Cell morphology impact on the set-up of electroporation protocols for in-suspension and adhered breast cancer cells.

In order to establish cancer-type-specific electroporation protocols for breast cancer, electroporation was performed in vitro in two modalities: in-suspension and adhered cells. Electroporation of cell suspensions was carried out through commercial electroporation cuvettes whereas a novel electrode for electroporation of adhered cells was designed and manufactured aimed to preserve cell structure, to provide a closer model to an in vivo scenario, and as a means to visualize the mechanical effects of electroporation on the cell membrane by using scanning electron microscopy. Electroporation protocols and electric field thresholds were predicted in silico and experimentally tuned through propidium iodide uptake and cell viability. Three breast-cancer cell lines (BT-20, MCF-7 and HCC1419) and a non-cancerous cell line (BEAS-2B) were used. Cancerous cells responded differently to electroporation depending on the electric parameters, cell histology, the cell culture modality, and the cell morphology (membrane thickness mainly), which was evaluated trough confocal and transmission electron microscopy. Particularly, it was found that electrochemotherapy may represent a promising alternative as an adjuvant treatment of metastatic breast tumours, and as a neoadjuvant therapy for Her2/neu tumours. Oppositely, triple negative breast tumours may show a high sensitivity to electroporation and therefore, they could be efficiently treated with irreversible electroporation. On the other hand, noncancerous cells demanded the highest voltage in both cell culture modalities in order to be electroporated. Hence, these cells in suspension may provide a reliable, easy-to-perform, low-cost model for the development of electroporation protocols for eradication of healthy tissue around a tumour in a safety margin.

Vorinostat, a possible alternative to metronidazole for the treatment of amebiasis caused by Entamoeba histolytica.

AbbreviationsSAHAsuberoylanilide hydroxamic acidEhHDACHistone Deacetylase from Entamoeba histolyticaRgRadius of gyrationRMSDroot-mean-square deviationRMSFroot-mean-square fluctuationMDSmolecular dynamics simulationVMDVisual Molecular DynamicsNAMDNanoscale Molecular DynamicsPBCperiodic boundary conditionsPMEParticle Mesh Ewald3Dthree-dimensionalCαalpha carbonFDAFood and Drug AdministrationnsnanosecondsGPU CUDAGraphics Processing Unit Compute Unified Device ArchitectureCommunicated by Ramaswamy H. Sarma.

Decanethiol functionalized silver nanoparticles are new powerful leishmanicidals in vitro.

We evaluated, for the first time, the leishmanicidal potential of decanethiol functionalized silver nanoparticles (AgNps-SCH) on promastigotes and amastigotes of different strains and species of Leishmania: L. mexicana and L. major isolated from different patients suffering from localized cutaneous leishmaniasis (CL) and L. mexicana isolated from a patient suffering from diffuse cutaneous leishmaniasis (DCL). We recorded the kinetics of promastigote growth by daily parasite counting for 5 days, promastigote mobility, parasite reproduction by CFSE staining's protocol and promastigote killing using the propidium iodide assay. We also recorded IC50's of promastigotes and amastigotes, therapeutic index, and cytotoxicity by co-culturing macrophages with AgNps-SCH or sodium stibogluconate (Sb) used as reference drug. We used Sb as a reference drug since it is used as the first line treatment for all different types of leishmaniasis. At concentrations 10,000 times lower than those used with Sb, AgNps-SCH had a remarkable leishmanicidal effect in all tested strains of parasites and there was no toxicity to J774A.1 macrophages since > 85% were viable at the concentrations used. Therapeutic index was about 20,000 fold greater than the corresponding one for Sb treated cells. AgNps-SCH inhibited > 80% promastigote proliferation in all tested parasites. These results demonstrate there is a high leishmanicidal potential of AgNps-SCH at concentrations of 0.04 µM. Although more studies are needed, including in vivo testing of AgNps-SCH against different types of leishmaniasis, they can be considered a potential new treatment alternative.

Haptoglobin and CCR2 receptor expression in ovarian cancer cells that were exposed to ascitic fluid: exploring a new role of haptoglobin in the tumoral microenvironment.

