Entamoeba histolytica: Diffuse Liver Inflammation in Gerbils (Meriones unguiculatus) with Experimentally Induced Amebic Liver Abscess

The histopathological changes that occur in the uninfected liver lobes of gerbils with a localized amebic liver abscess (left lobe) were studied from 5 to 60 days after inoculation. Early parenchymal changes (10 days) in the right and median liver lobes showed a lymphocytic infiltrate and bile duct proliferation in the portal areas. Maximum alternations occurred at 20 to 30 days after inoculation and were characterized by a dense portal infiltrate, hepatocyte degeneration, granulocytic infiltrate and marked glycogen depletion. In drug-treated infections with metronidazole, the diffuse liver inflammation was terminated. The results of this study suggest that the diffuse liver injury was directly or indirectly induced by the presence of a proliferating amebic liver abscess and that the pathology caused by amebic liver abscess is more extensive than previously thought.     

Entamoeba histolytica: diffuse liver inflammation in gerbils (Meriones unguiculatus) with experimentally induced amebic liver abscess

The histopathological changes that occur in the uninfected liver lobes of gerbils with a localized amebic liver abscess (left lobe) were studied from 5 to 60 days after inoculation. Early parenchymal changes (10 days) in the right and median liver lobes showed a lymphocytic infiltrate and bile duct proliferation in the portal areas. Maximum alterations occurred at 20 to 30 days after inoculation and were characterized by a dense portal infiltrate, hepatocyte degeneration, granulocytic infiltrate and marked glycogen depletion. In drug-treated infections with metronidazole, the diffuse liver inflammation was terminated. The results of this study suggest that the diffuse liver injury was directly or indirectly induced by the presence of a proliferating amebic liver abscess and that the pathology caused by amebic liver abscess is more extensive than previously thought.     

Entamoeba histolytica: antibody responses and lymphoreticular changes in gerbils (Meriones unguiculatus) in response to experimental liver abscess and amebic extract infection

Antibody responses and histological changes in hepatic lymph nodes and spleen of gerbils (Meriones unguiculatus) during the course of experimental hepatic amebiasis (5-60 days), or in those injected with extracts of Entamoeba histolytica, are described. Lymph node and spleen responses in infected animals paralleled the proliferation of the amebic liver abscess. However, spleen follicle responses were similar in animals that received low or high doses of the amebic extract and differed histologically from those with amebic liver abscess. Liver abscesses, up to 30 days postinfection (pi), doubled in weight between 10 and 15 and between 20 and 30 days pi. Early changes (10 days pi) in the lymphoreticular tissues were characterized by increased size and weight of the organs, hyperplastic follicles, and blastogenesis in the T-dependent areas. At 20 and 30 days pi, the size of spleen follicles increased and there was depletion of lymphocytes from the periarterial area (PAA), as well as gross extension of the red pulp, accompanied by extramedullary erythropoiesis and megakaryocytosis. The paracortical areas (PCA) of lymph nodes were depleted of lymphocytes and histiocytosis throughout the organ, and there was intense plasma cell activity in the medulla. At 60 days pi, lymphocyte repopulation was noted in the PCA and PAA; germinal centers were depleted of blast cells and the spleen red pulp had contracted. Antiamebic antibody titers were low throughout the infection. Changes in the cellularity of the lymphoid organs are discussed in relation to the proliferation of the amebic liver abscesses in infected animals and in those which were injected with the amebic extract.     

Entamoeba histolytica: identification of a distinct beta2 integrin-like molecule with a potential role in cellular adherence

Entamoeba histolytica infection causes dysentery, intestinal colitis, and hepatic abscess in an estimated 50 million people worldwide. Attachment of E. histolytica trophozoites to intestinal epithelium and vascular endothelium during liver metastasis results in an inflammatory process. We report the identification of a distinct amebic beta2 integrin (CD18)-like molecule which affords adherence to TNF-alpha-activated endothelial cells. Data from flow cytometry and indirect immunofluorescence assays suggest the amebic beta2 integrin was localized to focal adhesion plates and was present in both E. histolytica and Entamoeba dispar. The amebic beta2 integrin appeared to be distinct from the amebic Gal/GalNAc lectin based on recombinant expression, amebic colocalization, and ELISA studies. Trophozoite adherence to endothelial cells expressing ICAM-1 (CD54) following activation with TNF-alpha or ICAM-1-transfected CHO cells was specifically inhibited with anti-CD18 or anti-CD54 MAbs. In summary, evidence in support of a distinct beta2 integrin-like molecule participating in amebic adherence to TNF-alpha-activated endothelial cells expressing ICAM-1 is presented. The presence of integrin-dependent binding may allow trophozoites to opportunistically adhere to activated intestinal epithelium or vascular endothelium expressing ICAM-1 during amebic colitis or hepatic abscess.     

