Introduction: An amebic liver abscess is the most common presentation of extraintestinal amebiasis. This condition is the result of a parasite infection caused by Entamoeba histolytica. Materials and Methods: We report a case of a 53-year-old male who presented with abdominal pain in the right upper quadrant, jaundice, and a 10-kg weight loss within a 1-month span. Results and Conclusion: A wide range of symptoms and findings in the imaging tests suggestive of neoplasia, elevated levels of CA 19-9 and CA 125, and the presentation of biliary peritonitis as a complication makes this case a challenge for its approach and management. / Revisión por pares

Universiẗat, Diss., 2005--Würzburg.

Saposin-ähnliche Proteine (SAPLIPs) sind membraninteragierende Proteine, die sich durch die konservierte Position von drei Disulfidbrücken, einer typischen alpha-helikalen Proteinfaltung und der Fähigkeit mit Lipiden zu interagieren, auszeichnen. Ihre zellulären Funktionen sind äußerst vielfältig. Bis zum Beginn des Genomsequenzierungsprojektes waren die Amoebapores die einzigen bekannten und charakterisierten SAPLIPs von Entamoeba histolytica, dem Erreger der humanen Amöbenruhr. Aufgrund ihrer antimikrobiellen Aktivität stellen sie für diesen parasitischen Einzeller, der sich von phagozytierten Bakterien ernährt, wichtige Effektormoleküle dar. Sie können aber auch cytolytisch auf Wirtszellen wirken und werden deshalb als bedeutender Pathogenitätsfaktor angesehen. Die theoretische computergestützte Datenbankanalyse nach Abschluss der Genomsequenzierung ergab, dass es 16 weitere Gene kodierend für SAPLIPs zusätzlich zu den drei Amoebapore-Genen gibt. Die Sequenzen der neuen SAPLIPs sind abgesehen von dem Cysteinmotiv divers und auch die Größe der Proteine ist sehr unterschiedlich (77 - 1009 Aminosäuren). Alle besitzen sie jedoch eine einzige, C-terminal gelegene SAPLIP Domäne. Außer der SAPLIP-Domäne konnten keine weiteren bekannten funktionellen oder strukturellen Domänen in den relevanten Datenbanken identifiziert werden, die auf mögliche Funktionen hätten hinweisen können. Alle SAPLIP-Gene werden gleichzeitig in axenisch kultivierten Trophozoiten transkribiert wie durch reverse Transkriptions-PCR gezeigt wurde. Die vergleichende transkriptionelle Analyse im Mikroarray ergab, dass nach Kontakt mit menschlichen Kolonzellen keine Hochregulierung dieser Gene mit Ausnahme des Amoebapore A Gens stattfindet. Für die parallele Klonierung der verschiedenen SAPLIP-Domänen wurde ein ”Expressionsscreening” in E.coli mit dem grün fluoreszierenden Protein als Reporterprotein etabliert, das die erfolgreiche Klonierung und Expression eines Fragments aufgrund der Fluoreszenz der Bakterienkolonie bereits auf der Ebene der Transformation anzeigt. Die rekombinant exprimierte und bis zur Homogenität gereinigte SAPLIP-Domäne von SAPLIP 12 wies Amoebapore-ähnliche Aktivitäten auf. Unter Verwendung von Liposomen konnte porenbildende Aktivität nachgewiesen werden, wobei diese Aktivität stark an einen sauren pH-Wert gebunden ist. Die SAPLIP-Domäne 12 ist aber auch antibakteriell und dieses sogar mit vergleichbarer Selektivität wie Amoebapore A, nämlich Zelllyse von gram-positiven B. megaterium war nachweisbar, jedoch nicht von gram-negativen E. coli. Strukturell unterscheiden sich die SAPLIP-Domäne 12 und Amoebapore A bezüglich der Exposition positiver Ladungsansammlungen auf der Proteinoberfläche und des Fehlens des für den Mechanismus der Amoebapores essentiellen Histidinrestes an entsprechender Position in der Sequenz. Darüber hinaus übt die SAPLIP-Domäne 12 eine im Vergleich zum Amoebapore A geringere spezifische Aktivität aus. Diese Eigenschaften weisen darauf hin, dass es sich um einen anderen Wirkungsmechanismus handeln könnte. Für die SAPLIP-Domäne 12 wäre eine über die positiven Ladungen der Proteinoberfläche vermittelte Interaktion mit den negativ geladenen Phospholipidköpfen von Membranen denkbar, die bei Erreichen einer bestimmten Konzentration in einer Störung der Lipidordnung und letztendlich in der Auflösung der Membranstruktur resultieren könnte. SAPLIP 3 ähnelt den Amoebapores in der Größe und molekularen Architektur und kann somit als funktionelle Einheit angesehen werden, es unterscheidet sich aber durch eine hohe negative Nettoladung von den Amoebapores. Außerdem ist das rekombinante SAPLIP 3 nicht antibakteriell und die Membraninteraktionen dieses SAPLIPs unterscheiden sich grundlegend von denjenigen, die für die Amoebapores beschrieben sind. SAPLIP 3 zerstört nicht einfach die Liposomenstruktur wie von den porenbildenden Amoebapores bekannt, sondern es vermittelt die Fusion von multilamellaren Liposomen unter Freisetzung des Liposomeninhalts. Diese Aktivität ist abhängig von der Anwesenheit anionischer Lipide und von einem sauren pH-Wert. Die Fähigkeit zur Vesikelfusion sowie die Verteilung der negativen Ladungen von SAPLIP 3 auf der Proteinoberfläche ähneln Merkmalen des humanen Saposin C. Neben