Haptoglobin (Hp) is an acute-phase protein that is produced by the liver to capture the iron that is present in the blood circulation, thus avoiding its accumulation in the blood. Moreover, Hp has been detected in a wide variety of tissues, in which it performs various functions. In addition, this protein is considered a potential biomarker in many diseases, such as cancer, including ovarian carcinoma; however, its participation in the cancerous processes has not yet been determined. The objective of this work was to demonstrate the expression of Hp and its receptor CCR2 in the ovarian cancer cells and its possible involvement in the process of cell migration through changes in the rearrangement of the actin cytoskeleton using western blot and wound-healing assays and confirming by confocal microscopy. Ovarian cancer cells express both Hp and its receptor CCR2 but only after exposure to ascitic fluid, inducing moderated cell migration. However, when the cells are exposed to exogenous Hp, the expression of CCR2 is induced together with drastic changes in the actin cytoskeleton rearrangement. At the same time, Hp induced cell migration in a much more efficient manner than did ascitic fluid. These effects were blocked when the CCR2 synthetic antagonist RS102895 was used to pretreat the cells. These results suggest that Hp-induced changes in the cell morphology, actin cytoskeleton structure, and migration ability of tumor cells, is possibly "preparing" these cells for the potential induction of the metastatic phenotype.

Actin, RhoA, and Rab11 participation during encystment in Entamoeba invadens.

In the genus Entamoeba, actin reorganization is necessary for cyst differentiation; however, its role is still unknown. The aim of this work was to investigate the role of actin and encystation-related proteins during Entamoeba invadens encystation. Studied proteins were actin, RhoA, a small GTPase involved through its effectors in the rearrangement of the actin cytoskeleton; Rab11, a protein involved in the transport of encystation vesicles; and enolase, as an encystment vesicles marker. Results showed a high level of polymerized actin accompanied by increased levels of RhoA-GTP during cell rounding and loss of vacuoles. Cytochalasin D, an actin polymerization inhibitor, and Y27632, an inhibitor of RhoA activity, reduced encystment in 80%. These inhibitors also blocked cell rounding, disposal of vacuoles, and the proper formation of the cysts wall. At later times, F-actin and Rab11 colocalized with enolase, suggesting that Rab11 could participate in the transport of the cyst wall components through the F-actin cytoskeleton. These results suggest that actin cytoskeleton rearrangement is playing a decisive role in determining cell morphology changes and helping with the transport of cell wall components to the cell surface during encystment of E. invadens.

Exploring the possible role of lysine acetylation on Entamoeba histolytica virulence: a focus on the dynamics of the actin cytoskeleton.

Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

PPAR activation induces M1 macrophage polarization via cPLA2-COX-2 inhibition, activating ROS production against Leishmania mexicana.

Defence against Leishmania depends upon Th1 inflammatory response and, a major problem in susceptible models, is the turnoff of the leishmanicidal activity of macrophages with IL-10, IL-4, and COX-2 upregulation, as well as immunosuppressive PGE2, all together inhibiting the respiratory burst. Peroxisome proliferator-activated receptors (PPAR) activation is responsible for macrophages polarization on Leishmania susceptible models where microbicide functions are deactivated. In this paper, we demonstrated that, at least for L. mexicana, PPAR activation, mainly PPAR γ , induced macrophage activation through their polarization towards M1 profile with the increase of microbicide activity against intracellular pathogen L. mexicana. PPAR activation induced IL-10 downregulation, whereas the production of proinflammatory cytokines such as TNF- α , IL-1 ß , and IL-6 remained high. Moreover, PPAR agonists treatment induced the deactivation of cPLA2-COX-2-prostaglandins pathway together with an increase in TLR4 expression, all of whose criteria meet the M1 macrophage profile. Finally, parasite burden, in treated macrophages, was lower than that in infected nontreated macrophages, most probably associated with the increase of respiratory burst in these treated cells. Based on the above data, we conclude that PPAR agonists used in this work induces M1 macrophages polarization via inhibition of cPLA2 and the increase of aggressive microbicidal activity via reactive oxygen species (ROS) production.

CD4+ CD25+ FOXP3+ Treg cells induced by rSSP4 derived from T. cruzi amastigotes increase parasitemia in an experimental Chagas disease model.

