Overexpression of Fut 2, 4, and 8, and nuclear localization of Fut 4 in ovarian cancer cell lines induced by ascitic fluids from epithelial ovarian cancer patients.

Fucosyltransferases (Fut) regulate the fucosylation process associated with tumorogenesis in different cancer types. Ascitic fluid (AF) from patients diagnosed with advanced stage of epithelial ovarian cancer (EOC) is considered as a dynamic tumor microenvironment associated with poor prognosis. Previous studies from our laboratory showed increased fucosylation in SKOV-3 and OVCAR-3, cancer-derived cell lines, when these cells were incubated with AFs derived from patients diagnosed with EOC. In the present work we studied three fucosyltransferases (Fut 2, Fut 4, and Fut 8) in SKOV-3, OVCAR-3 and CAOV-3 cell lines in combination with five different AFs from patients diagnosed with this disease, confirming that all tested AFs increased fucosylation. Then, we demonstrate that mRNAs of these three enzymes were overexpressed in the three cell lines under treatment with AFs. SKOV-3 showed the higher overexpression of Fut 2, Fut 4, and Fut 8 in comparison with the control condition. We further confirmed, in the SKOV-3 cell line, by endpoint PCR, WB, and confocal microscopy, that the three enzymes were overexpressed, being Fut 4 the most overexpressed enzyme compared to Fut 2 and Fut 8. These enzymes were concentrated in vesicular structures with a homogeneous distribution pattern throughout the cytoplasm. Moreover, we found that among the three enzymes, only Fut 4 was located inside the nuclei. The nuclear location of Fut 4 was confirmed for the three cell lines. These results allow to propose Fut 2, Fut 4, and Fut 8 as potential targets for EOC treatment or as diagnostic tools for this disease.

A monoclonal antibody against a Leishmania mexicana COX-like enzymatic activity also recognizes similar proteins in different protozoa of clinical importance.

In Leishmania mexicana, the protease gp63 has been documented as the protein responsible for cyclooxygenase (COX) activity. The present work aimed to obtain a monoclonal antibody capable of recognizing this protein without blocking the COX-like enzymatic activity. The antibody produced by the selected hybridoma was named D12 mAb. The antigen recognized by the D12 mAb was characterized by the determination of COX activity associated with immune complexes in the presence of exogenous arachidonic acid (AA) using the commercial Activity Assay Abcam kit. LSM-SMS analysis validated the identity of the antigen associated with the D12 mAb as the L. mexicana protease gp63. Confocal microscopy assays with the D12 mAb detected, by cross-recognition, similar proteins in other protozoan parasites. COX-like molecules are located in vesicular structures, homogeneously distributed throughout the cytoplasm in amastigotes (intracellular infectious phase) and promastigotes of L. mexicana, and trophozoites of Entamoeba histolytica, Acanthamoeba castellanii, and Naegleria fowleri. However, in Giardia duodenalis trophozoites, the distribution of the COX-like molecule was also in perinuclear areas. In comparison, in Trypanosoma cruzi trypomastigotes, the distribution was mainly observed in the plasma membrane. Structural analyses of COX-2-like antigens revealed continuous and discontinuous epitopes for B cells, which could be relevant in the cross-reaction of D12 mAb with the analyzed parasites. These results indicate that the D12 mAb against the L. mexicana gp63 also recognizes a COX-like molecule in several protozoan parasites, suggesting that this D12 mAb could potentially be used in combined therapies against infectious diseases.

PHD finger protein 20-like protein 1 (PHF20L1) in ovarian cancer: from its overexpression in tissue to its upregulation by the ascites microenvironment.

BACKGROUND: Ovarian cancer is the most aggressive gynecological malignancy. Transcriptional regulators impact the tumor phenotype and, consequently, clinical progression and response to therapy. PHD finger protein 20-like protein 1 (PHF20L1) is a transcriptional regulator with several isoforms, and studies on its role in ovarian cancer are limited. We previously reported that PHF20L1 is expressed as a fucosylated protein in SKOV-3 cells stimulated with ascites from patients with ovarian cancer. METHODS: We decided to analyze the expression of PHF20L1 in ovarian cancer tissues, determine whether a correlation exists between PHF20L1 expression and patient clinical data, and analyze whether ascites can modulate the different isoforms of this protein. Ovarian cancer biopsies from 29 different patients were analyzed by immunohistochemistry, and the expression of the isoforms in ovarian cancer cells with or without exposure to the tumor microenvironment, i.e., the ascitic fluid, was determined by western blotting assays. RESULTS: Immunohistochemical results suggest that PHF20L1 exhibits increased expression in sections of tumor tissues from patients with ovarian cancer and that higher PHF20L1 expression correlates with shorter progression-free survival and shorter overall survival. Furthermore, western blotting assays determined that protein isoforms are differentially regulated in SKOV-3 cells in response to stimulation with ascites from patients with epithelial ovarian cancer. CONCLUSION: The results suggest that PHF20L1 could play a relevant role in ovarian cancer given that higher PHF20L1 protein expression is associated with lower overall patient survival.

Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding Proteins.

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.

Linearolactone and Kaempferol Disrupt the Actin Cytoskeleton in Entamoeba histolytica: Inhibition of Amoebic Liver Abscess Development.

Linearolactone (1) and kaempferol (2) have amebicidal activity in in vitro studies. The type of cell death induced by 1 and 2 and their effects on the virulence of E. histolytica were analyzed by transmission and confocal electron microscopy, reactive oxygen species (ROS) production, and apoptosis, detected by flow cytometry with dichlorofluorescein 2',7'-diacetate and annexin-V binding, respectively, and confirmed by TUNEL. The interaction of 1 and 2 with actin was analyzed by docking, and the in vivo amoebicidal activity was established with the Mesocricetus auratus model; amebic liver abscess (ALA) development was evaluated by magnetic resonance (MR) and validated post mortem. In vitro, compounds 1 and 2 caused chromatin condensation, intracellular ROS, and loss of actin structures. Coupling analysis showed that they bind to the allosteric and catalytic sites of actin with binding energies of -11.30 and -8.45 kcal/mol, respectively. Treatments with 1 and 2 induced a decrease in ALA formation without toxic effects on the liver and kidney. Thus, compound 1, but not 2, was able to induce apoptosis-like effects in E. histolytica trophozoites by intracellular production of ROS that affected the actin cytoskeleton structuration. In vivo, compound 1 was more active than compound 2 to reduce the development of ALA.

EhRab21 associates with the Golgi apparatus in Entamoeba histolytica.

Rab proteins constitute the largest group of small GTPases and act as molecular switches in a wide variety of cellular processes, including proliferation, cytoskeleton assembly, and membrane trafficking in all eukaryotic cells. Rab21 has been reported in several eukaryotic cells, and our results suggest that in Entamoeba histolytica, Rab21 is involved in the vesicular traffic associated with the Golgi apparatus, where its function appears to be important to maintain the structure of this organelle. In addition, proteins such as Rab1A and Sec24, identified in this work associated with EhRab21, participate in the traffic of COPII vesicles from the endoplasmic reticulum to the Golgi apparatus and are necessary to maintain the latter's structure in human cells. In addition, EhRab21 probably affects the lysosome biogenesis, as indicated by an increase in the number of lysosomes as a result of the increase in EhRab21 activity. The participation of EhRab21 in the pathogenesis of amebiasis was verified on the amoebic liver abscess formation model using hamsters (Mesocricetus auratus), in which the overexpression of EhRab21Q64L (positive dominant mutant protein) decreased the number of liver abscesses formed.

Functional Characterization of an Interferon Gamma Receptor-Like Protein on Entamoeba histolytica.

Entamoeba histolytica is an anaerobic parasitic protozoan and the causative agent of amoebiasis. E. histolytica expresses proteins that are structurally homologous to human proteins and uses them as virulence factors. We have previously shown that E. histolytica binds exogenous interferon gamma (IFN-γ) on its surface, and in this study, we explored whether exogenous IFN-γ could modulate parasite virulence. We identified an IFN-γ receptor-like protein on the surface of E. histolytica trophozoites by using anti-IFN-γ receptor 1 (IFN-γR1) antibody and performing immunofluorescence, Western blot, protein sequencing, and in silico analyses. Coupling of human IFN-γ to the IFN-γ receptor-like protein on live E. histolytica trophozoites significantly upregulated the expression of E. histolytica cysteine protease A1 (EhCP-A1), EhCP-A2, EhCP-A4, EhCP-A5, amebapore A (APA), cyclooxygenase 1 (Cox-1), Gal-lectin (Hgl), and peroxiredoxin (Prx) in a time-dependent fashion. IFN-γ signaling via the IFN-γ receptor-like protein enhanced E. histolytica's erythrophagocytosis of human red blood cells, which was abrogated by the STAT1 inhibitor fludarabine. Exogenous IFN-γ enhanced chemotaxis of E. histolytica, its killing of Caco-2 colonic and Hep G2 liver cells, and amebic liver abscess formation in hamsters. These results demonstrate that E. histolytica expresses a surface IFN-γ receptor-like protein that is functional and may play a role in disease pathogenesis and/or immune evasion.