der Funktion als Cofaktor von Exohydrolasen, die im Sphingolipid Katabolismus involviert sind, wird angenommen, dass die Fähigkeit von Saposin C, Vesikel zu fusionieren, wichtig für die Reorganisation der humanen lysosomalen Kompartimente ist. Die Saposin C-ähnlichen Charakteristika von SAPLIP 3 geben Grund zu der Annahme, dass es bereits in einem so basalen Organismus wie der Amöbe ein Protein mit Saposin-ähnlichen membranfusionierenden Aktivitäten gibt und dass dieses SAPLIP entsprechende Funktionen während endo- und exozytotischer Transportprozesse in der Amöbe übernehmen könnte. / Saposin-like proteins (SAPLIPs) are membrane-interacting proteins that are characterized by the conserved position of three disulphide bonds, a typical alpha-helical fold and the ability to interact with lipids. Their cellular functions are extremely diverse. Until the beginning of the genome-sequencing project, the amoebapores were the only known and characterized SAPLIPs of Entamoeba histolytica, which is the causative agent of human amoebiasis. Due to their antimicrobial activity, these proteins are important effector molecules of this unicellular parasite, which feeds on phagocytozed bacteria. However, they also exert cytolytic activity against host cells and therefore, they are considered to be a major pathogenicity factor of the amoeba. The theoretical computer-based data base analysis after the completion of genome sequencing revealed 16 genes coding for SAPLIPs in addition to the three amoebapore genes. The sequences of the novel SAPLIPs are diverse apart from the cysteine motif and furthermore, the sizes of the proteins are highly different (77 – 1009 amino acid residues). They all contain a single, C-terminally located SAPLIP domain. Beside the SAPLIP domain, no other functional or structural domains were identified in the relevant databases that would have pointed to possible functions. All SAPLIP-genes are transcribed in axenically cultured trophozoites at the same time as shown by reverse transcription PCR. The comparative transcriptional analysis using microarrays revealed that after six hours of contact with human cells and their phagocytosis none of these genes, with the exception of the amoebapore A gene, was upregulated. In order to clone the different fragments in parallel, an expression screening in E. coli with the green fluorescent protein as reporter protein was established, which indicates the successful cloning and expression of a fragment by the fluorescence of the bacterial colony already at the level of transformation. The SAPLIP domain 12, which has been recombinantly expressed and purified to homogeneity, exerts amoebapore-like activities. Using liposomes, pore-forming activity was detected, which is strongly dependent on acidic pH. Furthermore, the SAPLIP domain 12 is antibacterial even with similar selectivity as amoebapore A in that cell lysis of gram-positive B. megaterium was detectable but not of gram-negative E. coli. Structurally, the SAPLIP domain 12 differs from the amoebapores by exposing patches of positive charges on the protein surface und by lacking a histidine residue at a corresponding position in its sequence, which has been shown to be essential for the mechanism of amoebapore A. Moreover, the SAPLIP domain 12 displays a lower specific activity. These features indicate that this SAPLIP domain may act by means of another mechanism. It may interact with the negatively charged phospholipid head groups of membranes by its positively charged protein surface and after reaching a certain threshold concentration, possibly resulting in a disturbance of the lipid order and finally in the disintegration of the membrane structure. SAPLIP 3 resembles the amoebapores in size and molecular architecture and could therefore be considered as a functional unit, but it differs from the amoebapores by a highly negative net charge. Besides, the recombinant protein is not antibacterial and the membrane interactions of this SAPLIP are completely different from those described for the amoebapores. SAPLIP 3 does not simply destroy the liposome structure as known from the pore-forming amoebapores but induces leaky fusion of multilamellar liposomes. This activity is dependent on the presence of negatively charged lipids and on acidic pH. The capability of vesicle fusion as well as the negative charge distribution of SAPLIP 3 resembles features of the human saposin C. Beside its function as a cofactor of exohydrolases, which are involved in the sphingolipid catabolism, saposin C is considered to be involved in the reorganization of human lysosomal compartments due to its fusogenic activity. The saposin C-like characteristics of SAPLIP 3 suggest that a protein with saposin-like membrane fusogenic activity already exists in such a basal organism like an amoeba and that this SAPLIP fulfils corresponding functions during endo- and exocytotic transport processes in the amoeba.

