Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster

Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.     

Metabolomic profiling of amoebic and pyogenic liver abscesses: an in vitro NMR study

Pus samples obtained from 109 patients with liver abscess were examined by NMR spectroscopy. To our knowledge this is the first report on metabolic profiling of liver abscesses. Fifty metabolites were identified by combination of one (1D) and two-dimensional (2D) NMR spectra. Metabolic derangements were evaluated for differentiation between amoebic (ALA) and pyogenic liver abscess (PLA). The NMR results indicate that aspartate, asparagine and galactose, integral components of lipoproteophophoglycans (LPG) of the cell wall of Entamoeba histolytica are metabolic biomarkers of ALA. On the other hand, acetate, propionate, butyrate, succinate and formate, the fermentation products the facultative anaerobes are significantly prevalent in PLA. The NMR based metabolic profile of ALA and PLA are evaluated taking polymerase chain reaction (PCR) and bacterial culture as gold standard method. However, when NMR results were compared with culture and PCR methods, a correct diagnosis of 94.11% in ALA (n?=?85) and 100% in PLA (n?=?10) cases were observed. NMR spectroscopy in conjunction with PCR and culture can expedite in differentiating ALA from PLA.     

Cytopathogenicity of Entamoeba histolytica to human intestinal epithelial cells: inhibition by monoclonal antibodies and sera from amoebic patients.

Interactions of pathogenic Entamoeba histolytica (HM 1) with human intestinal epithelial cells (Henle-407) were investigated. The E. histolytica trophozoites adhered and cytolysed 87% of cultured epithelial cell monolayers. A significant (P less than 0.001) inhibition of cytopathic effect of amoebic trophozoites pretreated with monoclonal antibodies to a 29 kDa surface associated protein suggested utilization of the 29 kDa surface protein in recognition and cytolysis of epithelial target cells. The polyclonal sera from treated patients of amoebic liver abscess and anti-amoebic hyperimmune serum inhibited cytopathogenicity to a greater degree (P less than 0.001) than did the monoclonal antibodies. The data thus suggest involvement of several amoebic molecules in exercising cytopathogenicity to epithelial cells.     

Detergent dissection of membrane proteins of Entamoeba histolytica and its effect on lymphokine release in in vitro.

Fifty-two amoebic liver abscess cases were assessed for the release of lymphokines (LMIF) using detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200) obtained with sodium deoxycholate treatment. Lymphokines release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was tested by leukocyte migration inhibition test on blood samples from amoebic liver abscess cases. A significant increase was noted in the release of LMIF and 100% positivity was observed with DDMP compared to whole amoebic extract with a positivity of 73%. The difference between means of the above two with regards to release of LMIF was found to be highly significant (P less than 0.005). This shows the patients had high degree of leukocyte sensitization to surface antigens of E. histolytica compared to the whole amoebic lysate. These findings suggest that the antigens shed might have important role as a potent antigen in elicitation of CMI response in amoebic liver abscess cases.     

Role of pure and biologically active amoebal RNA in assessment of lymphokines: a report of 55 ALA cases.

Amoebic liver abscess cases (55) were assessed for release of lymphokines (LMIF) using pure and biologically active amoebal RNA of axenic Entamoeba histolytica (NIH: 200) obtained with cesium chloride centrifugation. Lymphokines released by T lymphocytes in response to both amoebal RNA and whole amoebic lysate (WAL) were tested by leukocyte migration inhibition test (LMIT) on blood samples from amoebic liver abscess cases. A significant increase was observed in the release of lymphokine and 100% positivity was observed with amoebal RNA compared to whole amoebic extract with a positivity of only 78%. The difference between means leukocyte migration inhibition of the above two with regards to release of lymphokine was highly significant (P less than 0.001). This shows that patients had high degree of leukocyte sensitization to amoebal RNA of E. histolytica compared to whole amoebic lysate. These findings suggest that the amoebal RNA plays an important role as a potent antigen in the elicitation of cell mediated immune responses in amoebic liver abscess cases.     

Entamoeba histolytica: rapid detection of indian isolates by cysteine proteinase gene-specific polymerase chain reaction

Amoebiasis, caused by Entamoeba histolytica, is still one of the major problems for developing countries like India. Early detection of the parasite is a must for its prevention and control. In this study, PCR analysis of the cysteine proteinase gene from clinical isolates of symptomatic intestinal and amoebic liver abscess (ALA) cases has been compared with the stool microscopy, serology, and ultrasonography methods. The clinical isolates negative for E. histolytica by stool microscopy demonstrated the presence of the cysteine proteinase gene by PCR amplification. Also the gene copy number was increased in ALA samples compared with intestinal cases. Hence an accurate, early, and easier detection was possible by cysteine proteinase gene amplification directly from the clinical samples.     

