence assembly, improved the gene predictions and refreshed the functional gene assignments. As a result, this effort has led to a more accurate gene annotation, and the discovery of novel features, such as the presence of genome segmental duplications and the close association of some gene families with transposable elements. We believe that continuing efforts to improve genomic data will undoubtedly help to identify and characterize potential targets for amoebiasis control, as well as to contribute to a better understanding of genome evolution and pathogenesis for this parasite.
PMCID: PMC2886108  PMID: 20559563
12.  Entamoeba histolytica and E. dispar trophozoites in the liver of hamsters: in vivo binding of antibodies and complement 
Parasites & Vectors  2010;3:23.
Human amoebiasis is caused by the parasitic protozoan Entamoeba histolytica that lives in the large intestine of hosts, where can produce asymptomatic colonization until severe invasive infections with blood diarrhea and spreading to other organs. The amoebic abscesses in liver are the most frequent form of amoebiasis outside intestine and still there are doubts about the pathogenic mechanisms involved in their formation. In this study we evaluated the in situ binding of antibodies, C3 and C9 complement components on trophozoites, in livers of hamsters infected with E. histolytica or E. dispar. These parameters were correlated with the extension of the hepatic lesions observed in these animals and with trophozoites survivor.
Hamsters were inoculated intra-hepatically with 100,000 trophozoites of E. histolytica or E. dispar strain and necropsied 12, 24, 48, 72, 144 and 192 h after inoculation. Antibodies, C3 and C9 binding to trophozoites were detected by immunohistochemistry. The estimation of the necrosis area and the number of labeled trophozoites was performed using digital morphometry analysis.
In the liver sections of animals inoculated with the amoebas, the binding of antibodies to E. histolytica trophozoites was significantly lower than to E. dispar trophozoites. Trophozoites of E. dispar were also more frequently vacuolated and high labeled cellular debris observed in the lesions. Positive diffuse reaction to C3 complement component was more intense in livers of animals inoculated with E. histolytica after 24 and 72 h of infection. C3(+) and C9(+) trophozoites were detected in the vascular lumen, granulomas and inside and in the border of necrotic areas of both infected group animals. C3(+) and C9(+) trophozoite debris immunostaining was higher in livers of E. dispar than in livers of E. histolytica. A positive correlation between necrotic areas and number of C9(+) trophozoites was observed in animals inoculated with E. dispar.
Morphological and immunohistochemical results suggest that antibodies and complement are able to bind and destroy some trophozoites in the liver of experimentally infected hamsters, perhaps selecting the more resistant parasites which are responsible by progression of amoebic abscesses. The findings indicate that E. histolytica possesses an enhanced ability in vivo to evade the immune responses compared to E. dispar, although it also causes experimental hepatic lesions.
PMCID: PMC2861030  PMID: 20338063
13.  Pulmonary amoebiasis presenting as superior vena cava syndrome 
Thorax  2005;60(4):350-352.
Pulmonary amoebiasis without liver involvement occurs sporadically as a result of haematogenous spread from a primary site, the colon. The case history is presented of a patient who developed superior vena cava syndrome due to a pulmonary amoebic abscess without liver involvement. He was initially suspected of having a neoplasm but a combination of tests including histological examination of the H&E stained excised tissue, immunofluorescence using anti-Entamoeba histolytica antibodies, and serology confirmed the diagnosis of amoebiasis. To our knowledge this is the first description of pulmonary amoebiasis presenting as superior vena cava syndrome.