Invasive amebiasis, caused by the protozoan parasite Entamoeba histolytica, is one of the leading parasitic causes of mortality worldwide, and there are no vaccines available to control the disease. The heavy subunit of the E. histolytica Gal-lectin is regarded as a potential subunit vaccine candidate. A Th1 (cell-mediated) immune response is protective against invasive amebiasis, and DNA vaccination is a strategy to induce such a response against specific antigens. The objective of this study was to construct and test a Gal-lectin-based DNA vaccine against E. histolytica. DNA encoding as 894--1081 of the Gal-lectin heavy subunit was resynthesized using a gerbil codon frequency bias and inserted in a mammalian expression vector to generate the DNA vaccine pCISToGL6. Balb/c mice vaccinated intradermally developed a Gal-lectin-specific cellular immune response, as well as an anti-Gal-lectin humoral immune response. Serum antibodies recognized a recombinant portion of the Gal-lectin heavy subunit by immunoblot and ELISA, and bound to native Gal-lectin on the surface of live trophozoites, inhibiting adherence to target cells. The Gal-lectin-specific serum antibodies were of the IgG2a isotype, indicating that a Th1 response was stimulated by the vaccine. We were also interested in using DNA encoding IL-12, IL-18 or GM-CSF as genetic adjuvants co-injected with pCISToGL6 to potentiate the immune response. Since the DNA vaccine was destined to confer protection in the gerbil model of invasive amebiasis, we cloned gerbil IL-12 (p35 and p40), IL-18 and its convertase caspase-1, and GM-CSF. The proteins were expressed in mammalian cells and showed bioactivity in vitro. Taken together, these results have laid the foundation to optimize and test a working Gal-lectin with co-stimulatory molecules to elicit a Th1 immune response for protective immunity against invasive amebiasis.