Partial characterization of a 36-kDa antigen of Entamoeba histolytica and its recognition by sera from patients with amoebiasis

A 36-kDa antigen of axenically grown pathogenic Entamoeba histolytica (HM1-IMSS) was eluted from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved crude amoebic extract antigens. The immunoreactivity of this partially purified 36-kDa antigen with monoclonal antibody (MoAb) 3D(10) altered significantly (P<0.01) after heat and trypsin treatment but remained unaltered after treatment with sodium metaperiodate (P0.5), thereby indicating the protein nature of the epitope recognized by MoAb 3D(10). The epitope was found to be localized on the surface as well as in the cytoplasm of the E. histolytica trophozoites with the majority of it in the cytoplasm. In addition, this epitope was also found to be present on the cyst form of the parasite. The 36-kDa molecule was recognized by the sera from 29 (85%) of the 34 patients with amoebic liver abscess and five (83%) of the six patients with amoebic colitis. No serum samples from asymptomatic cyst passers, from patients with non-amoebic hepatic or intestinal disorders and apparently healthy subjects had antibodies that reacted with this 36-kDa molecule. The immune responses in man to this 36-kDa amoebic molecule indicate a potential specific role for this molecule in invasive amoebiasis.     

Detection of autoreactive antibodies to serum IgG and IgA in amoebic liver abscess cases.

Assessment of autoreactive antibodies in response to healthy human serum IgA and IgG was performed by indirect haemagglutination assay on serum samples from 81 amoebic liver abscess cases for IgA and 70 for IgG. Appropriate controls were taken simultaneously. IgA, IgG were isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography. These immunoglobulins were used for the detection of its own antibodies in amoebic liver abscess cases. This revealed that 43.20% and 48.50% of the cases were positive for IgA and IgG respectively, where as only 19.35% and 28.30% of the controls were in positive category (IgA and IgG respectively). The mean titres with standard deviation of the autoreactive antibodies to serum IgA both in ALA cases and controls shows a highly significant difference between tests and controls (P less than 0.001). Similarly the mean titres with standard deviation both in ALA and controls for the serum IgG differed significantly (P less than 0.001). This suggests the presence of autoreactive antibodies against serum IgA and IgG in amoebic liver abscess cases.     

Entamoeba histolytica: inflammatory process during amoebic liver abscess formation involves cyclooxygenase-2 expression in macrophages and trophozoites

It has been demonstrated that expression of cyclooxygenase-2 (COX-2) isoform is induced by Entamoeba histolytica in macrophages and polymorphonuclear cells during amoebic liver abscess (ALA) formation in hamsters. Trophozoites present in the lesion were also positive for COX-2 signal. However, no cross reactivity of the anti-COX-2 antibody with protein extract of cultivated trophozoites was found. To clarify if trophozoites are involved in PGE(2) production during ALA development, COX-2 expression was detected by in situ hybridization and RT-PCR in liver tissue from intrahepatically infected hamsters. COX-2 mRNA was in polymorphonuclear cells since 4h postinfection, and subsequently, local macrophages expressed COX-2 mRNA in a similar way. Additionally, a positive signal for COX-2 mRNA expression was detected in E. histolytica trophozoites, suggesting that, in vivo, parasite COX expression may be an important mechanism to promote inflammation.     

Effective human defense against E. histolytica: high amoebicidal activity of lymphocytes and monocytes in amoebic liver abscess patients until 3 months follow-up

Adherence of pathogenic Entamoeba histolytica trophozoites mediated by Gal/GalNAc lectin is a prerequisite for killing na?ve T cells and monocytes but the activated T cells and monocyte derived macrophages (MDMs) not only resist the attack but can kill the parasite. In the present study, we have analysed the adherence and cytotoxicity of the immunecompetent cells from patients of amoebic liver abscess at the time of their diagnosis and after 3 months to elucidate the development of cell mediated cytotoxicity, a major mechanism of resistance to amoebic infection. The results show that CD3+ cells from amoebic liver abscess cases, when stimulated, in vitro, bound E. histolytica trophozoites with increased intensity and their viability was also increased. The activated lymphocytes (taken at 3 months post treatment) were also able to kill amoebae. MDMs bound amoebae with greater intensity than lymphocytes, until 3 months post infection. These MDMs were effective in killing approximately 40% amoebae which was significantly less than at the time of diagnosis but was very significant as compared to the controls. The data suggest that cell mediated cytotoxic responses are maximum until 1 month post treatment and are significantly reduced thereafter.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. The immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica. The monoclonal antibody belonged to IgG1 isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant (P less than 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant (P less than 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant (P less than 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

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.     