Entamoeba histolytica: collagenolytic activity and virulence

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Cytopathologic and genetic diagnosis of pulmonary amebiasis: a case report

BACKGROUND: Amebiasis is a parasitic infection with Entamoeba histolytica. Pulmonary amebiasis is rare since the infection is commonly manifested as amebic colitis or liver abscess. Most pleuropulmonary amebiasis is seen in patients with amebic liver abscesses. A pulmonary amebic lesion without either a liver abscess or amebic colitis is extremely rare. Thus, reported cases of sputum cytologic diagnosis of a pulmonary amebic lesion from a patient without a liver abscess are also very rare. CASE: A 53-year-old man presented with a dry cough and mild fever. Chest radiography revealed an abnormal solitary mass lesion in the right upper lung field. The clinical diagnosis was a bacterial lung abscess. Sputum cytologic examination demonstrated many trophozoites of E. histolytica. Following sputum cytodiagnosis, serologic tests revealed a slightly high but almost normal titer of IgG antibodies to E. histolytica, indicating the possible presence of the pathogen. Polymerase chain reaction (PCR) using E. histolytica-specific primers for DNA extracted from the sputum sample revealed specific DNA product. CONCLUSION: Pulmonary amebiasis without either a liver abscess or amebic colitis must be distinguished from bacterial abscesses and neoplastic disease. A sputum cytologic examination combined with PCR for DNA extracted from a sputum sample is a good approach to the diagnosis of a pulmonary amebic abscess.     

Entamoeba histolytica: Collagenolytic Activity and Virulence1

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS, and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.     

Monoclonal antibodies directed against the galactose-binding lectin of Entamoeba histolytica enhance adherence.

The Entamoeba histolytica galactose-binding lectin is a surface glycoprotein composed of 170- and 35-kDa subunits. Inhibition of this lectin with galactose or anti-170 kDa subunit polyclonal antibody blocks amebic adherence to target cells and colonic mucin glycoproteins. We describe the properties of 10 mAb with specificity for the 170-kDa subunit. Based on competitive binding studies, six nonoverlapping antigenic determinants on the lectin were identified. The effect of the mAb on adherence of amebic trophozoites to both Chinese hamster ovary (CHO) cells and human colonic mucins was measured. Antilectin antibodies directed against epitopes 1 and 2 enhanced adherence, with the number of amebae having at least three adherent CHO cells increasing with the addition of epitope 1 mAb from 26 +/- 9 to 88 +/- 2% and the binding of colonic mucins increasing from 34 +/- 1 to 164 +/- 3 pg/10(5) amebae. Antibody-enhanced adherence remained 90 to 100% galactose inhibitable, occurred at 4 degrees C and was not Fc mediated. Univalent Fab fragments of epitope 1 mAb augmented mucin binding by 238% and CHO cell adherence by 338%. The binding of purified lectin to CHO cells was increased from 1.1 +/- 0.1 to 2.4 +/- 0.3 ng/10(3) CHO cells by mAb directed to epitope 1, demonstrating that enhanced adherence was due to direct activation of the lectin. mAb to epitope 3 bound to the lectin only upon its solubilization from the membrane and had no effect on adherence. Adherence to CHO cells and mucins was inhibited from 50 to 75% by mAb to epitopes 4 and 5; epitope 6 mAb inhibited amebic adherence to CHO cells but not mucins. The pooled sera from 10 patients with amebic liver abscess blocked the binding to the 170-kDa subunit of mAb directed to all six epitopes. Striking individual variations in the effects of immune sera on adherence were observed. Although the sera of all 44 South African patients with amebic liver abscess had high titer anti-lectin antibodies, 16 patients' sera significantly (more than 3 SEM) enhanced adherence whereas 25 patients' sera significantly inhibited adherence. Antilectin antibodies exert profound functional effects on the interaction of E. histolytica with target cells and human colonic mucins. Exploration of the clinical consequences of adherence-enhancing and inhibitory antibody responses may give insight into the role of antilectin antibodies in immunity to invasive amebiasis.     

