Trichomonas vaginalis exosome-like vesicles modify the cytokine profile and reduce inflammation in parasite-infected mice.

Trichomonas vaginalis (Tv) is a flagellated parasite commonly spread through sexual transmission. This protozoan initiates a severe inflammatory process, inducing nitric oxide, interleukin-6 (IL-6), IL-8, IL-10, IL-17 and IL-22 production by host immune cells. The parasites elicit these responses by releasing surface lipophosphoglycan, small extracellular vesicles (exosomes) and other factors. Tv exosomes are similar to mammalian exosomes and have been implicated in the modulation of IL-8 secretion by epithelial cells. Here, we report that exosome-like vesicles from T. vaginalis (Tv-ELVs) induced a more than 15-fold increase in IL-10 expression in RAW264.7 macrophages but only a two fold increase in IL-6 and TNF-α expression levels measured by RT-PCR. Because Tv-ELVs modulated the macrophage response, we also explored the effect of Tv-ELVs in a murine model of infection. Pretreatment with Tv-ELVs significantly increased IL-10 production as measured in vaginal washes by days 8 and 16 post-infection. Remarkably, Tv-ELVs-pretreated mice exhibited a decrease in IL-17 production and a significant decrease in vulvar inflammation. In addition, IL-6 and IL-13 were decreased during infection. Our results suggest that Tv-ELVs have an immunomodulatory role on the cytokine profile induced by the parasite and promote a decrease in the inflammatory process in mice infected with T. vaginalis.

Recognition of carbohydrate epitopes specific for electron-dense granule antigens from Entamoeba histolytica by monoclonal antibodies in the cecal content of infected hamsters.

The pathogenic trophozoites of Entamoeba histolytica produce and secrete electron-dense granules (EDG) containing collagenase, considered a virulence factor. Two monoclonal antibodies (MAbs) (L7.1 and L1.1) anti-EDG antigens were raised. MAb L7.1 has been reported to recognize proteic EDG antigens and MAb L1.1 reacted with a carbohydrate epitope. These epitopes were present in axenic and xenic amoebas. To detect EDG antigens by a enzyme-linked immunosorbent assay (ELISA) in a experimental model of early intestinal amoebiasis, both MAbs were employed. E. histolytica HM1 axenic and monoxenic trophozoites were inoculated into the cecum according to the washed-closed cecal loop technique. The cecal content was recovered at 8, 24, and 48 h post-inoculation. Antigens from EDG in whole trophozoites and cell-free supernatants were detected. Our results indicate that it is possible to detect EDG antigens in the cecal content of hamsters in the early phase of the invasive amoebiasis.

Entamoeba histolytica: surface proteolytic activity and its relationship with in vitro virulence.

We determined the surface-associated proteolytic activity in three Entamoeba histolytica Schaudinn, 1903 strains (monoxenic HM1, axenic HM1, and HK9) of known virulence and its relationship with collagenase activity. Both activities were also determined in axenic HM1 amoebae trophozoites which were sensitive and resistant to complement-mediated lysis. Surface proteolytic activity was determined in glutaraldehyde-fixed E. histolytica trophozoites, which degraded the insoluble substrate, hide powder azure, and cleaved the human immunoglobulin G heavy chain in a time-dependent fashion, at neutral pH, in presence of 2-mercaptoethanol as cysteine protease activator. Surface proteolytic activity was strain dependent: monoxenic HM1 > axenic HM1 > axenic HK9. This activity correlated with collagenolytic activity (p < 0.05). Acquisition of resistance to complement-mediated lysis by axenic HM1 strain did not modify either surface proteases or collagenase expression. Our results suggest that this surface proteolytic activity could be used as an in vitro virulence marker for E. histolytica.

Expression of in vitro virulence by Entamoeba histolytica: effect of calmodulin inhibitors.

The possible role of calmodulin (CaM) in functions such as cytotoxicity, phagocytosis, and amoebic growth was studied. These are important factors for the in vitro pathogenicity of Entamoeba histolytica HM1 strain. The CaM inhibitors trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) and N-(6-aminohexyl)-naphthalenesulfonamide (W-5) were used for these purposes. Cytotoxicity was determined as the ability of amoebae to kill and/or lyse nucleated mouse spleen cells. Phagocytosis by amoebae was assessed by calculating the number of endocytosed human red blood cells. Cytotoxicity by E. histolytica showed a decrease dependent upon the concentration of W-7 and TFP, whereas W-5 did not show a significant inhibiting effect. Phagocytosis was roughly sensitive to W-7; TFP and W-5 did not have any significant effect. Amoebic growth was sensitive to TFP and W-7 CaM inhibitors. These results suggest that CaM participates in the expression of in vitro cytotoxicity of E. histolytica.

Proinflammatory cytokines regulate cyclooxygenase-2 mRNA expression in human macrophages.