Amebic liver abscess: report of three cases

Amebic abscess is a common manifestation of extraintestinal amebiasis and it is associated with relatively high morbidity and mortality. We present three cases seen in Bari, Southern Italy, one of which was autochthonous and the other two were not. Diagnosis was performed by elevated antibody titre for E. histolytica through immunofluorescence assay and positive antigen determination by ELISA in stools and in abscess aspirate. Fever often accompanied by chills, abdominal pain, weight loss and hepatomegaly were present. Laboratory findings also revealed leukocytosis with neutrophilia. Pleural effusion was observed in two patients. In all our patients multiple abscesses were observed. All the patients were treated with metronidazole and two of them also underwent the aspiration of the amoebic abscess. In all of them there was improvement of the clinical picture, as demonstrated by computerized tomography.     

Initiation of inflammation and cell death during liver abscess formation by Entamoeba histolytica depends on activity of the galactose/N-acetyl-D-galactosamine lectin

The parasite Entamoeba histolytica colonizes the human intestine causing amoebic colitis and disseminates through the vascular route to form liver abscesses. The Gal/GalNAc lectin is an adhesion protein complex which sustains tissue invasion by E. histolytica. Disruption of the Gal/GalNAc lectin function in engineered parasites (HGL-2 trophozoites) changed the pathophysiology of hamster liver abscess formation. HGL-2 trophozoites produced numerous small inflammatory foci located in the vicinity of blood vessels. The low penetration of HGL-2 trophozoites into hepatic tissue was shown to be associated with weak attraction of neutrophils and macrophages to the infiltrated areas and absence of pro-inflammatory tumour necrosis factor, in contrast to wild type or control vector infections. The low host inflammatory response in HGL-2 infections correlated with a delay in apoptosis of hepatic cells, whereas apoptosis of endothelial cells was not detected. Triggering of apoptosis in both host cell types most likely has a central role in modulating inflammation, a major landmark in hepatic amoebiasis. These data highlight the key role of the Gal/GalNAc lectin in initiation of E. histolytica hepatic infection.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Abstract Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. THe immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica . The monoclonal antibody belonged to IgG₁ isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant ( P < 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant ( P < 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant ( P < 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

An experimental model for amoebic abscess production in the cheek pouch of the Syrian golden hamster, Mesocricetus auratus

A new experimental model was developed in hamsters for amoebic abscess caused by Entamoeba histolytica. E. histolytica trophozoites were cultured in a liquid axenic medium, and then injected intradermally into the cheek pouch of the Syrian golden hamster, Mesocricetus auratus. Inoculation consistently resulted in abscess formation at the site in 20 of 22 (91%) study animals. The amoebic nature of the abscesses was confirmed by light microscopy and histopathologic examination. Abscess formation was maximal at day 12 post-inoculation. Potential applications of this simple and reliable model include further elucidation of the pathogenesis of invasive amoebiasis, studies of the host response to amoebae, and in vivo evaluation of chemotherapeutic agents that show in vitro efficacy against E. histolytica.     

Recent developments in amoebiasis:the Gal/GalNAc lectins of Entamoeba histolytica and Entamoeba dispar

Amoebiasis is responsible for 50000-100000 deaths annually. Invasive amoebic disease begins with the attachment of Entamoeba histolytica trophozoites to colonic mucin, a process mediated by the amoebic Gal/GalNAc lectin. The non-pathogenic counterpart, E. dispar, is morphologically identical but genetically distinct. Investigations comparing the Gal/GalNac lectin from these two organisms are under way.     

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.     

Caspase 3-dependent killing of host cells by the parasite Entamoeba histolytica

The parasite Entamoeba histolytica is named for its ability to lyse host tissues. To determine the factors responsible, we have initiated an examination of the contribution of parasite virulence factors and host caspases to cellular destruction by the parasite. Amoebic colitis in C3H/HeJ mice was associated with extensive host apoptosis at sites of E. histolytica invasion. In vitro studies of E. histolytica –Jurkat T-cell interactions demonstrated that apoptosis required contact via the amoebic Gal/GalNAc lectin, but was unaffected by 75% inhibition of the amoebic cysteine proteinases. Parasite-induced DNA fragmentation was unaffected in caspase 8-deficient Jurkat cells treated with the caspase 9 inhibitor Ac-LEHD-fmk. In contrast, caspase 3-like activity was observed within minutes of E. histolytica contact and the caspase 3 inhibitor Ac-DEVD-CHO blocked Jurkat T cell death, as measured by both DNA fragmentation and ⁵¹Cr release. These data demonstrate rapid parasite-induced activation of caspase 3-like caspases, independent of the upstream caspases 8 and 9, which is required for host cell death.