Biological and toxicological evaluation of Rhus trilobata Nutt. (Anacardiaceae) used traditionally in mexico against cancer.

BACKGROUND: Rhus trilobata Nutt. (Anacardiaceae) (RHTR) is a plant of Mexico that is traditionally used as an alternative treatment for several types of cancer. However, the phytochemical composition and potential toxicity of this plant have not been evaluated to support its therapeutic use. Therefore, this study aimed to evaluate the biological activity of RHTR against colorectal adenocarcinoma cells, determine its possible acute toxicity, and analyze its phytochemical composition. METHODS: The traditional preparation was performed by decoction of stems in distilled water (aqueous extract, AE), and flavonoids were concentrated with C18-cartridges and ethyl acetate (flavonoid fraction, FF). The biological activity was evaluated by MTT viability curves and the TUNEL assay in colorectal adenocarcinoma (CACO-2), ovarian epithelium (CHO-K1) and lung/bronchus epithelium (BEAS-2B) cells. The toxicological effect was determined in female BALB/c mice after 24 h and 14 days of intraperitoneal administration of 200 mg/kg AE and FF, respectively. Later, the animals were sacrificed for histopathological observation of organs and sera obtained by retro-orbital bleeding for biochemical marker analysis. Finally, the phytochemical characterization of AE and FF was conducted by UPLC-MSE. RESULTS: In the MTT assays, AE and FF at 5 and 18 µg/mL decreased the viability of CACO-2 cells compared with cells treated with vehicle or normal cells (p ≤ 0.05, ANOVA), with changes in cell morphology and the induction of apoptosis. Anatomical and histological analysis of organs did not reveal important pathological lesions at the time of assessment. Additionally, biochemical markers remained normal and showed no differences from those of the control group after 24 h and 14 days of treatment (p ≤ 0.05, ANOVA). Finally, UPLC-MSE analysis revealed 173 compounds in AE-RHTR, primarily flavonoids, fatty acids and phenolic acids. The most abundant compounds in AE and FF were quercetin and myricetin derivates (glycosides), methyl gallate, epigallocatechin-3-cinnamate, ß-PGG, fisetin and margaric acid, which might be related to the anticancer properties of RHTR. CONCLUSION: RHTR exhibits biological activity against cancer cells and does not present adverse toxicological effects during its in vivo administration, supporting its traditional use.

Effect of ovarian cancer ascites on SKOV-3 cells proteome: new proteins associated with aggressive phenotype in epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer is the second most lethal gynecological cancer worldwide. Ascites can be found in all clinical stages, however in advanced disease stages IIIC and IV it is more frequent and could be massive, associated with worse prognosis. Due to the above, it was our interest to understanding how the ascites of ovarian cancer patients induces the mechanisms by which the cells present in it acquire a more aggressive phenotype and to know new proteins associated to this process. METHODS: A proteomic analysis of SKOV-3 cells treated with five different EOC ascites was performed by two-dimensional electrophoresis coupled to MALDI-TOF. The level of expression of the proteins of interest was validated by RT-PCR because several of these proteins have only been reported at the messenger level. RESULTS: Among the proteins identified that increased their expression in ascites-treated SKOV-3 cells, were Ran GTPase, ZNF268, and Synaptotagmin like-3. On the other hand, proteins that were negatively regulated by ascites were HLA-I, HSPB1, ARF1, Synaptotagmin 1, and hnRNPH1, among others. Furthermore, an interactome for every one of these proteins was done in order to identify biological processes, molecular actions, and cellular components in which they may participate. CONCLUSIONS: Identified proteins participate in cellular processes highly relevant to the aggressive phenotype such as nuclear transport, regulation of gene expression, vesicular trafficking, evasion of the immune response, invasion, metastasis, and in resistance to chemotherapy. These proteins may represent a source of information which has the potential to be evaluated for the design of therapies directed against these malignant cells that reside on ovarian cancer ascites.

The translational blocking of α5 and α6 integrin subunits affects migration and invasion, and increases sensitivity to carboplatin of SKOV-3 ovarian cancer cell line.