Entamoeba histolytica: lymphoreticular changes in gerbils (Meriones unguiculatus) with experimentally induced cecal amebiasis

Antibody responses and changes in the lymphoreticular tissues of gerbils with experimental cecal amebiasis were studied from 5 to 60 days PI. Changes in the cecum consisted of lymphoid follicle hyperplasia and depletion of lymphocytes, followed by follicle atrophy and histiocytosis. Mesenteric lymphadenopathy, and histologic alterations in the lymph nodes paralleled the progressive development of amebic cecal lesions. Early in the infection (5 to 10 days PI) mesenteric lymph nodes showed cortical follicle hyperplasia, blastogenesis in the paracortical areas (PCA) and intense lymphoblast and plasma cell activity in the medullary cords. At 20 to 30 days PI, the cortical follicles, the PCA and the medulla were depleted of lymphocytes and there was histiocytosis throughout the organ. At 60 days PI, lymphocyte repopulation took place in the PCA, and cortical follicles had active germinal centers. Spleen follicles did not increase in number as the infection progressed, but became hyperplastic. Antibody titers to ameba were low throughout the cecal infection but rose whenever amebic metastasis to the liver occurred. The results of this study indicate that lymphocytes from the submucosal lymphoid follicles and the draining lymph nodes may control the pathogenesis of the infection. Lymphoreticular tissue alterations could result from antigenic stimulation and migration of cells to the sites of infection.     

Pleuropulmonary manifestations of hepatic amebiasis.

Pleuropulmonary manifestations of hepatic amebiasis occurred in 30 patients; 18 (60%) presented with at least 1 pulmonary complaint and 10 (33%) had multiple pulmonary symptoms. In 14 patients (47%), abnormalities were found on examination of the chest. In 16 chest roentgenograms (53%), there was at least 1 abnormality: right-sided pleural effusion (9 patients) and elevated right hemidiaphragm (8 patients) were the most common. All patients were treated with metronidazole (Flagyl) and had resolution of the amebic liver abscess and pulmonary disease. Pleuropulmonary disease is a common complication of amebic liver abscess. The clinical presentation and chest roentgenograms are virtually diagnostic and obviate the need for invasive procedures to confirm the diagnosis. Pleuropulmonary disease resolves with amebicidal treatment of the hepatic abscess.     

Patients treated for amebic liver abscess develop cell-mediated immune responses effective in vitro against Entamoeba histolytica

We studied the afferent and efferent cell-mediated immune response in 15 patients treated for amebic liver abscess. Patients had a lower T4 to T8 ratio (1.25 +/- 0.65) compared with age- and sex-matched controls (1.89 +/- 0.44, p less than 0.01) due to a decrease in T4-"helper" cells and an increase in T8-"suppressor" cells (p less than 0.01). The in vitro proliferative response of patient T lymphocytes to the plant mitogen concanavalin A (Con A) was depressed; responses to phytohemagglutinin were not. The proliferative response of patient lymphocytes to an amebic soluble protein preparation (SPP) was greater than the mitogenic response seen in control lymphocytes (mean of 68,300 delta cpm and 22,300 delta cpm, respectively, p less than 0.001), correlated with the T4 to T8 ratio (p less than 0.05) and the duration of time from initiation of antiamebic therapy (p less than 0.01). Supernatants from patient lymphocytes exposed to the amebic SPP activated normal monocyte-derived macrophages to kill virulent axenic E. histolytica trophozoites (p less than 0.001); patient monocyte-derived macrophages activated by Con A-elicited lymphokine could also kill amebae. Finally, when incubated with the amebic SPP for 5 days, T lymphocytes from patients were able to kill virulent amebae (p less than 0.005); patient T lymphocytes not exposed to the amebic SPP or control T lymphocytes incubated for 5 days with the amebic SPP were not cytotoxic to E. histolytica trophozoites. In summary, after cure of amebic liver abscess, specific cell-mediated immune mechanisms develop that are effective in vitro against the parasite.     

Natural Killer T Cells Activated by a Lipopeptidophosphoglycan from Entamoeba histolytica Are Critically Important To Control Amebic Liver Abscess

The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jα18^-/- mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using α-galactosylceramide (α-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d^−/− mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-γ but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28∶0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-γ production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to α-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess.     

Entamoeba histolytica: impedance measurements and cytotoxicity in the presence of bepridil, verapamil, and cytochalasin D

Entamoeba histolytica, and invasive enteric protozoa, kills mammalian target cells by sequential adherence and cytolytic events. Using platinum plate electrodes with an alternating current source placed in a Wheatstone bridge circuit, the impedance (resistance to ion flow) of a cell suspension of axenic amebae (strain HM1-IMSS) was measured. The impedance of the amebic cell suspension, expressed as resistivity (in ohm-cm), was significantly greater than the test solution and increased with decreasing temperature or greater cell packing (P less than 0.01), indicating that the resistivity measurements reflected the impedance of the amebic surface membrane. Cytochalasin D (10 micrograms/ml), a microfilament inhibitor which inhibited amebic in vitro adherence and cytolysis of target Chinese hamster ovary (CHO) cells (P less than 0.001), also increased resistivity of the amebic suspension (P less than 0.01). Exposure of amebae to bepridil (10(5) M), a slow-channel blocker, inhibited amebic killing of target cells (P less than 0.01) and also increased the resistivity of the amebic suspension (P less than 0.01), but both to a lesser degree than cytochalasin D (P less than 0.001). In contrast, exposure of amebae to verapamil followed by washing had no effect on amebic killing of target cells or resistivity of the amebic suspension. The increased resistivity measured in cytochalasin D or following exposure to bepridil was not due to a change in cell density of the amebic suspension. These studies indicate that changes in impedance of the amebic surface membrane are produced by bepridil and cytochalasin D. The effect of these agents on membrane impedance may contribute directly to the concurrent observed alteration in amebic cytopathogenic capacity or may serve as a parallel marker for the cell membrane alterations induced by such pharmacologic agents which inhibit amebic microfilament function or calcium flux.     

