Acanthamoeba castellanii: Effect of neuroactive substances on trophozoite migration.

Acanthamoeba castellanii is the etiological agent of granulomatous amebic encephalitis, amebic keratitis, and skin lesions. In vitro and in vivo studies have demonstrated that Acanthamoeba trophozoites induce contact-dependent, and contact-independent pathogenic mechanisms. We have explored the potential role neuroactive substances may have in the migration of Acanthamoeba castellanii trophozoites using Transwell permeable supports in the presence of physiological concentrations of dopamine, glutamate, serotonin, or taurine diluted in PBS. Quantitation of migrated amoebae was carried out in scanning electron micrographs of the upper and under compartments sides of the chamber membranes. Our results showed that at 2 h of interaction, a statistically significant larger proportion of A. castellanii trophozoites migrated through the chamber membranes when neurotransmitters were placed in the lower compartments of the chambers compared to control. This migration effect was more evident under the presence of glutamate and taurine on the three surfaces (upper/lower membrane and bottom compartment) when the percentage of migrated trophozoites was analyzed. Scanning electron microscopy of trophozoites revealed that glutamate and taurine induced the formation of large adhesion lamellas and phagocytic stomas. These observations suggest that certain neuroactive substances could stimulate the migration of A. castellanii trophozoites in the central nervous system.

T-Cell Response against Varicella Zoster Virus in Patients with Multiple Sclerosis during Relapse and Remission.

An association between varicella zoster virus (VZV) and multiple sclerosis (MS) has been reported in Mexican populations. The aim of this study was to compare the response of T cells from MS patients, during relapse and remission, to in vitro stimulation with VZV, adenovirus (AV) and Epstein-Barr virus (EBV). Proliferation and cytokine secretion of T cells from 29 relapsing-remitting MS patients and 38 healthy controls (HC) were analyzed by flow cytometry after stimulating with VZV, AV or EBV. IgG and IgM levels against VZV and EBV were quantified using Enzyme-Linked Immunosorbent Assay. Relapsing MS patients showed a higher percentage of responding CD4+ and CD8+ T cells against VZV compared to AV. In HC and remitting MS patients, proliferation of CD4+ T cells was higher when stimulated with VZV as compared to EBV. Moreover, T cells isolated from remitting patients secreted predominantly Th1 cytokines when cell cultures were stimulated with VZV. Finally, high concentration of anti-VZV IgG was found in sera from patients and controls. The results support previous studies of an VZV-MS association in the particular population studied and provide additional information about the possible role of this virus in the pathogenesis of MS.

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.

Giardia lamblia: Identification of peroxisomal-like proteins.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.

Acanthamoeba culbertsoni isolated from a clinical case with intraocular dissemination: Structure and in vitro analysis of the interaction with hamster cornea and MDCK epithelial cell monolayers.

Acanthamoeba culbertsoni trophozoites, previously isolated from a human keratitis case with severe intraocular damage, were maintained in axenic culture. Co-incubation of amoebae with MDCK cell monolayers demonstrated an apparent preference of the amoebae to introduce themselves between the cells. The trophozoites appeared to cross the cell monolayer through the tight junctions, which resulted in decreased trans-epithelial resistance (TER) measurements. Unexpectedly, after co-incubation of amoebae with hamster corneas, we observed that the trophozoites were able to cross the different cell layers and reach the corneal stroma after only 12 h of interaction, in contrast to other Acanthamoeba species. These observations suggest that this A. culbertsoni isolate is particularly pathogenic. Further research with diverse methodologies needs to be performed to explain the unique behavior of this Acanthamoeba strain.

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.

Acanthamoeba culbertsoni: Electron-Dense Granules in a Highly Virulent Clinical Isolate.