Isoenzyme characterisation of Entamoeba histolytica into pathogenic and non-pathogenic zymodemes substantiated previously held views that this parasite con5titutes two distinct strains or even sub-species that are morphologically identical but vary in their pathogenicity. A reappraisal of the epidemiology of amoebiasis and investigation of the patho-physiological relationships between these pathogenic and non-pathogenic zymodemes and their host was therefore indicated. Only pathogenic zymodemes were isolated from hospitalised patients with amoebic liver abscess (ALA) and amoebic dysentery (AD). In the amoebiasis endemic peri-urban population of Durban, I. histolytica occurred at an overall prevalence of 10%. Carriers of non-pathogenic zymodemes constituted 9% of the population. A key observation was that asymptomatic infections with pathogenic zymodemes occurred at a prevalence of 1%. Higher prevalence of E. histolytica occurred in association with poor sanitary conditions. Furthermore., both pathogenic and non-pathogenic zymodemes tended to cluster into family units suggesting person-to-person transmission of the parasite by the faecal-oral route. Although invasive amoebiasis occurs far more frequently in males than females (8:1) both pathogenic and non-pathogenic zymodemes are equally distributed in male and female E. histolytica cyst passers. Ninety percent of carriers of pathogenic zymodemes spontaneously cleared their infections and remained asymptomatic throughout the study period of 2 years while 10% developed AD which required treatment with metronidazole. No spontaneous changes in zymodemes from the non-pathogenicto the pathogenic type was observed in a longitudinal study. The serological response of asymptomatic carriers of pathogenic zymodemes (100% seropositive) was identical to that of patients with ALA or AD with a high proportion (94-100%) of them being strongly seropositive. The prevalence of seropositivity amongst subjects who were not infected by E. histolytica (13% seropositive) was not statistically different (p>0,5) from that of the random population of this endemic area (19% seropositive) and carriers of non-pathogenic zymodemes (21% positive); the prevalence of strongly seropositive reactions among this group was only between 2-4%. It is concluded that a positive serological response is directly due to past or present contact with pathogenic zymodemes. This is further substantiated by the observation that the proportion of seropositive subjects was found to increase dramatically in a population near Cape Town where an outbreak of invasive amoebiasis (ALA and AD) occurred indicating a high prevalence of pathogenic zymodemes in this community. Another community in northern Transvaal (Gazankulu) where ALA and AD does not occur was, as expected, uniformly seronegative. Axenic growth of pathogenic zymodemes was possible but could not be accomplished with the non-pathogenic zymodemes. Even though monaxenic growth together with Trypanosoma cruzi was possible with both strains, the pathogenic zymodemes tended to grow more prolificly. No zymodeme changes from non-pathogenic to pathogenic and vice versa were observed with such changes in culture conditions. Cyst production by the pathogenic zymodemes in vivo was confirmed experimentally, thereby demonstrating the ability of pathogenic E. histolytica to independently complete their life-cycle thus giving it the ability to propagate itself successfully as a species. / Thesis (M.D.)-University of Natal, Durban, 1989.

Entamoeba histolytica, etiological agent of amebiasis, continues to be a significant threat to human health worldwide. The disease affects 10% of the world's population and leads to an estimated 100, 000 deaths a year. The parasite's surface Gal-lectin is an immunodominant protein that also mediates colonization and pathogenicity. The Gal-lectin is the most promising vaccine candidate against amebiasis. However, the immune mechanisms involved in protection against disease remain unclear. The objective of this study was to characterize the immunological basis of the host defense mechanisms using a Gal-lectin based vaccine. Exposure of the Gal-lectin with immature dendritic cells increased cell maturation and activation and upregulated co-stimulatory molecules and pro-inflammatory cytokines production. Dendritic cell activation was dependent on NF-kappaB and MAPK activation. In vaccination studies, the adjuvant effect of CpG-ODN, a synthetic oligodeoxynucleotide capable of stimulating Th1 immune responses enhanced the immune response to the Gal-lectin when administered systemically or mucosally. Protected animals had elevated anti-Gal-lectin serum and stool IgA antibodies capable of blocking parasite adherence in vitro. Analysis of cytokine responses in vaccinated and protected animals revealed increased IFN-gamma production compared to controls. Finally, E. histolytica DNA was shown to activate macrophages in a TLR9 and MYD88-dependent manner. Immunized gerbils with Gal-lectin and E. histolytica DNA induced protective immunity against a challenge infection. Taken together, these findings underscore the importance of multivalent subunit vaccines in Th1 mediated immune responses in host defense against amebiasis.