Detection of pathogenic Entamoeba histolytica DNA in liver abscess fluid by polymerase chain reaction.

, , and 1992. Detection of pathogenic Entamoeba histolytica DNA in liver abscess fluid by polymerase chain reaction. International Journal for Parasitology 22: 1193–1196. A sensitive method for detection of pathogenic Entamoeba histolytica DNA in drained fluids from liver abscess patients, using the polymerase chain reaction (PCR), has been developed. The PCR employs oligonucleotide primers specific for the gene encoding the 30 kDa molecule of pathogenic E. histolytica. Liver abscess fluids (19 samples), from 14 patients with a presumptive amebic liver abscess, were examined microscopically and by the PCR method. Only two of the 19 samples were positive microscopically, whereas all 19 samples tested positive by PCR. This technique can be used to confirm the diagnosis of amebic liver abscess.     

Tissue invasion by Entamoeba histolytica: evidence of genetic selection and/or DNA reorganization events in organ tropism

Entamoeba histolytica infection may have various clinical manifestations. Nine out of ten E. histolytica infections remain asymptomatic, while the remainder become invasive and cause disease. The most common form of invasive infection is amebic diarrhea and colitis, whereas the most common extra-intestinal disease is amebic liver abscess. The underlying reasons for the different outcomes are unclear, but a recent study has shown that the parasite genotype is a contributor. To investigate this link further we have examined the genotypes of E. histolytica in stool- and liver abscess-derived samples from the same patients. Analysis of all 18 paired samples (16 from Bangladesh, one from the United States of America, and one from Italy) revealed that the intestinal and liver abscess amebae are genetically distinct. The results suggest either that E. histolytica subpopulations in the same infection show varying organ tropism, or that a DNA reorganization event takes place prior to or during metastasis from intestine to liver.     

Efficacy and safety of paromomycin for treating amebiasis in Japan

The clinical management of amebiasis is a growing concern, particularly among human immunodeficiency virus (HIV)-infected individuals who are predisposed to severe illness. Treatment with a luminal amebicide is strongly recommended following acute-stage treatment with a nitroimidazole. In 2004, the Japanese Research Group on Chemotherapy of Tropical Diseases introduced paromomycin, which was not nationally licensed, and offered it to a number of patients. From 2004 to 2011, 143 case records of amebiasis (123 with amebic colitis, 16 with amebic liver abscess, and 4 with both) in which patients were treated with paromomycin, mainly 1500 mg/day for 9 or 10 days following metronidazole treatment, were submitted. Among 123 evaluable cases, 23 (18.7%) experienced possible adverse effects, the most common being diarrhea (17/123, 13.8%) and other gastrointestinal problems that were resolved after the completion or discontinuation of treatment. In addition, single cases of bloody stools associated with Clostridium difficile colitis, skin rash, and the elevation of liver enzymes were also reported, although the causal relationship was not clear. HIV infection did not appear to increase the incidence of adverse drug effects. Each of the 11 asymptomatic or mildly symptomatic amebic colitis cases became negative for stool cysts after paromomycin treatment. Paromomycin was shown to be safe and well tolerated, as well as effective in a special subset of amebic colitis cases.     

Experimental infection of rhesus monkeys with Entamoeba histolytica mimics human infection

Experimental infection of Entamoeba histolytica was successfully established in rhesus monkeys (Macaca mulatta). Parasites of proven pathogenicity maintained through liver passage in hamsters were used in this study. Laparotomized monkeys were inoculated intracecally/intrahepatically with trophozoites, or by oral administration of cysts (2000 cysts/ml). The cysts were isolated from an infected human stool sample. Formation of typical amebic lesions in the inoculated tissues along with alterations in hematologic indices were studied in the infected monkeys. All the experimentally inoculated monkeys showed leukocytosis and mild neutrophilia, while the hemoglobin content and levels of blood sugar and blood urea remained unaltered during the post-infection period. Specific antiamebic antibodies were readily detectable in post-infected sera.     

TNFα-Mediated Liver Destruction by Kupffer Cells and Ly6Chi Monocytes during Entamoeba histolytica Infection

Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis.