Destruction Of Monolayer Research Articles

Study of the Antiviral Activity and Toxicity of Dextrin - In Vitro Study

Objective: This study was to in vitro study of the antiviral effect of "Dextrin" on the replication of the EMCV virus in HeLa cells and toxicity of "Dextrin". Methods: For this purpose, the virus at a dose of 100 TCD50 is applied to a 24-hour monolayer of HeLa cells. The experiment is carried out under conditions of 5% CO2 saturation and 100% humidity. The virus dose used was expressed as a tissue cytopathic dose of 100 TCD50/ml. Cultivation of HeLa cells, to study toxicity, was performed on 96-well plates under conditions of 5% CO2 saturation and 100% humidity. The toxic effect of "Dextrin" on HeLa cells was studied by the degree of destruction of the monolayer and at the population level. Results: Peak levels of the virus reached after 48 hours of replication accompanied by complete destruction of the cell monolayer (7-8 cycles of replication). As follows from the graph, “Dextrin significantly” (significance is indicated by asterisks.) reduces the dose of the virus when introduced into the medium with HeLa cells to, or at the early stages of EMCV virus replication (-1,0 and 1 hour). Based on the data obtained, it was decided to use a dose of 1:100 (1%), which is the highest non-toxic dose. Conclusion: “The investigated Dextrin has an antiviral effect against the EMCV virus, when applied in the early stages of infection. This may open up the prospect of studying the antiviral spectrum of Dextrin in more future studies”.

FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration

BackgroundForkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood–retina barrier of the immune privileged eye.MethodsWe investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1β and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing.ResultsRPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1β to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells.ConclusionOur data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.

EhRho6-mediated actin degradation in Entamoeba histolytica is associated with compromised pathogenicity.

Entamoeba histolytica causes amoebiasis which is a major health concern in developing countries. E. histolytica pathogenicity has been implicated to a large repertoire of small GTPases which switch between the inactive GDP bound state and the active GTP bound state with the help of guanine nucleotide exchange factors (GEFs) and GTPase activating protein (GAPs). Rho family of small GTPases are well known to modulate the actin cytoskeletal dynamics which plays a major role in E. histolytica pathogenicity. Here, we report an atypical amoebic RhoGEF, and its preferred substrate EhRho6, which, upon overexpression abrogated the pathogenic behavior of the amoeba such as adhesion to host cell, monolayer destruction, erythrophagocytosis, and formation of actin dots. A causative immunoblot analysis revealed actin degradation in the EhRho6 overexpressing trophozoites that could be inhibited by blocking the amoebic proteasomal pathway. A careful analysis of the results from a previously published transcriptomics study, in conjunction with our observations, led to the identification of a clade of Rho GTPases in this pathogenic amoeba which we hypothesize to have implications during the amoebic encystation.

Plant isoflavones can affect accumulation and impact of silver and titania nanoparticles on ovarian cells.