The virulence of various amoebic parasites has been correlated with the presence of electron-dense granules (EDGs) in the cytoplasm of trophozoites. Here, we report the finding by transmission electron microscopy of a large number of EDGs in a recent culture of Acanthamoeba culbertsoni, isolated from a severe case of human keratitis. When this isolate was maintained in culture for 6 mo, the granules almost disappeared. However, after induction of mice brain lesions with the long-term cultured isolate, recovered amoebas had abundant EDGs. Trophozoites of the original isolate, or those recovered from experimental lesions, secreted EDGs into the medium when incubated with MDCK cells. To analyze a possible cytotoxic effect the conditioned medium was incubated with MDCK monolayers. After 5 h, the media containing EDGs produced opening of the tight junctions; at 24 h, cell viability was compromised, and at 48 h most of the cells were detached from the monolayer. In contrast, trophozoites in long-term cultures did not release EDGs to the medium during incubation with MDCK cells, and the corresponding conditioned medium did not have any effect on MDCK monolayers. Our observations further support the hypothesis that EDGs play a role in the cytopathogenic mechanisms of A. culbertsoni.

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.

Acanthamoeba genotypes T3 and T4 as causative agents of amoebic keratitis in Mexico.

Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection.

Balamuthia mandrillaris: Further morphological observations of trophozoites by light, scanning and transmission electron microscopy.

Additional morphological features of Balamuthia mandrillaris observed by light and electron microscopy are reported. Trophozoites were extremely pleomorphic: their cell shapes ranged from rounded to elongated and sometimes they appeared exceptionally stretched out and branched. By transmission electron microscopy it was possible to observe two different cytoplasmic areas, the ectoplasm and the endoplasm and often sections of rough endoplasmic reticulum were found in the transition zone. The cytoplasm was very fibrogranular and most of the organelles typically found in eukaryotic cells were observed. A particular finding was the presence of numerous mitochondria with a different structure from those of other free-living amoebae. The observations reported here may reinforce the morphological knowledge of this amoeba and provide a background for further analyses.

Naegleria fowleri: contact-dependent secretion of electrondense granules (EDG).

The free living amoeba Naegleria fowleri is pathogenic to humans but also to other mammalians. These amoebae may invade the nasal mucosa and migrate into the brain causing cerebral hemorrhagic necrosis, a rapidly fatal infection. Knowledge of the cytolytic mechanism involved in the destruction of brain tissues by Naegleria trophozoites is limited. In other amoebic species, such as Entamoeba histolytica, we have previously reported the possible lytic role of small cytoplasmic components endowed with proteolytic activities, known as electrondense granules (EDG). Using transmission electron microscopy we now report that EDG, seldom found in long term cultured N. fowleri, are present in abundance in trophozoites recovered from experimental mice brain lesions. Numerous EDG were also observed in amoebae incubated with collagen substrates or cultured epithelial cells. SDS-PAGE assays of concentrated supernatants of these trophozoites, containing EDG, revealed proteolytic activities. These results suggest that EDG may have a clear role in the cytopathic mechanisms of this pathogenic amoeba.

Silicone hydrogel contact lenses surface promote Acanthamoeba castellanii trophozoites adherence: qualitative and quantitative analysis.

PURPOSE: To describe the adhesion properties of Acanthamoeba castellanii trophozoites to silicone hydrogel contact lenses of first generation (lotrafilcon A), second generation (galyfilcon A), and third generation (comfilcon A) and correlate the results with their specific surface characteristics, time of interaction, and suspension media. METHODS: Qualitative and quantitative assessments of the adhesion of 200 trophozoites of A. castellanii on contact lenses in culture medium (Bacto Casitone) and isotonic saline (IS) at different time points (15 minutes and 6 hours) were determined. RESULTS: By scanning electron microscopy, A. castellanii trophozoites were observed firmly adhered to the surface of hydrogel lenses after 15 minutes of interaction. The surface of lotrafilcon A lenses on which amoebae adhere better (16.4±10.2 amoebae/lens section) is rough and folded, which increases the contact surface with trophozoites, allowing acanthopodia to attach firmly. Contrarily, galyfilcon A lenses have a smoother surface, and lower numbers of amoebae were observed adhered to these lenses (4.7±2.9 amoebae/lens section). Even fewer amoebae adhered to the smoother surface of the comfilcon A lens (2.2±1.7 amoebae/lens section). Trophozoites showed similar behavior in both Bacto Casitone medium and IS. CONCLUSION: A rough surface may contribute to better adhesion of amoebae to silicone hydrogel lenses. Although a reduced numbers of trophozoites adhered to smooth lenses, trophozoites are a risk factor for amoebic keratitis. Isotonic saline facilitated trophozoite survival, suggesting that homemade saline solutions may contribute to the persistence of trophozoites, especially when there is no proper hygiene regimen used with the contact lens cases.