PMCID: PMC1747354  PMID: 15790993
14.  Antiamoebic and Toxicity Studies of a Carbamic Acid Derivative and Its Therapeutic Effect in a Hamster Model of Hepatic Amoebiasis 
Amoebiasis is an important public health problem in developing countries. Entamoeba histolytica, the causative agent of amoebiasis, may develop resistance to nitroimidazoles, a group of drugs considered to be the most effective against this parasitic disease. Therefore, research on new drugs for the treatment of this common infection still constitutes an important therapeutic demand. In the present study we determined the effects of a carbamate derivative, ethyl 4-chlorophenylcarbamate (C4), on trophozoites of E. histolytica strain HM-1:IMSS. C4 was subject to various toxicity tests, including the determination of mutagenicity for bacterial DNA and changes in the enzymatic activities of eukaryotic cells. Genotoxicity studies were performed by the mutagenicity Ames test (plate incorporation and preincubation methods) with Salmonella enterica serovar Typhimurium, with or without metabolic activation produced by the S9 fraction of rat liver. C4 toxicity studies were performed by measuring enzymatic activity in eukaryotic cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-formazan test with Fischer 344 rat hepatocytes. C4 did not induce either frame-shift mutations in S. enterica serovar Typhimurium TA97 or TA98 or base pair substitutions in strains TA100 and TA102. The compound was not toxic for cultured rat hepatic cells. Trophozoites treated with 100 μg of C4 per ml were inhibited 97.88% at 48 h of culture; moreover, damage to the amoebae was also confirmed by electron microscopy. The antiamoebic activity of C4 was evaluated by using an in vivo model of amoebic liver abscess in hamsters. Doses of 75 and 100 mg/100 g of body weight reduced the extent of the amoebic liver abscess by 84 and 94%, respectively. These results justify further studies to clearly validate whether C4 is a new suitable antiamoebic drug.
PMCID: PMC549235  PMID: 15728919
15.  Evaluation of antigen detection and polymerase chain reaction for diagnosis of amoebic liver abscess in patients on anti-amoebic treatment 
BMC Research Notes  2012;5:416.
Diagnosis of amoebic liver abscess (ALA) in patients on anti-amoebic drugs is difficult. There is scanty data on this issue using Entamoeba histolytica (E. histolytica) lectin antigen and polymerase chain reaction (PCR). We studied utility of lectin antigen, PCR, and IgG antibody in diagnosis of liver abscess in patients on anti-amoebic treatment. Liver aspirate of 200 patients, of which 170 had anti-amoebic drug prior to drainage, was tested for E. histolytica lectin antigen by (ELISA), PCR, bacterial culture, and serum IgG antibody by (ELISA). Classification of abscesses was based on result of anti-amoebic IgG antibody and bacterial culture, E. histolytica PCR and bacterial culture, and E. histolytica lectin antigen and bacterial culture.
Using anti-amoebic IgG antibody and bacterial culture, 136/200 (68.0%) were classified as ALA, 12/200 (6.0%) as pyogenic liver abscess (PLA), 29/200 (14.5%) as mixed infection, and 23/200 (11.5%) remained unclassified. Using amoebic PCR and bacterial culture 151/200 (75.5%) were classified as ALA, 25/200 (12.5%) as PLA, 16/200 (8.0%) as mixed infection, and 8/200 (4.0%) remained unclassified. With E. histolytica lectin antigen and bacterial culture, 22/200 (11.0%) patients were classified as ALA, 39/200 (19.5%) as PLA, 2/200 (1.0%) as mixed infection, and 137/200 (68.5%) remained unclassified.
E. histolytica lectin antigen was not suitable for classification of ALA patients who had prior anti-amoebic treatment. However, PCR may be used as alternative test to anti-amoebic antibody in diagnosis of ALA.
PMCID: PMC3477060  PMID: 22870930
Entamoeba histolytica; Amoebiasis; Pyogenic liver abscess; Anti-amoebic IgG antibody
16.  Corticosteroids and liver amoebiasis. 
British Medical Journal  1978;2(6134):394-395.
Patients with amoebiasis who receive steroid treatment may suffer adverse affects including acute amoebic dysentery and exacerbation of the amoebiasis. In some cases the presenting symptoms are initially misdiagnosed and steroids prescribed, which provokes fulminating progression of hepatic amoebiasis. Repeated stool examinations often yield negative results. Any patient being considered for treatment with corticosteroids who has lived in the tropics should be investigated for amoebiasis serologically and by repeated stool examination. Even after negative results the possibility of amoebiasis should be reconsidered if diarrhoea or fever develops during or after steroid treatment.
PMCID: PMC1609038  PMID: 687936
17.  Evaluation of Entamoeba histolytica recombinant phosphoglucomutase protein for serodiagnosis of amoebic liver abscess 
BMC Infectious Diseases  2013;13:144.