Objectives. The application of nanoparticles is experiencing a rapid growth, but it faces a problem of their toxicity, especially adverse effects on female reproduction. Food and medicinal plants and their isoflavones can be protectors against environmental stressors, but their ability to abate the adverse effects of nanoparticles has not been studied yet. In the present study, we examined the effect of silver (AgNPs) and titanium dioxide (titania, TiO2NPs) nanoparticles alone or in combination with plant phytoestrogens/antioxidants (resveratrol, diosgenin, and quercetin) on accumulation of nanoparticles, and progesterone release by cultured porcine ovarian granulosa cells.Methods. Porcine granulosa cells were incubated in the presence of AgNPs or TiO2NPs (0.1, 1, 10 or 100 µg/ml) alone or in combination with resveratrol, diosgenin or quercetin (10 µg/ml) for 48 h. The accumulation of tested nanoparticles by granulosa cells was assessed under light microscope. Progesterone concentration in culture media was measured by ELISA kit.Results. Cells accumulated both AgNPs and TiO2NPs in a dose-dependent manner. AgNPs, but not TiO2NPs, at highest dose (100 µg/ml) resulted in a destruction of cell monolayer. Both Ag-NPs and TiO2NPs reduced progesterone release. Resveratrol, diosgenin, and quercetin promoted accumulation of both AgNPs and TiO2NPs in ovarian cells and inhibited the progesterone output. Furthermore, resveratrol and diosgenin, but not quercetin, prevented the suppressive action of both AgNPs, and TiO2NPs on progesterone release.Conclusions. These observations (1) demonstrate accumulation of AgNPs and TiO2NPs in ovarian cells, (2) confirm the toxic impact of AgNPs, and TiO2NPs on these cells, (3) confirm the inhibitory effects of plant polyphenols/phytoestrogens on ovarian steroidogenesis, (4) show the ability of these isoflavones to increase the accumulation of AgNPs and TiO2NPs, and (5) show their ability to reduce the suppressive effect of AgNPs and TiO2NPs on ovarian progesterone release. The suppressive effect of AgNPs and TiO2NPs on ovarian functions should be taken into account by their exposition. However, these adverse effects could be mitigated by some plant isoflavones.

Entamoeba histolytica Interaction with Enteropathogenic Escherichia coli Increases Parasite Virulence and Inflammation in Amebiasis.

Epidemiological studies suggest frequent association of enteropathogenic bacteria with Entamoeba histolytica during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic Escherichia coli (strain DH5α) could modify the virulence of E. histolytica to cause disease in animal models of amebiasis. In vitro studies showed a 2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted with EPEC but not with E. coli DH5α for 2.5 h. This was associated with increased E. histolytica proteolytic activity as revealed by zymogram analysis and degradation of the E. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 substrate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP-A1, -A2, -A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked upregulation of E. histolytica virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inoculated with either E. histolytica-EPEC or E. histolytica-E. coli DH5α. Histopathology of livers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reactions, ischemia, and necrosis with high expression of il-1β, ifn-γ, and tnf-α proinflammatory cytokine genes compared with that in livers of E. histolytica-E. coli DH5α-inoculated animals. In closed colonic loops from mice, intense inflammation was observed with E. histolytica-EPEC manifested by downregulation of Math1 mRNA with a corresponding increase in the expression of Muc2 mucin and proinflammatory cytokine genes il-6, il-12, and mcp-1 These results demonstrate that E. histolytica/EPEC interaction enhanced the expression and production of key molecules associated with E. histolytica virulence, critical in pathogenesis and progression of disease.

Conditional reprogrammed human limbal epithelial cells represent a novel in vitro cell model for drug responses

In this study, we established human limbal epithelial cells (LECs) from normal limbal tissues by using Conditional Reprogramming (CR) technology (refer to CR-LEC cells in this study). We have successfully established CR-LEC cell strains from three human donors (3 out of 3), and normal rabbits (2 out of 2) and pig (1 out of 1) as well. CR-LEC cells sustained a continuous and stable proliferation status with a normal karyotype, normal response to DNA damage, well-defined structured spheres in matrigel 3D culture. Responses of CR-LEC cells to IFN α2b, Ganciclovir and 5-Fluorouracil were different, suggesting that these drugs had different toxicities to these cells as expected. More important, there was no significant difference of responses to drugs between early and late passages of CR-LEC cells (p>0.05), indicating CR-LEC cells can serve a stable normal human cell model for toxicity assessment. Toxicity tests with monolayer cultures of CR-LEC cells were measured by staining the F-actin and Dsg-1 expression. Toxicity of three drugs at LD50 concentration resulted in a gradually increased destruction of monolayer, which is, in accordance with the irritation grade of three drugs on human cornea epithelium. Therefore, CR-LEC cells provide a novel and reliable in vitro physiological cell model for corneal toxicity assessment.