Acanthamoeba royreba: morphological features and in vitro cytopathic effect.

Observations on cultured Acanthamoeba royreba trophozoites and in vitro cytopathogenicity of this amoeba are described. In culture, amoebae were active, pleomorphic and moved on the substrate by producing endocytic structures and emitting slight cytoplasmic microprojections from the cell surface. These projections were formed by hyaline cytoplasm and they were related to motion structures such as acanthopodia and lamellipodia, in which actin provides a framework that allows rapid changes in morphology. In the cytoplasm abundant vacuoles of different size and content were seen. By means of electron microscopy, it was possible to observe the compact fibrogranular appearance of the cytoplasm, along with the main cellular organelles such as the Golgi complex, the endoplasmic reticulum, digestive vacuoles, mitochondria and contractile vacuoles. Incubation of MDCK epithelial cell monolayers with conditioned medium did not produce a significant structural damage to the monolayer, even after 24h of incubation. When the trophozoites were incubated with the target cells the monolayer exhibited a clear injury created by the amoebae, which produced focal damage. Nevertheless, the rest of the monolayer appeared to remain intact, suggesting that a contact-dependent interaction is necessary to damage the target cells. These observations demonstrate the low invasive capacity of this amoeba.

Acanthamoeba castellanii cysts: new ultrastructural findings.

During Acanthamoeba castellanii trophozoite-cysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst, immature cysts, and mature cysts. Fluorescence microscopy reveals the presence of small cumulus of actin in the cytoplasm of precysts after treatment with rhodamine phalloidin. By the contrary, in mature cysts, fluorescence was not observed. However, when excystation was induced, large fluorescent patches were present. By transmission electron microscopy, encysting amebas showed small cytoplasmic vesicles containing fibrillar material, surrounded by a narrow area of thin fibrils. Similar appearance was observed in pseudopods and phagocytic invaginations. In addition, large aggregates of rod-shape elements, similar to the chromatoid bodies, described in other amebas, were present in the cytoplasm. These cysts presented large areas with orange fluorescence after treatment with acridine orange.

Vahlkampfia sp: structural observations of cultured trophozoites.

Some structural observations on cultured Vahlkampfia sp. trophozoites are reported. Trophozoites are active and pleomorphic, producing large cell protrusions related to locomotion such as lamellipodia, filopodia and endocytic structures formed by hyaline cytoplasm, in which actin provides a framework that allows rapid changes in morphology. As observed by transmission electron microscopy, the cytoplasm is highly granular masking some cell organelles and the major cytoplasmic membrane systems. The structure of cell organelles such as the nucleus, endoplasmic reticulum, and digestive vacuoles is described. A common finding was the presence of 50 nm electron-dense round granules that are not limited by a membrane and that appear scattered in the cytoplasm, and whose function remains unknown. Apparently, the cell reserve material is glycogen, since complete trophozoites were positive to Schiff periodic-acid technique.

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.

[Health research]. / La investigación en salud.

Research in Mexico has had an important advance, including health research. However, despite great efforts to include medical research in decision making, it is necessary to strengthen existing actions and programs, generating new research agendas in health.

High-resolution electron microscopical study of cyst walls of Entamoeba spp.

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.

Naegleria fowleri: enolase is expressed during cyst differentiation.

Cysts of Naegleria fowleri present an external single-layered cyst wall. To date, little information exists on the biochemical components of this cyst wall. Knowledge of the cyst wall composition is important to understand its resistance capacity under adverse environmental conditions. We have used of a monoclonal antibody (B4F2 mAb) that specifically recognizes enolase in the cyst wall of Entamoeba invadens. By Western blot assays this antibody recognized in soluble extracts of N. fowleri cysts a 48-kDa protein with similar molecular weight to the enolase reported in E. invadens cysts. Immunofluorescence with the B4F2 mAb revealed positive cytoplasmic vesicles in encysting amebas, as well as a positive reaction at the cell wall of mature cysts. Immunoelectron microscopy using the same monoclonal antibody confirmed the presence of enolase in the cell wall of N. fowleri cysts and in cytoplasmic vesicular structures. In addition, the B4F2 mAb had a clear inhibitory effect on encystation of N. fowleri.

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.