Currently, there is a considerable controversy over the participation of Treg cells during Trypanosoma cruzi infection, the main point being whether these cells play a negative or a positive role. In this work, we found that the adoptive transfer of CD4(+)CD25(+)FOXP3(+) T cells from rSSP4- (a recombinant Trypanosoma cruzi amastigote derived protein, previously shown to have immunomodulatory properties on macrophages) immunized BALB/c donors into syngenic recipients simultaneously with T. cruzi challenge reduces cardiac inflammation and prolongs hosts' survival but increases blood parasitemia and parasite loads in the heart. These CD4(+)CD25(+)FOXP3(+) Treg cells from immunized mice have a relatively TGF-ß-dependent suppressive activity on CD4(+) T cells. Therefore, regulatory CD4(+)CD25(+) T cells play a positive role in the development of acute T. cruzi infection by inducing immunosuppressive activity that controls early cardiac inflammation during acute Chagas disease, prolonging survival, but at the same time promoting parasite growth.

Differential immune response in mice immunized with the A, R or C domain from TcSP protein of Trypanosoma cruzi or with the coding DNAs.

In a murine model of experimental Trypanosoma cruzi (H8 strain) infection, we investigated the induction of protective immunity against the domains [amino (A), repeats (R) and carboxyl (C)] of the surface protein (SP), a member of the trans-sialidase (TS) superfamily. Recombinant proteins and plasmid DNA coding for the respective proteins were used to immunize BALB/c mice, and the humoral response and cytokine levels were analysed. Immunization with the recombinant proteins induced higher levels of anti-TcSP antibodies than immunization with the corresponding DNAs, and analysis of serum cytokines showed that immunization with both recombinant proteins and naked DNA resulted in a Th1-Th2 mixed T-cell response. Mice immunized with either recombinant proteins or plasmid DNA were infected with blood trypomastigotes. The recombinant protein-immunized mice showed a variable reduction in peak parasitemia, and most died by day 60. Only the pBKTcSPR-immunized mice exhibited a significant reduction in peak parasitemia and survived the lethal challenge. DNA-based immunization with DNA coding for the repeats domain of TcSP is a good candidate for the development of a vaccine against experimental T. cruzi infection.

Src and PI3 K inhibitors affect the virulence factors of Entamoeba histolytica.

Protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. A large number of protein kinases within the protein kinome of Entamoeba histolytica strongly suggest that protein phosphorylation is a key component of pathogenesis regulation by this parasite. PI3 K and Src are kinases previously described in this parasite, but their role is poorly understood. Here, the effect of Src-1-inhibitor and PI3 K inhibitor (Wortmannin) on the virulence factors of E. histolytica was evaluated. Results show that both inhibitors affect the actin cytoskeleton and the amoebic movement. Also, the proteolytic activity is diminished by Wortmannin, but not by Src-inhibitor-1; however, the phagocytic capacity is diminished by Wortmannin and Src-1-inhibitor. Finally, we found that the virulence in vivo of E. histolytica is affected by Wortmannin but not by Src-1-inhibitor. This study opens the way for the design of anti-amoebic drugs based on kinase inhibition.

Rab7 and actin cytoskeleton participate during mobilization of ß1EHFNR in fibronectin-stimulated Entamoeba histolyticatrophozoites.

In vitro interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces redistribution of the amoebic fibronectin receptor (ß1EhFNR). Trafficking of beta1 integrins is important for cell adhesion and migration in higher eukaryotes and requires the participation of Rab proteins. In E. histolytica, the machinery involved in integrin trafficking is not completely known. EhRab7 is a 24.5-kDa protein whose alignment with other Rab7 proteins demonstrated that it shared significant homology with Rab7 proteins from other organisms, including humans. Using different types of microscopy (fluorescence and confocal microscopy), it was established that Rab7 and the actin cytoskeleton participated in the mobilization of ß1EhFNR in FN-stimulated trophozoites. ß1EhFNR and Rab7 co-localized only in vesicular structures at 5 min, and at longer time (1 h), both co-localized in both plasma membrane and in vesicular structures; at the same time, Rab7 co-localized with specific actin structures (phagocytic vacuoles). At 5 h the ß1EhFNR, Rab7, and actin co-localized at the plasma membrane, and only ß1EhFNR and Rab7 decorated vesicles of different sizes. Actin and Rab7 co-localized in a cap-like structure at one end of the cell. Fluorescence resonance energy transfer and electron microscopy confirmed the close interaction between ß1EhFNR and Rab7. Moreover, the use of a lysosome-specific marker (LysoTracker) and a Golgi-specific marker (NBD C(6)-ceramide) allowed us to establish that, at some point within the endocytic route, ß1EhFNR and Rab7 co-localized within a lysosome-type organelle, but not a Golgi-like organelle, which indicated that this integrin-like molecule was returned to the plasma membrane via exocytic or secretory vesicles.

Absence of CD38 delays arrival of neutrophils to the liver and innate immune response development during hepatic amoebiasis by Entamoeba histolytica.

To define the role of CD38 in the migration of neutrophils to the liver and consequently in the induction of an innate immune response during murine hepatic amoebiasis by Entamoeba histolytica, we examined amoebic liver abscess development (ALA), presence of amoebae and neutrophils, and expression levels of cytokines and other inflammation mediators mRNA, in infected wild-type and CD38 Knockout (CD38KO) C57BL/6J mice. Results showed that CD38KO mice undergo a delay in ALA development in comparison with the wild-type strain. The presence of amoebae lasted longer in CD38(-/-), and although neutrophils arrived to the liver in both strains, there was a clear difference in the time between the two strains; whereas in the wild-type strain, neutrophils arrived at early times (6-12 h), in the CD38KO strain, neutrophils arrived later (48-72 h). Cytokines profile during the innate immune response development (TNF-α, IL-1ß, IL-6) was, for WT mice concomitant with, and preceded, for CD38KO mice, the time in which neutrophils were present in the liver lesion. In conclusion, CD38 is important for neutrophils migration during hepatic amoebiasis, and in turn, these cells play an important role in the innate immune response.

Subcellular distribution of the Entamoeba histolytica 140 kDa FN-binding molecule during host-parasite interaction.

Entamoeba histolytica trophozoites recovered from the host-parasite interface during abscess development obtain different stimuli compared with long-term cultured cells. In order to have a better understanding about the mechanisms in which the 140 kDa fibronectin (FN)-binding molecule (EhFNR) is involved during the invasive process, we decided to compare the regulation process of this molecule among long-term cultured trophozoites, FN-stimulated trophozoites, and trophozoites recently recovered from a liver abscess. A cDNA clone (5A) containing a fragment of the EhFNR that shows identity to the C-terminal region of the intermediate galactose lectin subunit Igl, was selected with a mAb (3C10). Identity of EhFNR with Igl was confirmed by immunoprecipitation with 3C10 and EH3015 (against the Gal/GalNAc intermediate subunit) mAbs. The 3C10 mAb was used as a tool to explore the modulation of the amoebic receptor (EhFNR). Our results showed specific regulation of the EhFNR in FN-interacted amoebas, as well as in trophozoites recovered at different stages of abscess development. This regulation involved mobilization of the receptor molecule from internal vesicles to the plasma membrane. Therefore, we suggest that in the host-parasite interface, the EhFNR (Igl) plays an important role in the adhesion process during abscess development.

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.

Cytoskeleton disruption in chondrocytes from a rat osteoarthrosic (OA) -induced model: its potential role in OA pathogenesis.

Morphological and functional changes of chondrocytes are typical in OA cartilage. In this work, we have described noteworthy changes in intermediate filaments cytoskeleton evidenced by transmission electron microscopy. Alterations in the distribution as well as in the content of vimentin, actin, and tubulin have been described by specific fluorescence labelling of each cytoskeletal component and confocal analysis. Normal vs OA cartilages showed a reduction in the percentage of labelled chondrocytes of 37.1% for vimentin, 4.7% for actin, and 20.1% for tubulin. Statistical analysis of fluorescence intensities (mean % +/- SEM) between normal and OA rat cartilage revealed a highly significant difference in vimentin, a significant difference in tubulin, and a non-significant difference in actin. Moreover, by western blot, altered electrophoretic patterns were observed mainly for vimentin and tubulin in OA cartilage in comparison with normal cartilage. These results allow us to suggest that substantial changes in vimentin and tubulin cytoskeleton of chondrocytes might be involved in OA pathogenesis.

Entamoeba histolytica: induction of cyclooxygenase-2 expression during amoebic liver abscess formation in hamsters (Mesocricetus auratus).

Experimental amoebic liver abscess in hamsters curses with an increase in both, systemic levels of prostaglandin E2 (PGE(2)) and local cyclooxygenase activity in liver microsomes. The cellular source of PGE(2) and the isoform of cyclooxygenase responsible are not completely evidenced. Cyclooxygenase-2 (COX-2) protein and gene expression were demonstrated on macrophages and polymorphonuclear cells as a result of Entamoeba histolytica infection in hamsters at 2, 4, and 7 days postinfection by immunohistochemistry and RT-PCR. E. histolytica trophozoites located in the lesion showed a strong positive signal for COX-2, however the enzyme was not detected in cultured trophozoites by Western blot. Our results indicate that the increment in PGE(2) is the result of COX-2 activity from cells of the reticuloendothelial system and reinforce the possibility that PGE(2) production by enzyme induction in macrophages may be a mechanism by which E. histolytica modulates the host immune response in this parasitic infection.

Recombinant SSP4 protein from Trypanosoma cruzi amastigotes regulates nitric oxide production by macrophages.

Acute infection with Trypanosoma cruzi is characterized by immunosuppression mediated by T cells and macrophages (Mphis). Nitric oxide (NO) production during the initial phase of acute infection might participate in the clearance of parasites by Mphis, whereas its overproduction during the late phase of acute infection would account for the immunosuppression observed. Trypanosoma cruzi molecules that might regulate the host responses have not been fully identified. Here, we demonstrate that active immunization with MBP::SSP4, a recombinant protein derived from a surface antigen specific of T. cruzi amastigotes (TcSSP4), was able to stimulate Ab production (IgG1, IgG2a, and IgG2b). On the other hand, MBP::SSP4 was able to stimulate NO production by peritoneal Mphis from BALB/c mice and Mphis from the J774 cell line. This effect was also observed at the level of inducible nitric oxide synthase (iNOS) detected by Western Blot. Furthermore, MBP::SSP4 was also shown to induce the expression of IL-1alpha, IL-6, IL-12, IFN-gamma, and TNF-alpha in normal animals, and IL-10 in immunized animals. In addition the protein MBP::SSP4 was able to bind to the surface of PMphis and J774 Mphis. These results suggest that TcSSP4 could modulate Mphi NO production and this may represent a mechanism participating in the immunoregulatory processes during Chagas' disease.

An ecto-protein tyrosine phosphatase of Entamoeba histolytica induces cellular detachment by disruption of actin filaments in HeLa cells.

Actin cytoskeleton disruption in host cells has been demonstrated for PTPases from pathogenic microorganisms. In this work, we analysed whether the secreted acid phosphatase from Entamoeba histolytica has phosphotyrosine phosphatase activity and the possibility that this activity may participate in damaging host cells. The secreted acid phosphatase of E. histolytica, which catalyses p-nitrophenyl phosphate hydrolysis at acid pH values, was found to have phosphotyrosine phosphatase activity. The enzymatic properties of phosphotyrosine phosphatase and acid phosphatase were virtually identical and included: Km values of 10 x 10(-4) M, no requirement for divalent cations, and sensitivity to molybdate, vanadate, and tungstate. The phosphotyrosyl phosphatase activity caused significant levels of cell rounding and detachment correlating with disruption of the actin stress fibres in HeLa cells. Thus, our data suggest that secreted phosphotyrosine phosphatase could play a cytotoxic role during amoebic infection.

Membrane-bound acid phosphatase (MAP) from Entamoeba histolytica has phosphotyrosine phosphatase activity and disrupts the actin cytoskeleton of host cells.

Protein tyrosine phosphatases (PTPases) have been described as virulence factors in different pathogenic microorganisms. The pathogenic process by Enatamoeba histolytica is a multifactorial phenomenon that occurs in 3 steps: adhesion, cytolytic and cytotoxic effect, and phagocytosis. Lytic enzymes may participate during the second part of this process. In this work, we determined that purified membrane-bound acid phosphatase (MAP) from E. histolytica trophozoites has PTPase activity. The enzyme specifically dephosphorylated O-phospho-L-tyrosine at optimum pH of 5.0, with little activity towards O-phospho-L-serine, O-phospho-L-threonine, and ATP. It was inhibited by ammonium molybdate and sodium tungstate, and trifluoperazine did not show any effect. A monoclonal antibody against the catalytic domain of the human placental PTPase 1B, cross-reacted with a 55 kDa molecule present in the solubilized fraction. The interaction of the amoebic PTPase with HeLa cells resulted in the alteration of the cell actin cytoskeleton by disruption of the actin stress fibres.