Establishment of a cell-based wound healing assay for bio-relevant testing of wound therapeutics

IntroductionPredictive in vitro testing of novel wound therapeutics requires adequate cell-based bio-assays. Such assays represent an integral part during preclinical development as pre-step before entering in vivo studies. Simple “scratch tests” based on defected skin cell monolayers exist, however these can solely be used for testing liquids, as cell monolayer destruction and excessive hydration limit their applicability for (semi-)solid systems like wound dressings. In this context, a cell-based wound healing assay is introduced for rapid and predictive testing of wound therapeutics independent of their physical state in a bio-relevant environment. MethodsA novel wound healing assay was established for bio-relevant and predictive testing of (semi-) solid wound therapeutics. ResultsThe assay allows for physiologically relevant hydration of the tested wound therapeutics at the air-liquid interface and their removal without cell monolayer disruption. In a proof-of-concept study, the applicability and discriminative power could be demonstrated by examining unloaded and drug-loaded wound dressings with two different established wound healing actives (dexpanthenol and metyrapone) and their effect on skin cell behavior. The influence of the released drug on the cells´ healing behavior could successfully be monitored over time. Wound size assessment after 96h resulted in an eight fold smaller wound area for drug treated models compared to the ones treated with unloaded fibers and non-treated wounds. DiscussionThis assay provides valuable first insights towards the establishment of a valid screening and evaluation tool for preclinical wound therapeutic development from liquid to (semi-)solid systems to improve predictability in a simple, yet standardized way.

Cytotoxicity of Odorous Compounds from Poultry Manure.

Long-term exposure and inhalation of odorous compounds from poultry manure can be harmful to farm workers and the surrounding residents as well as animals. The aim of the present study was to determine the cytotoxicity and IC50 values of common odorous compounds such as ammonium, dimethylamine, trimethylamine, butyric acid, phenol, and indole in the chick liver hepatocellular carcinoma cell line LMH (Leghorn Male Hepatoma), in vitro, using MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) and PrestoBlue cytotoxicity assays. The cells were microscopically examined for any morphological changes post treatment. Dimethylamine exhibited the strongest cytotoxic effect on LMH cells with an IC50 value of 0.06% and 0.04% after an exposure of 24 h and 48 h, respectively. Both ammonium and trimethylamine had comparable cytotoxicity and their IC50 values were 0.08% and 0.04% after 24 h and 48 h, respectively. Of note, indole had the lowest cytotoxicity as the majority of cells were viable even after 72 h exposure. Thus, the IC50 for indole was not calculated. Results achieved from both MTT and PrestoBlue assays were comparable. Moreover, the morphological changes induced by the tested odours in LMH cells resulted in monolayer destruction, cytoplasm vacuolisation, chromatin condensation, and changes in nucleus and cell shape. Our study showed harmful effects of odorous compounds in chick tissues.

Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD+poly I:C, lipopolysaccharide (LPS), LPS+poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P<0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.

Some but not All Tetrahymena Species Destroy Monolayer Cultures of Cells from a Wide Range of Tissues and Species.

The activities of Tetrahymena corlissi, Tetrahymena thermophila, and Tetrahymena canadensis were studied in coculture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not carryout phagocytosis and did not destroy monolayers, whereas T. canadensis was able to carryout phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. In addition, the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore, differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not.

Replication of Infectious Pancreatic Necrosis Virus in Different Cell Lines and in Rainbow Trout (Oncorhynchus Mykiss) Fingerlings

Abstract The results of a study of Infectious Pancreatic Necrosis Virus (IPNV) isolated in natural reservoirs in Ukraine are presented. The pathogenicity of isolates was investigated in vitro on cell cultures and in vivo on rainbow trout, Oncorhynchus mykiss (Walbaum), fingerlings. Experimental indications were that the Ukrainian IPNV isolates have affinity with reference European strains. During the reproduction of these isolates in cell cultures of FHM (fat head minnow), RTG-2 (rainbow trout gonads), and BF-2 (bluegill caudal peduncle), complicated degenerative changes were visible that finally led to the full destruction of cell monolayers. The experimental infection of rainbow trout fingerlings resulted in typical disease symptoms that were systemic. However, obvious evidence of viral infection was noted in single individuals only, and the majority of experimental fish died without visible disease symptoms. During the study of physicochemical properties, it was noted that Ukrainian isolates completely lost their infectivity with chloroform treatment and heating to 60°C. This proved that IPNV isolates are resistant to Ion concentrations in the range of pH 3.0 to 12.0.