Amoebic liver abscess (ALA) is the most frequent clinical presentation of extra-intestinal amoebiasis. The diagnosis of ALA is typically based on the developing clinical symptoms, characteristic changes on radiological imaging and serology. Numerous serological tests have been introduced for the diagnosis of ALA, either detecting circulating amoebic antigens or antibodies. However those tests show some pitfalls in their efficacy and/or the preparation of the tests are costly and tedious. The commercial IHA kit that used crude antigen was reported to be useful in diagnosis of ALA, however high antibody background in endemic areas may cause problems in its interpretation. Thus, discovery of well-defined antigen(s) is urgently needed to improve the weaknesses of current serodiagnostic tests.
Crude antigen of E. histolytica was analysed by 2-DE and Western blot to identify a protein of diagnostic potential for ALA. The corresponding gene of the antigenic protein was then cloned, expressed and the purified recombinant protein was subsequently evaluated for serodiagnosis of ALA in an indirect ELISA format.
Analysis of crude antigen showed that phosphoglucomutase (PGM) has the diagnostic potential. Recombinant PGM (rPGM) showed 79.17% (19/24) sensitivity and 86.67% (195/225) specificity in diagnosis of ALA based on the COV of mean +1SD. There was no significant difference between rPGM-ELISA and IHA diagnostic kit in the diagnosis of ALA in terms of sensitivity and specificity at p-value < 0.05.
In conclusion, rPGM-ELISA is found to be useful for serodiagnosis of ALA. Future studies will determine whether rPGM-ELISA also detects antibodies produced in amoebic dysentery and asymptomatic cases.
PMCID: PMC3606622  PMID: 23514636
Entamoeba histolytica; Amoebic liver abscess; 2-DE; Western blot; Phosphoglucomutase; Diagnosis; rPGM-ELISA
18.  Differences in the transcriptome signatures of two genetically related Entamoeba histolytica cell lines derived from the same isolate with different pathogenic properties 
BMC Genomics  2010;11:63.
The availability of two genetically very similar cell lines (A and B) derived from the laboratory isolate Entamoeba histolytica HM-1:IMSS, which differ in their virulence properties, provides a powerful tool for identifying pathogenicity factors of the causative agent of human amoebiasis. Cell line A is incapable inducing liver abscesses in gerbils, whereas interaction with cell line B leads to considerable abscess formation. Phenotypic characterization of both cell lines revealed that trophozoites from the pathogenic cell line B have a larger cell size, an increased growth rate in vitro, an increased cysteine peptidase activity and higher resistance to nitric oxide stress. To find proteins that may serve as virulence factors, the proteomes of both cell lines were previously studied, resulting in the identification of a limited number of differentially synthesized proteins. This study aims to identify additional genes, serving as virulence factors, or virulence markers.
To obtain a comprehensive picture of the differences between the cell lines, we compared their transcriptomes using an oligonucleotide-based microarray and confirmed findings with quantitative real-time PCR. Out of 6242 genes represented on the array, 87 are differentially transcribed (≥two-fold) in the two cell lines. Approximately 50% code for hypothetical proteins. Interestingly, only 19 genes show a five-fold or higher differential expression. These include three rab7 GTPases, which were found with a higher abundance in the non-pathogenic cell line A. The aig1-like GTPasesare of special interest because the majority of them show higher levels of transcription in the pathogenic cell line B. Only two molecules were found to be differentially expressed between the two cell lines in both this study and our previous proteomic approach.
In this study we have identified a defined set of genes that are differentially transcribed between the non-pathogenic cell line A and the pathogenic cell line B of E. histolytica. The identification of transcription profiles unique for amoebic cell lines with pathogenic phenotypes may help to elucidate the transcriptional framework of E. histolytica pathogenicity and serve as a basis for identifying transcriptional markers and virulence factors.
PMCID: PMC2823695  PMID: 20102605
19.  Serological diagnosis of amoebiasis by immunofluorescence 
Journal of Clinical Pathology  1967;20(6):875-878.