The enteric protozoan parasite Entamoeba histolytica is the etiologic agent of the disease amebiasis which is characterized by colitis or hepatic lesions. Amebae colonize the colon by binding to mucous glycoproteins (mucins). Secretory mucins provide the gel nature to mucus and are a vital component of epithelial barrier function. Mucins prevent contact-dependent cytolysis of colonic cells by E. histolytica. To possibly circumvent this barrier, the parasite secretes a potent yet unidentified mucin secretagogue, which could deplete the stored mucin pool and render the mucous layer less protective. The objective of this study was to investigate the molecular mechanisms by which E. histolytica modulates colonic mucin exocytosis. We showed that E. histolytica converts exogenous arachidonic acid to prostaglandin E2 (PGE2), a known mucin secretagogue and potential mechanism by which the parasite evokes mucin secretion. Conversion was via a novel cyclooxygenase-like activity and was inhibitable with the known cyclooxygenase inhibitor aspirin. To study E. histolytica-mucin interactions, we developed an in vitro model of LS174T human colonic epithelial cells that secrete mucin constitutively and in response to mucin agonists. Highly purified mucins isolated from LS174T cells markedly inhibited amebic adherence to target cells and the mucous barrier protected the LS174T monolayers from amebic cytolysis. We have identified that Gal and GalNAc residues (O-linked sugars) of mucins are the protective moiety as O- but not N-linked glycosylation inhibitors decreased their protective effect. To understand how mucins are regulated during intestinal amebiasis and in the inflamed gut, we determined that PGE2 binds the EP4 receptor on LS174T cells and in rat colon to stimulate cyclic adenosine monophosphate-dependent mucin exocytosis. Taken together, these studies delineate how E. histolytica modulates host responses during infection to allow the parasite to survive and persist in th

The pathogenesis of the enteric protozoan parasite Entamoeba histolytica remains poorly understood. Moreover, the host responses during the early periods of interaction in the gut remain to be clarified. In this study I investigated the cell specific responses to the parasite and the importance of cross talk between epithelial-immune cells that could potentially influence the outcome of infection, with a central focus on Nuclear factor (NF)-kappaB. NF-kappaB is a ubiquitous transcription factor that plays a critical role in mucosal inflammation and its regulation by E. histolytica has not been studied so far. Gal-lectin is a well characterized parasite virulence factor and vaccine candidate. I first characterized the interactions between Gal-lectin and macrophages and found that several proinflammatory genes are upregulated as early as 2h. The Gal-lectin activated NF-kappaB and up-regulated Toll like receptor-2 expression in an NF-kappaB- and p38 Mitogen Activated Protein (MAP) kinase-dependent manner. As intestinal epithelial cells (IEC) form the first line of active host defense against mucosal pathogens, I determined the interaction between ameba soluble proteins and naive IEC. I observed that the parasite could elicit a chemokine response via activation of PI3 kinase and phosphorylation of p65 subunit to induce monocyte chemoattractant protein-1. The consequent recruitment of immune cells could be responsible for colonic inflammation. Finally, I made the novel observation that in macrophage-primed IEC, ameba proteins elicited a cytoprotective stress response. Using a combination of siRNA and over expression studies, I demonstrated that amebic proteins increased the expression and phosphorylation of Heat shock protein (Hsp) 27 thereby enhancing its association with and subsequent inhibition of Inhibitory kappaB kinase (IKK). The resulting inhibition of NF-kappaB could be a potential mechanism that explains the absence of inflammation in the majority of infected individuals. Taken together, the findings of this study open up a new facet in the pathogenesis of amebiasis and unravel a novel paradigm to study host-parasite interactions in the gut.

The heat shock response was studied in the intestinal parasitic protozoan Entamoeba histolytica. Temperature shifts from 37$ sp circ$C to 44$ sp circ$C enhanced the synthesis of five major heat shock (or stress) proteins (HSP) of 100, 50, 42, 37, and 28 kDa. Similarly, exposure of amebae to lymphokine activated macrophages and hydrogen peroxide caused HSP expression. Heat shock caused the reversible inhibition of amebic adherence to Chinese hamster ovary cells and human colonic mucin binding to trophozoites by ${>80 %}$. This was due to a decrease in the surface expression of the Gal/GalNAc adherence lectin and a marked reduction in the lectin mRNA expression. However, the presence of target Chinese hamster ovary cells during recovery at 37$ sp circ$C augmented amebic adherence. These results suggest that E. histolytica trophozoites produce a variety of HSP in response to different stimuli and can modulate the expression of the surface adherence lectin which maybe important in pathogenesis.

Thesis (Ph. D.)--University of Virginia, 1998. / Spine title: Control of E. histolytica lectin. Includes bibliographical references (p. 93-105). Also available online through Digital Dissertations.