Epithelial ovarian cancer is the most lethal gynecologic malignancy. Integrins, overexpressed in cancer, are involved in various processes that favor the development of the disease. This study focused on determining the degree of involvement of α5, α6 and ß3 integrin subunits in the establishment/development of epithelial ovarian cancer (EOC), such as proliferation, migration, invasion, and response to carboplatin. The translation of the α5, α6 and ß3 integrins was blocked using morpholines, generating morphant cells for these proteins, which were corroborated by immunofluorescence assays. WST-1 proliferation assay showed that silencing of α5, α6, and ß3 integrins does not affect the survival of morphants. Wound healing and transwell chamber assays showed that blocking α5 and α6 integrins decrease, in lesser and greater level respectively, the migratory and the invasive capacity of SKOV-3 cells. Finally, blocking α5 and α6 integrins partially sensitized the cells response to carboplatin, while blocking integrin ß3 generated resistance to this drug. Statistical analyses were performed with the GraphPad Prism 5.0 software employing one way and two-way ANOVA tests; data are shown as average±SD. Results suggest that α5 and α6 integrins could become good candidates for chemotherapy targets in EOC.

Acanthamoeba (T4) trophozoites cross the MDCK epithelium without cell damage but increase paracellular permeability and transepithelial resistance by modifying tight junction composition.

Free-living amoebae of the genus Acanthamoeba are protozoa ubiquitously found in nature. Some species of the genus are potentially pathogenic for humans provoking keratitis in healthy individuals, often in contact lens wearers and opportunistic infections such as pneumonitis, fatal granulomatous encephalitis and skin infections, particularly in immunocompromised individuals. The pathogenic mechanisms of these amoebae are poorly understood, however it had been suggested that contact dependent mechanisms are important during invasion, regardless of the epithelia type, since amoebae penetrate epithelia separating tight junction (TJ). This study was undertaken to determine whether Acanthamoeba sp. (T4) damages the barrier function of the TJ in MDCK epithelial monolayers. Actin cytoskeleton staining and electron microscopy analyses were performed; paracellular permeability and TJ sealing were evaluated by apicobasolateral diffusion of ruthenium red and transepithelial resistance (TER) measurements; immunofluorescence and Western blot assays were performed to locate and estimate expression of TJ protein claudins 2 (Cldn2) and 4 (Cldn4). The results show that Acanthamoeba sp. crosses the MDCK monolayer without altering the actin cytoskeleton or the morphology of the cells. When trophozoites or conditioned medium interact with the monolayer, paracellular diffusion of ruthenium red increases. After 6 h, the amoebae, but not their conditioned medium, increase the TER, and Cldn2 is removed from the TJ, and its overall content in the cells diminishes, while Cldn4 is targeted to the TJ without changing its expression level. In conclusion Acanthamoeba (T4) crosses MDCK monolayer without damaging the cells, increasing permeability and TER through Cldn2 degradation, and redirecting Cldn4 to TJ. These results strongly suggest that contact-dependent mechanisms are relevant during amoebae invasion.

Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy.

Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica.

Isoindoline Derivatives of α-Amino Acids as Cyclooxygenase 1 and 2 Inhibitors.

IC50 values were obtained for two series of isoindolines derived from α-amino acids over cyclooxygenase 1 and 2 (COX-1 and COX-2). In order to explain the biological activity observed, a structure-activity relationship (SAR) model was achieved for the tested compounds and 19 reference compounds with known selective inhibitory activity, through the correlation of the binding energies calculated from rigid docking of the best conformations into the catalytic sites of COX-1 and COX-2, as well as their molecular descriptors: Log P, molecular weight (MW), volume (V), and solvation energy (Esol) versus their experimental IC50 values by MLR and LS-SVM methods. The model probed whether the COX-1 and COX-2 inhibitory activities of the isoindolines correlate with steric, hydrophobic, and thermodynamic parameters. The correlation values with MLR for COX-1 and COX-2 (r(2) = 0.4193 and r(2) = 0.5929) were optimized with LS-SVM until r(2) = 0.6818 for COX-1 and r(2) = 0.8985 for COX-2, resulting in a good predictive ability for COX-1 and -2 inhibition with this model. In conclusion, the data suggests that the physicochemical descriptors evaluated have an impact on the inhibitory activity and selectivity of isoindolines over COX-1 and COX-2.

The Feline calicivirus Leader of the Capsid (LC) Protein Contains a Putative Transmembrane Domain, Binds to the Cytoplasmic Membrane, and Exogenously Permeates Cells.

Feline calicivirus (FCV), an important model for studying the biology of the Caliciviridae family, encodes the leader of the capsid (LC) protein, a viral factor known to induce apoptosis when expressed in a virus-free system. Our research has shown that the FCV LC protein forms disulfide bond-dependent homo-oligomers and exhibits intrinsic toxicity; however, it lacked a polybasic region and a transmembrane domain (TMD); thus, it was initially classified as a non-classical viroporin. The unique nature of the FCV LC protein, with no similarity to other proteins beyond the Vesivirus genus, has posed challenges for bioinformatic analysis reliant on sequence similarity. In this study, we continued characterizing the LC protein using the AlphaFold 2 and the recently released AlphaFold 3 artificial intelligence tools to predict the LC protein tertiary structure. We compared it to other molecular modeling algorithms, such as I-Tasser's QUARK, offering new insights into its putative TMD. Through exogenous interaction, we found that the recombinant LC protein associates with the CrFK plasmatic membrane and can permeate cell membranes in a disulfide bond-independent manner, suggesting that this interaction might occur through a TMD. Additionally, we examined its potential to activate the intrinsic apoptosis pathway in murine and human ovarian cancer cell lines, overexpressing survivin, an anti-apoptotic protein. All these results enhance our understanding of the LC protein's mechanism of action and suggest its role as a class-I viroporin.

Myo1f has an essential role in γδT intraepithelial lymphocyte adhesion and migration.

γδT intraepithelial lymphocyte represents up to 60% of the small intestine intraepithelial compartment. They are highly migrating cells and constantly interact with the epithelial cell layer and lamina propria cells. This migratory phenotype is related to the homeostasis of the small intestine, the control of bacterial and parasitic infections, and the epithelial shedding induced by LPS. Here, we demonstrate that Myo1f participates in the adhesion and migration of intraepithelial lymphocytes. Using long-tailed class I myosins KO mice, we identified the requirement of Myo1f for their migration to the small intestine intraepithelial compartment. The absence of Myo1f affects intraepithelial lymphocytes' homing due to reduced CCR9 and α4ß7 surface expression. In vitro, we confirm that adhesion to integrin ligands and CCL25-dependent and independent migration of intraepithelial lymphocytes are Myo1f-dependent. Mechanistically, Myo1f deficiency prevents correct chemokine receptor and integrin polarization, leading to reduced tyrosine phosphorylation which could impact in signal transduction. Overall, we demonstrate that Myo1f has an essential role in the adhesion and migration in γδT intraepithelial lymphocytes.

Extracellular Vesicles Secreted by Acanthamoeba culbertsoni Have COX and Proteolytic Activity and Induce Hemolysis.

Several species of Acanthamoeba genus are potential pathogens and etiological agents of several diseases. The pathogenic mechanisms carried out by these amoebae in different target tissues have been documented, evidencing the relevant role of contact-dependent mechanisms. With the purpose of describing the pathogenic processes carried out by these protozoans more precisely, we considered it important to determine the emission of extracellular vesicles (EVs) as part of the contact-independent pathogenicity mechanisms of A. culbertsoni, a highly pathogenic strain. Through transmission electronic microscopy (TEM) and nanoparticle tracking analysis (NTA), EVs were characterized. EVs showed lipid membrane and a size between 60 and 855 nm. The secretion of large vesicles was corroborated by confocal and TEM microscopy. The SDS-PAGE of EVs showed proteins of 45 to 200 kDa. Antigenic recognition was determined by Western Blot, and the internalization of EVs by trophozoites was observed through Dil-labeled EVs. In addition, some EVs biological characteristics were determined, such as proteolytic, hemolytic and COX activity. Furthermore, we highlighted the presence of leishmanolysin in trophozites and EVs. These results suggest that EVs are part of a contact-independent mechanism, which, together with contact-dependent ones, allow for a better understanding of the pathogenicity carried out by Acanthamoeba culbertsoni.

Effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis.

Entamoeba histolytica is the causative agent of amoebiasis, a disease that produces dysentery as a result of the perforation of the large intestine. This parasite often invades other organs, primarily the liver, leading to an amoebic liver abscess (ALA), which can cause death. Metronidazole is the drug of choice for the treatment of ALA; however, it produces toxic side effects in patients. Lactoferrin (Lf) is a glycoprotein of the innate immune response that sequesters iron in the mucosae. Lf possesses immune-regulatory properties, such as antiinflammatory and antioxidant activities. Moreover, the microbicidal activity of apoLf, which lacks bound iron, has been shown. In this study, we evaluated the therapeutic effect of bovine Lf (bLf) against ALA in a model of hepatic amoebiasis in hamsters. Interestingly, hamsters treated intragastrically with Lf (2.5 mg/100 g mass) over a period of 8 days showed no clinical signs of disease and ALA was effectively decreased, with only 0.63% detectable lesion, compared with 63% in untreated animals. Furthermore, liver function and blood cells approached normal levels among those receiving bLf treatment. These results suggest that bLf may aid in the therapy of amoebiasis, likely without producing undesirable effects in patients.

Cytotoxicity of functionalized carbon nanotubes in J774A macrophages.

Cytotoxicity of carbon nanotubes (CNTs) is a prime concern for its use as antigen carriers. Here we evaluated the cytotoxic effect of unpurified (UP-CNTs), purified (P-CNTs), fluorescein isothiocyanate-functionalized (FITC-CNTs), and Entamoeba histolytica 220-kDa lectin-functionalized CNTs (L220-CNTs) in J774A macrophage (MOs) cell line. Cell viability and apoptosis were analyzed by MTT and TUNEL assays, respectively. Cyclooxygenase-2 (COX-2) was analyzed by reverse transcription-polymerase chain reaction. Cytotoxicity at 6.0 mg/L was higher with UP-CNTs > P-CNTs > FITC-CNTs, showing a decrease in cell viability and an increase in apoptosis. In contrast, MOs interacted with L220-CNTs showed an increase in cell viability without signs of apoptosis. Although UP-CNTs and P-CNTs exhibited COX-2 induction with 6.0 mg/L, functionalized CNTs were able to induce COX-2 at concentrations as low as 0.06 mg/L. These results suggest that functionalization decreases toxicity, and that L220-CNTs may be an excellent candidate for the production of a nanovaccine against amebiasis.

Differences in cap formation between invasive Entamoeba histolytica and non-invasive Entamoeba dispar.

The rapid redistribution of surface antigen-antibody complexes in trophozoites of the human protozoan parasite Entamoeba histolytica, in a process known as capping, has been considered as a means of the parasite to evade the host immune response. So far, capping has been documented in the invasive E. histolytica, whereas the mobility of surface components in the non-invasive Entamoeba dispar is not known. E. dispar does not induce liver lesions in rodent experimental models, in contrast to the liver abscesses produced by E. histolytica in the same animal model. We have therefore analyzed the mobility of surface receptors to the lectin concanavalin A and of Rab11, a membrane-associated protein, in both species of Entamoebae by confocal fluorescence microscopy and transmission and scanning electron microscopy. The great majority of E. histolytica trophozoites became morphologically polarized through the formation of well-defined caps at the posterior pole of the parasite. Actin colocalized with the lectin caps. Antibodies against the membrane protein Rab 11 also produced capping. In striking contrast, in E. dispar, the mobility of concanavalin A surface receptors was restricted to the formation of irregular surface patches that did no progress to constitute well-defined caps. Also, anti-Rab 11 antibodies did not result in capping in E. dispar. Whether the failure of E. dispar to efficiently mobilize surface molecules in response to lectin or antibodies as shown in the present results is related to its non-invasive character represents an interesting hypothesis requiring further analysis.

Clinical case of cerebral amebiasis caused by E. histolytica.

Although amebic brain abscess is a rare form of invasive amebiasis, when present, it is frequently lethal. This disorder always begins with the infection of the colon by Entamoeba histolytica trophozoites, which then travel to extra-intestinal tissues through the bloodstream. Amebic brain abscesses are produced when trophozoites invade the central nervous system. Computerized axial tomography scans can be used to diagnose the presence or absence of a brain abscess with a certainty of 100%. However, this diagnostic tool does not reveal the etiological agent of disease. By analyzing the clinical case of a patient that died due to untimely treatment of this malady, the present study aims to identify a diagnostic tool that can give a precise determination of the etiological agent and therefore permit adequate and opportune treatment. Currently, diagnosis of amebic brain abscess is often done by identification of the ameba in a biopsy or autopsy. By immunohistochemistry and immunofluorescence with specific antibodies, we identified the existence of E. histolytica, which presents proteins similar to Naegleria fowleri in its membrane.