Delineating cellular interactions between ciliates and fish by co-culturing Tetrahymena thermophila with fish cells.

Although several species of Tetrahymena are often described as histophagous and opportunistic pathogens of fish, little is known about ciliate/fish cell interactions, but one approach for studying these is in vitro with cell lines. In this study, T. thermophila, B1975 (wild type) and NP1 (temperature sensitive mutant for phagocytosis) were cultured on monolayers of 3 fish epithelial cell lines, CHSE-214, RTgill-W1, and ZEB2J, and the rabbit kidney epithelial cell line, RK-13. Generally the ciliates flourished, whereas the monolayers died, being completely consumed over several days. The destruction of monolayers required that the ciliates could make contact with the animal cells through swimming, which appeared to dislodge or loosen cells so that they could be phagocytosed. The ciliates internalized into food vacuoles ZEB2J from cell monolayers as well as from cell suspensions. Phagocytosis was essential for monolayer destruction as monolayers remained intact under conditions where phagocytosis was impeded, such as 37°C for NP1 and 4°C for B1975. Monolayers of fish cells supported the proliferation of ciliates. Thus T. thermophila can 'eat' animal cells or be histophagous in vitro, with the potential to be histophagous in vivo.

Cysteine protease activity of feline Tritrichomonas foetus promotes adhesion-dependent cytotoxicity to intestinal epithelial cells.

Trichomonads are obligate protozoan parasites most renowned as venereal pathogens of the reproductive tract of humans and cattle. Recently, a trichomonad highly similar to bovine venereal Tritrichomonas foetus but having a unique tropism for the intestinal tract was recognized as a significant cause of colitis in domestic cats. Despite a high prevalence, worldwide distribution, and lack of consistently effective drugs for treatment of the infection, the cellular mechanisms of T. foetus pathogenicity in the intestinal tract have not been examined. The aims of this study were to determine the pathogenic effect of feline T. foetus on porcine intestinal epithelial cells, the dependence of T. foetus pathogenicity on adhesion of T. foetus to the intestinal epithelium, and the identity of mediators responsible for these effects. Using an in vitro coculture approach to model feline T. foetus infection of the intestinal epithelium, these studies demonstrate that T. foetus promotes a direct contact-dependent activation of intestinal epithelial cell apoptosis signaling and progressive monolayer destruction. Moreover, these pathological effects were demonstrated to be largely dependent on T. foetus cell-associated cysteine protease activity. Finally, T. foetus cysteine proteases were identified as enabling cytopathic effects by promoting adhesion of T. foetus to the intestinal epithelium. The present studies are the first to examine the cellular mechanisms of pathogenicity of T. foetus toward the intestinal epithelium and support further investigation of the cysteine proteases as virulence factors in vivo and as potential therapeutic targets for ameliorating the pathological effects of intestinal trichomonosis.

Grass carp reovirus induces apoptosis and oxidative stress in grass carp (Ctenopharyngodon idellus) kidney cell line

Grass carp hemorrhage is an acute contagious disease caused by grass carp reovirus (GCRV). The pathogenesis of GCRV and the relationship between GCRV and the host cells remain unclear. The aim of the present study was to investigate the relations among apoptosis, intracellular oxidative stress and virus replication in GCRV infected-cells. The results showed that GCRV induced activation of caspase proteases as early as 12h, and reached maximum activities at 24h or 48h post-infection in a grass carp kidney cell line (CIK cells). Meanwhile, the levels of tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) also were increased in GCRV-infected CIK cells and showed a statistically significant difference from 24h to 96h post-infection. The infection of GCRV caused the destruction of entire monolayer and the death of host cells. Accompanied by the infection, a severe oxidative stress occurred, which led to extensive loss of antioxidants and formation of lipid peroxidation after 48h post-infection. These data suggested that the apoptosis which was triggered at an early stage (12–24h) in the viral infection cycle, might be independent of virus replication, while the oxidative stress induced by GCRV was mostly related to the virus replication.

Characteristics of spring viremia of carp virus strains isolated in different regions of Ukraine

The paper contains results of studies of spring viremia of carp virus (SVCV) strains isolated in specialized fish farms in different regions of Ukraine. Pathogenicity of these isolates has been determined in vitro on cell culture and in vivo on young-of-the-year (YOY) carp (Cyprinus carpio L.). These SVCV isolates showed different pathogenicity towards YOY carp and caused mortality from 35 to 60%. Reproduction of these isolates in FHM, EPC, and BF-2 cell lines caused complex degenerative processes in cells, ultimately resulting in complete monolayer destruction. During experimental infection, it was found that the disease had a systemic nature. YOY carp had signs typical of SVCV infection, and affection of internal organs by the virus resulted in fish death. Clear clinical signs of the viral infection were observed in solitary individuals only, while the majority of fish died without any visible external clinical signs. A similarity between isolated SVCV strains and the reference strain in their physical–...

Wild-type Measles Virus Infection Upregulates Poliovirus Receptor-Related 4 and Causes Apoptosis in Brain Endothelial Cells by Induction of Tumor Necrosis Factor-Related Apoptosis-lnducing Ligand

Small numbers of brain endothelial cells (BECs) are infected in children with neurologic complications of measles virus (MV) infection. This may provide a mechanism for virus entry into the central nervous system, but the mechanisms are unclear. Both in vitro culture systems and animal models are required to elucidate events in the endothelium. We compared the ability of wild-type (WT), vaccine, and rodent-adapted MV strains to infect, replicate, and induce apoptosis in human and murine brain endothelial cells (HBECs and MBECs, respectively). Mice also were infected intracerebrally. All MV stains productively infected HBECs and induced the MV receptor PVRL4. Efficient WT MV production also occurred in MBECs. Extensive monolayer destruction associated with activated caspase 3 staining was observed in HBECs and MBECs, most markedly with WT MV. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not Fas ligand, was induced by MV infection. Treatment of MBECs with supernatants from MV-infected MBEC cultures with an anti-TRAIL antibody blocked caspase 3 expression and monolayer destruction. TRAIL was also expressed in the endothelium and other cell types in infected murine brains. This is the first demonstration that infection of low numbers of BECs with WT MV allows efficient virus production, induction of TRAIL, and subsequent widespread apoptosis.

Cytotoxic activity of extracts from Hypochaeris radicata

Pasture-associated stringhalt is an acquired equine disease characterized by peripheral neuropathy and hyperflexion of the pelvic limbs. The disease occurs most commonly during periods of drought in horses grazing pastures heavily contaminated by Hypochaeris radicata. We hypothesized that stringhalt is caused by neurotoxins elaborated by H. radicata in response to the stress of drought conditions. Supernates were collected from H. radicata that were stressed (or not) by immersion in copper chloride solution, then extracted with ethyl acetate and dried. Dilutions of extracts from stressed (SE) and control, unstressed (UE) plants were incubated with myelinating spinal cord cultures (MSCC) established from fetal Swiss mice, and with spinal ganglion cultures (SGC) and dermal fibroblast cultures derived from neonatal mouse tissues. Cytotoxicity in culture monolayers was evaluated both morphologically by microscopy and by release of lactate dehydrogenase activity into culture supernates. Three different SGC preparations were exposed to a single H. radicata extract and single preparations of fibroblasts and MSCC were exposed to three different extracts. Repin, a plant-derived sesquiterpene lactone neurotoxin, was included as a positive control. Significant dose-dependent cytotoxicity was seen within 24 h in all three culture types when incubated with SE or repin. Complete morphologic destruction of culture monolayers was induced by the highest concentrations tested of SE (100 μg/mL) and repin (30 μg/mL). Cytotoxic effect of SE was significantly greater than that of UE for all three cell types and was not due to copper contamination of the extract. This study has identified a cytotoxic activity in leaf exudates of H. radicata that was upregulated by the model stressor, copper chloride.

Cysteine Peptidases, Secreted by Trichomonas gallinae, Are Involved in the Cytopathogenic Effects on a Permanent Chicken Liver Cell Culture

Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.

Entamoeba histolytica Cell Surface Calreticulin Binds Human C1q and Functions in Amebic Phagocytosis of Host Cells

Phagocytosis of host cells is characteristic of tissue invasion by the intestinal ameba Entamoeba histolytica, which causes amebic dysentery and liver abscesses. Entamoeba histolytica induces host cell apoptosis and uses ligands, including C1q, on apoptotic cells to engulf them. Two mass spectrometry analyses identified calreticulin in amebic phagosome preparations, and, in addition to its function as an endoplasmic reticulum chaperone, calreticulin is believed to be the macrophage receptor for C1q. The purpose of this study was to determine if calreticulin functions as an E. histolytica C1q receptor during phagocytosis of host cells. Calreticulin was localized to the surface of E. histolytica during interaction with both Jurkat lymphocytes and erythrocytes and was present in over 75% of phagocytic cups during amebic erythrophagocytosis. Presence of calreticulin on the cell surface was further demonstrated using a method that selectively biotinylated cell surface proteins and by flow cytometry using trophozoites overexpressing epitope-tagged calreticulin. Regulated overexpression of calreticulin increased E. histolytica's ability to phagocytose apoptotic lymphocytes and calcium ionophore-treated erythrocytes but had no effect on amebic adherence to or destruction of cell monolayers or surface expression of the GalNAc lectin and serine-rich E. histolytica protein (SREHP) receptors. Finally, E. histolytica calreticulin bound specifically to apoptotic lymphocytes and to human C1q. Collectively, these data implicate cell surface calreticulin as a receptor for C1q during E. histolytica phagocytosis of host cells.

Control of Entamoeba histolytica Adherence Involves Metallosurface Protease 1, an M8 Family Surface Metalloprotease with Homology to Leishmanolysin

Invasive amebiasis due to Entamoeba histolytica infection is an important cause of morbidity in developing countries. The E. histolytica genome contains two homologues to the metalloprotease leishmanolysin gene, Entamoeba histolytica MSP-1 (EhMSP-1) and EhMSP-2, while the commensal ameba Entamoeba dispar has lost EhMSP-1. In this study, we sought to characterize E. histolytica metallosurface protease 1 (EhMSP-1). Using immunoprecipitation and a model substrate, we found that EhMSP-1 was a functional metalloprotease. Confocal microscopy and flow cytometry revealed that EhMSP-1 localized to the cell surface and revealed the existence of distinct, nonclonal trophozoite populations with high and low EhMSP-1 surface abundance that became synchronized following serum starvation. Phenotypic assays were performed after silencing EhMSP-1. Adherence of EhMSP-1-deficient trophozoites to tissue culture cell monolayers was more than five times greater than that of control amebas, but surface staining of several antigens, including the galactose adherence lectin, was unchanged. EhMSP-1 silencing similarly increased adherence to both viable and apoptotic Jurkat lymphocytes. Tissue culture cell monolayer destruction was reduced by EhMSP-1 silencing, although it was blocked almost completely by inhibiting cysteine proteases. Consistent with a primary defect in regulation of amebic adherence, EhMSP-1 silencing also resulted in reduced mobility on tissue culture cell monolayers and in increased phagocytosis. In conclusion, EhMSP-1 was shown to be a surface metalloprotease involved in regulation of amebic adherence, with additional effects on cell motility, cell monolayer destruction, and phagocytosis.