A positive serological diagnosis of amoebiasis could be made by immunofluorescence in 66 of 78 established cases, taking a serum titre of 16 or higher as diagnostic: at this level there were no false positives among 94 control sera. The test is simple and may be carried out on amoebic smears stored for several months in 2-octanol. The serological activity is largely confined to the IgG immunoglobulin fraction and is specific for Entamoeba histolytica; cross reactions were not detected with other protozoa. Gel diffusion serological analysis permitted a positive diagnosis of amoebiasis in 60 of the 78 cases, and, combining this with the immunofluorescence test, raised the diagnostic score to 71 cases.
PMCID: PMC473630  PMID: 4302225
20.  Antigens of diagnostic and prophylactic importance inEntamoeba histolytica 
Amoebiasis, caused by an enteric protozoanEntamoeba histolytica, is one of the major parasitic diseases of mankind. Current estimate suggests that the parasite infects about 10% of the world population at any given time. There is an urgent need to characterize the antigenic molecules ofE. histolytica, and find out antigens which have both immunodiagnostic and prophylactic potential against amoebiasis. The results of somatic antigen analysis, using sera from immune or infected individuals, indicated that the wholeE. histolytica trophozoites, are highly complex and heterogeneous in nature and both immunodiagnostic and immuno prophylaxis activity remain mainly in a surface associated 29 kDa glycoprotein ofE. histolytica. Future studies at molecular level particularly, genes responsible for expression of this protein, their homology with other proteins and structure analysis will give better understanding about this polypeptide. Studies on excretory secretory antigens, clearly demonstrated thatE. histolytica like many organisms, also liberates certain antigenic moieties into the culture medium during in vitro cultivation and this antigen has similar diagnostic values like the conventional somatic antigens. It is important that the ESA should be prepared from the supernatant after collecting the cell and use for immunodiagnosis of amoebiasis. This is an additional source of antigen which will help in carrying out more tests using the same reagents. Further studies are needed to clarify the location of these molecules on the parasite, along with detailed biochemical and immunological characterization and their relation with the pathogenesis.
PMCID: PMC3453534  PMID: 23105180
Enzyme linked immunosorbent assay; immunoblot; somatic antigen; excretorysecretory antigens; protection
21.  New insights into host–pathogen interactions during Entamoeba histolytica liver infection 
Amoebiasis is the third worldwide disease due to a parasite. The causative agent of this disease, the unicellular eukaryote Entamoeba histolytica, causes dysentery and liver abscesses associated with inflammation and human cell death. During liver invasion, before entering the parenchyma, E. histolytica trophozoites are in contact with liver sinusoidal endothelial cells (LSEC). We present data characterizing human LSEC responses to interaction with E. histolytica and identifying amoebic factors involved in the process of cell death in this cell culture model potentially relevant for early steps of hepatic amoebiasis. E. histolytica interferes with host cell adhesion signalling and leads to diminished adhesion and target cell death. Contact with parasites induces disruption of actin stress fibers and focal adhesion complexes. We conclude that interference with LSEC signalling may result from amoeba-triggered changes in the mechanical forces in the vicinity of cells in contact with parasites, sensed and transmitted by focal adhesion complexes. The study highlights for the first time the potential role in the onset of hepatic amoebiasis of the loss of liver endothelium integrity by disturbance of focal adhesion function and adhesion signalling. Among the amoebic factors required for changed LSEC adherence properties we identified the Gal/GalNAC lectin, cysteine proteases and KERP1.
PMCID: PMC3894810  PMID: 24466432
amoebiasis; cell death; Entamoeba; focal adhesions; integrins; KERP1; liver sinusoidal endothelial cells
22.  Surgery in amoebic colitis 
Gut  1970;11(11):941-946.
The feasibility of surgery in amoebic colitis is presented by illustrative case histories in two patients. Subtotal colectomy and ileostomy were carried out for fulminating colitis in the one and exteriorization, excision, and subsequent re-anastomosis of a localized perforated area of bowel in the other.
It is suggested that the place of surgery in amoebic colitis should be reappraised, particularly with the advent of modern medical and surgical methods now available. The indications for contemplating surgery in abdominal amoebiasis are outlined and a workable approach is presented.
PMCID: PMC1553135  PMID: 5492250
23.  Lactobacillus plantarum (VR1) isolated from an Ayurvedic medicine (Kutajarista) ameliorates in vitro cellular damage caused by Aeromonas veronii 
BMC Microbiology  2011;11:152.