Prostaglandins play a major role in the inflammatory and immune response. Macrophages constitutively produce prostaglandins by the enzyme cyclooxygenase-1 (cox) whereas inflammatory mediators and cytokines stimulate the inducible enzyme cox-2 for high output prostaglandin production. In this study, we investigated the effect of LPS, IFN-gamma and TNF-alpha in the regulation of cox-1 and cox-2 mRNA expression in PMA-differentiated U937 human macrophages. LPS, but not IFN-gamma or TNF-alpha, caused the induction of cox-2 mRNA in a dose- and time-dependent fashion. In IFN-gamma-primed macrophages, LPS stimulated a marked increase in the accumulation of cox-2 mRNA, whereas TNF-alpha and IFN-gamma induced a moderate level. However, IFN-gamma in combination with either LPS or TNF-alpha induced a synergistic increase in the accumulation of cox-2 mRNA after 24 hours. Regardless of the conditions, cox-1 mRNA remained unchanged. These results indicate that IFN-gamma priming is required for full expression of cox-2 mRNA in response to LPS or TNF-alpha stimulation and suggest that cox-2 mRNA is highly regulated by cytokines during macrophage activation.

Biochemical analysis of Entamoeba histolytica HM1 strain resistant to complement lysis.

In this study, quantitation of several hydrolases and analysis of electrophoretic patterns of E. histolytica HM1 axenic strain periodically exposed to lytic-human serum (lytic-HS), heat-inactivated human (HI-HS) or bovine (HI-BS) sera were performed. The N-acetylglucosaminidase (NAGAse) levels increased in amebas resistant to complement. The acid phosphatase (AP) and proteolytic activity (PA) levels did not vary significantly in all groups of treated amebas, as compared with amebas from axenic cultures. Moreover, amebas exposed to either HI-HS or lytic-HS sera showed a discrete quantitative increase as compared with HI-BS group, in some peptides with a M(r) between 116 and 45 kDa. These results indicate that, besides resistance to complement lysis, exposure of E. histolytica to lytic-HS induces an augmentation in NAGAse levels.

Entamoeba histolytica: a simplified method to quantify its cytotoxicity.

A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized. Mice spleen leucocytes were utilized as target cells. Interaction time was reduced to 1 h by pelleting interacting cells. To assess target-cell killing by amoebae, a nigrosine exclusion test was employed. Fixation with glutaraldehyde stabilized the percentage of stained target cells. Similar results were obtained when cytotoxicity of the E. histolytica HM1 strain was tested by the traditional and proposed methods. The new method allowed quantification of the contribution of cytolysis and cytophagocytosis to amoebic cytotoxicity. It was also demonstrated that uncloned E. histolytica HM1 strain is a heterogeneous population with respect to cytotoxicity expression.

In vitro activity of nalidixic acid and its iron (III) complex on Entamoeba histolytica.

The in vitro activity of the antibacterial agent nalidixic acid (HNal) and its iron (III) complex (FeNal) against Entamoeba histolytica HM1 strain trophozoites in axenic or monoxenic (associated with Clostridium symbiosum) cultures was investigated. Using a dilution test with TYI-S-33 medium, this protozoan was found to be susceptible to both drugs, but FeNal showed amoebicidal activity only at concentrations higher than those used with HNal.

Entamoeba histolytica: collagenolytic activity and virulence.

Several axenic strains of pathogenic and nonpathogenic Entamoeba histolytica were tested for their capacity to digest native radioactive type I collagen gels and to produce liver abscesses when injected into the liver of newborn hamsters. The results demonstrate that the pathogenic strains of amebas (HM1:IMSS, HM3:IMSS, HM38:IMSS and HK9) have a collagenolytic activity that closely correlates with their in vivo capacity to produce liver lesions. The nonpathogenic isolate (Laredo) did not show collagenolytic activity and failed to produce lesions in the liver of newborn hamsters. The results also demonstrate that type I collagen obtained from rodents and cats is degraded less by amebic collagenase than is bovine collagen, which is similar to human collagen. These findings suggest that species susceptibility to invasive infection may depend, among other factors, on the characteristics of the extracellular components of host tissues.

Actividad citotoxica de Entamoeba histolytica sobre celulas linfoides de bazo de raton. / Cytotoxic activity of Entamoeba histolytica on lymphoid cells of rat spleen

Se analizo la capacidad citotoxica de Entamoeba histolytica empleando el metodo de liberacion de 51Cr de celulas linfoides de bazo de raton como celulas blanco. Se utilizaron trofozoitos de las cepas HM1 y HK-9 y de la clona 2C1 obtenida esta en nuestro laboratorio a partir de la cepa HM1. Las celulas linfoides de bazo de raton se marcaron con 51Cr y se mezclaron con los trofozoitos de las diferentes cepas en el medio TYI-S-33 para lisis. Se calculo el porcentaje de lisis especifica a partir de la medicion de 51 Cr liberado al sobrenadante. La actividad citotoxica en los trofozoitos se determino en las diferentes fases de la curva de crecimiento. Se observo que la actividad citotoxica es dependiente de la cepa utilizada, de la fase de crecimiento y de la proporcion de amiba por celula blanco probada. La actividad citotoxica medida fue mayor para la cepa HM1 y menor para la cepa HK-9