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.

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.

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.

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.

Alpha-terthienyl increases filamentous actin of Entamoeba histolytica.

This study aimed to know if alpha terthienyl (α-T) affects E. histolytica viability and to analyze its effect on the actin cytoskeleton. Trophozoites of E. histolytica HM1-IMSS were treated with α-T, then, cell viability and morphology were evaluated using tetrazolium salts and scanning electron microscopy, respectively; while actin filaments (F-actin) were stained with rhodamine-phalloidin, observed by confocal microscopy and quantified by fluorometry. Data showed that α-T inhibited cell viability of trophozoites (IC50, 19.43 µg / mL), affected the cell morphology, and increased the F-actin in a dose-dependent manner. Production of reactive oxygen species and RhoA-GTP levels remained normal in α-T-treated amebas. Two inhibitors that affect the organization of the trophozoites cytoskeleton, one that interacts directly with actin, Cytochalasin D (CD), and one that affects the Rho signaling pathway by inhibiting the downstream effector Rock, Y27632, were tested. Y27632 did not affect the increase of polymerized actin observed with α-T, this compound partially ameliorates the potent disrupting effects of CD on actin filaments. Docking results suggest that α-T could be an antagonist of CD for the same interaction zone in actin, however, more studies are needed to define the action mechanism of this compound.

Differentiation of Entamoeba histolytica: a possible role for enolase.

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process.

Protein Phosphatase PP2C Identification in Entamoeba spp.

Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.

Antineoplastic Activity of Rhus trilobata Nutt. (Anacardiaceae) against Ovarian Cancer and Identification of Active Metabolites in This Pathology.

Rhus trilobata (RHTR) is a medicinal plant with cytotoxic activity in different cancer cell lines. However, the active compounds in this plant against ovarian cancer are unknown. In this study, we aimed to evaluate the antineoplastic activity of RHTR and identify its active metabolites against ovarian cancer. The aqueous extract (AE) and an active fraction (AF02) purified on C18-cartridges/ethyl acetate decreased the viability of SKOV-3 cells at 50 and 38 µg/mL, respectively, compared with CHO-K1 (>50 µg/mL) in MTT assays and generated changes in the cell morphology with apoptosis induction in Hemacolor® and TUNEL assays (p ≤ 0.05, ANOVA). The metabolite profile of AF02 showed a higher abundance of flavonoid and lipid compounds compared with AE by UPLC-MSE. Gallic acid and myricetin were the most active compounds in RHTR against SKOV-3 cells at 50 and 166 µg/mL, respectively (p ≤ 0.05, ANOVA). Antineoplastic studies in Nu/Nu female mice with subcutaneous SKOV-3 cells xenotransplant revealed that 200 mg/kg/i.p. of AE and AF02 inhibited ovarian tumor lesions from 37.6% to 49% after 28 days (p ≤ 0.05, ANOVA). In conclusion, RHTR has antineoplastic activity against ovarian cancer through a cytostatic effect related to gallic acid and myricetin. Therefore, RHTR could be a complementary treatment for this pathology.

Entamoeba invadens, encystation process and enolase.

The reptilian parasite Entamoeba invadens is accepted as a model for the study of the Entamoeba encystation process. Here we describe the production and characterization of a mAb (B4F2), generated against a component of the E. invadens cyst wall. This mAb specifically recognizes a 48-kDa protein present in cytoplasmic vesicles of cells encysting for 24 h. In mature cysts (96 h), the antigen was detected on the cyst surface. By two-dimensional electrophoresis and mass spectrometry analysis, the B4F2 specific antigen was identified as enolase. Levels of enolase mRNA were increased in encysting cells and the B4F2 mAb was found to inhibit cyst formation. Therefore, these results strongly suggest a new role for enolase in E. invadens encystation, and the B4F2 mAb will be useful tool to study its role in the differentiation process.

Effect of Gallic acid and Myricetin on ovarian cancer models: a possible alternative antitumoral treatment.

BACKGROUND: Ovarian cancer is the leading cause of mortality among malignant gynecological tumors. Surgical resection and chemotherapy with intravenous platinum/taxanes drugs are the treatments of choice, with little effectiveness in later stages and severe toxicological effects. Therefore, this study aimed to evaluate the antineoplastic activity of gallic acid (GA) and myricetin (Myr) administrated peritumorally in Nu/Nu mice xenotransplanted with SKOV-3 cells. METHODS: Biological activity of GA and MYR was evaluated in SKOV-3 and OVCAR-3 cells (ovarian adenocarcinomas) by confocal/transmission electron microscopy, PI-flow cytometry, H2-DCF-DA stain, MTT, and Annexin V/PI assays. Molecular targets of compounds were determined with ACD/I-Labs and SEA. Antineoplastic activity was performed in SKOV-3 cells subcutaneously xenotransplanted into female Nu/Nu mice treated peritumorally with 50 mg/kg of each compound (2 alternate days/week) for 28 days. Controls used were paclitaxel (5 mg/kg) and 20 µL of vehicle (0.5% DMSO in 1X PBS). Tumor lesions, organs and sera were evaluated with NMR, USG, histopathological, and paraclinical studies. RESULTS: In vitro studies showed a decrease of cell viability with GA and Myr in SKOV-3 (50 and 166 µg/mL) and OVCAR-3 (43 and 94 µg/mL) cells respectively, as well as morphological changes, cell cycle arrest, and apoptosis induction due to ROS generation (p ≤ 0.05, ANOVA). In silico studies suggest that GA and MYR could interact with carbonic anhydrase IX and PI3K, respectively. In vivo studies revealed inhibitory effects on tumor lesions development with GA and MYR up to 50% (p ≤ 0.05, ANOVA), with decreased vascularity, necrotic/fibrotic areas, neoplastic stroma retraction and apoptosis. However, toxicological effects were observed with GA treatment, such as leukocyte infiltrate and hepatic parenchyma loss, hypertransaminasemia (ALT: 150.7 ± 25.60 U/L), and hypoazotemia (urea: 33.4 ± 7.4 mg/dL), due to the development of chronic hepatitis (p ≤ 0.05, ANOVA). CONCLUSION: GA and Myr (50 mg/kg) administered by peritumoral route, inhibit ovarian tumor lesions development in rodents with some toxicological effects. Additional studies will be necessary to find the appropriate therapeutic dose for GA. Therefore, GA and Myr could be considered as a starting point for the development of novel anticancer agents.

Ascites from Ovarian Cancer Induces Novel Fucosylated Proteins.

Ovarian cancer is considered to be the most lethal type of gynecological cancer. During the advanced stages of ovarian cancer, an accumulation of ascites is observed. Fucosylation has been classified as an abnormal post-translational modification that is present in many diseases, including ovarian cancer. Ovarian cancer cells that are cultured with ascites stimulation change their morphology; concomitantly, the fucosylation process is altered. However, it is not known which fucosylated proteins are modified. The goal of this work was to identify the differentially fucosylated proteins that are expressed by ovarian cancer cell lines that are cultured with ovarian cancer patients' ascites. Aleuria aurantia lectin was used to detect fucosylation, and some changes were observed, especially in the cell membrane. Affinity chromatography and mass spectrometry (MALDI-TOF) were used to identify 6 fucosylated proteins. Four proteins (Intermediate filament family orphan 1 [IFFO1], PHD finger protein 20-like protein 1 [PHF20L1], immunoglobulin gamma 1 heavy chain variable region partial [IGHV1-2], and Zinc finger protein 224 [ZNF224]) were obtained from cell cultures stimulated with ascites, and the other two proteins (Peregrin [BRPF1] and Dystrobrevin alpha [DTNA]) were obtained under normal culture conditions. The fucosylated state of some of these proteins was further analyzed. The experimental results show that the ascites of ovarian cancer patients modulated the fucosylation process. The PHD finger protein 20-like protein 1, Zinc finger protein 224 and Peregrin proteins colocalize with fucosylation at different levels.

Acanthamoeba castellanii: identification and distribution of actin cytoskeleton.

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparison between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.

EhRab21 mobilization during erythrophagocytosis in Entamoeba histolytica.

Rab proteins are present in all eukaryotic lineages and regulate vesicular trafficking. Entamoeba histolytica has approximately 100 genes encoding Rab proteins, among which 16 have homology with human Rab proteins. Human Rab21 participates in integrin recycling, and thus amoebic Rab21 was believed to regulate the mobilization of Ehß1FNR (integrin-like fibronectin receptor related with human integrin ß1). We analyzed the distribution of EhRab21 using a polyclonal antibody produced with a specific peptide against the amoebic Rab protein, using confocal microscopy and specific probes for different organelles. EhRab21 was not associated with Ehß1FNR in fibronectin-stimulated trophozoites. However, EhRab21 was relocalized to lysosomes in erythrophagocytosis assays and was also found in Golgi-positive structures and the nuclear periphery. These results suggest that EhRab21, unlike its human homologue, is not present in the recycling pathway. However, according to the results, EhRab21 may regulate the trafficking between lysosomes and the Golgi apparatus.

Leishmania mexicana gp63 is the enzyme responsible for cyclooxygenase (COX) activity in this parasitic protozoa.

Cyclooxygenase-2 (COX-2) is an enzyme responsible of prostaglandins production, such as prostaglandin E2 (PGE2), an immune response modulator that regulates the immune system to inhibit Th1 and to promote Th2 cytokines production. Many parasites modulate their host immune response through PGE2 effects; however, in parasites, only one protein with COX activity has been described, the α-actinin of Entamoeba histolytica. Prostanoids production has been reported in some species of Leishmania but not the enzymes responsible of their production. To identify the protein responsible for COX activity in Leishmania mexicana, we examined total extracts of promastigotes and samples with COX activity were subjected to ion exchange column purification and precipitation with ammonium sulphate; fractions with activity were analyzed by SDS-PAGE and Western blot using an anti-mouse COX-2 polyclonal antibody. Results showed that in those samples with enzymatic activity, the anti-mouse COX-2 polyclonal antibody recognized a protein with an approximate molecular weight of 66 KDa. Bands recognized by the antibody were subjected to mass spectrometry analysis and the results showed that several peptides from the bands purified by two different methods, and that were recognized by the anti-mouse COX-2 polyclonal antibody corresponded to the Leishmania mexicana gp63 surface protease. L. mexicana gp63 was purified by a Concanavalin A (Con-A) affinity column and subjected to immunoprecipitation with a commercial anti-Leishmania gp63 polyclonal antibody; the immunoprecipitated sample was analyzed for COX activity showing that the anti-gp63 antibody did immunoprecipitate the COX activity. The presence of COX activity was further confirmed in amastigotes extracts of L. mexicana. Moreover, a recombinant gp63 protein was produced and its COX activity tested, confirming that gp63 is the molecule responsible for COX activity.

Entamoeba histolytica L220 induces the in vitro activation of macrophages and neutrophils and is modulated by neurotransmitters.

The neuroimmunoregulation of inflammation has been well characterized. Entamoeba histolytica provokes an inflammatory response in the host in which macrophages and neutrophils are the first line of defense. The aim of this study was to analyze the effect of the 220 kDa lectin of Entamoeba histolytica on stimulation of human macrophages and neutrophils, especially the secretion of cytokines and the relation of these to neurotransmitters. Human cells were interacted with L220, epinephrine, nicotine, esmolol and vecuronium bromide. The concentrations of IL-1ß, IFN-γ, TNF-α and IL-10 were determined by ELISA at, 4 h of interaction. L220 has a cytokine stimulating function of macrophages and neutrophils for secretion of IL-1ß, and IL-10 only by macrophages, which was modulated by the effect of vecuronium on cholinergic receptors in this immune cells.