Lactobacillus plantarum is considered as a safe and effective probiotic microorganism. Among various sources of isolation, traditionally fermented foods are considered to be rich in Lactobacillus spp., which can be exploited for their probiotic attribute. Antibacterial property of L. plantarum has been demonstrated against various enteric pathogens in both in vitro and in vivo systems. This study was aimed at characterizing L. plantarum isolated from Kutajarista, an ayurvedic fermented biomedicine, and assessing its antagonistic property against a common enteropathogen Aeromonas veronii.
We report the isolation of L. plantarum (VR1) from Kutajarista, and efficacy of its cell free supernatant (CFS) in amelioration of cytotoxicity caused by Aeromonas veronii. On the part of probiotic attributes, VR1 was tolerant to pH 2, 0.3% bile salts and simulated gastric juice. Additionally, VR1 also exhibited adhesive property to human intestinal HT-29 cell line. Furthermore, CFS of VR1 was antibacterial to enteric pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Aeromonas veronii and clinical isolates of P. aeruginosa and E. coli. Detailed study regarding the effect of VR1 CFS on A. veronii cytotoxicity showed a significant decrease in vacuole formation and detrimental cellular changes in Vero cells. On the other hand, A. veronii CFS caused disruption of tight junction proteins ZO-1 and actin in MDCK cell line, which was prevented by pre-incubation with CFS of VR1.
This is the first study to report isolation of L. plantarum (VR1) from Kutajarista and characterisation for its probiotic attributes. Our study demonstrates the antagonistic property of VR1 to A. veronii and effect of VR1 CFS in reduction of cellular damage caused by A. veronii in both Vero and MDCK cell lines.
PMCID: PMC3145568  PMID: 21707995
24.  Bioinformatics and Functional Analysis of an Entamoeba histolytica Mannosyltransferase Necessary for Parasite Complement Resistance and Hepatical Infection 
The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis.
Author Summary
The causative agent of the infectious disease, amoebiasis, is the parasite Entamoeba histolytica, which targets human intestine and liver. Once in the host, this parasite attaches to human cells and matrix components via factors at its surface such as the Gal/GalNAc lectin and proteophosphoglycans (PPGs). These factors are themselves anchored to the parasite surface by a glycosylphosphatidylinositol (GPI) moiety. To synthesise the GPI, a cascade of enzymes are necessary including the mannosyltransferase 1 (PIG-M1). A homolog of the PIG-M1 enzyme was shown to be present in E. histolytica (EhPIG-M1). To study the role of EhPIG-M1 in E. histolytica, parasites were constructed that had a reduced amount of mannosyltransferase. These parasites displayed a diminished production of GPI molecules and a lower amount of PPGs at the cell surface. Interestingly, the parasites were highly sensitive to the host blood complement and the formation of liver abscesses in hamsters was dramatically impaired. These results suggest that molecules anchored to the cell surface with the GPI moiety have a pivotal role in the survival of E. histolytica during pathogenesis.
PMCID: PMC2239303  PMID: 18270556
25.  Hepatic manifestations during amoebic dysentery 
Postgraduate Medical Journal  1973;49(570):261-264.
A variety of hepatic manifestations may occur during attacks of acute amoebic dysentery, conforming to clearly defined clinical groups.
The incidence of definite hepatic involvement presenting as cases of hepatic amoebiasis with or without demonstrable pus or with tender hepatomegaly was 57·5%.
While it is possible that the cases presenting with hepatic manifestations without demonstrable pus are due to small deep-seated abscesses, the non-specific nature of the histological lesions observed in the study and the inability to demonstrate the presence of amoebae in the lesions support the contention that these hepatic manifestations may represent a non-specific reaction in the liver to amoebic ulceration of the colon.
Chest radiology may be helpful in confirming the clinical evidence of hepatic involvement but the leucocyte count, ESR, and the serum enzymes are of limited value in indicating both its presence and type.
Patients manifesting hepatic involvement during attacks of amoebic dysentery should be adequately treated with tissue amoebicidals. This therapeutic approach would be useful in reducing the incidence of hepatic amoebiasis.
PMCID: PMC2495693  PMID: 4760721

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