scholarly journals Adherence of Trichomonas vaginalis to SiHa Cells is Inhibited by Diphenyleneiodonium

2020 ◽  
Vol 8 (10) ◽  
pp. 1570
Author(s):  
Yeeun Kim ◽  
Young Ha Lee ◽  
In-Wook Choi ◽  
Bu Yeon Heo ◽  
Ju-Gyeong Kang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Trichomonas Vaginalis ◽  
Parasitic Infection ◽  
Oxidase Inhibitor ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Siha Cells ◽  
Parasite Infections

Microbial adhesion is critical for parasitic infection and colonization of host cells. To study the host–parasite interaction in vitro, we established a flow cytometry-based assay to measure the adherence of Trichomonas vaginalis to epithelial cell line SiHa. SiHa cells and T. vaginalis were detected as clearly separated, quantifiable populations by flow cytometry. We found that T. vaginalis attached to SiHa cells as early as 30 min after infection and the binding remained stable up to several hours, allowing for analysis of drug treatment efficacy. Importantly, NADPH oxidase inhibitor DPI treatment induced the detachment of T. vaginalis from SiHa cells in a dose-dependent manner without affecting host cell viability. Thus, this study may provide an understanding for the potential development of therapies against T. vaginalis and other parasite infections.

scholarly journals Trichomonas vaginalis Induces SiHa Cell Apoptosis by NF-κB Inactivation via Reactive Oxygen Species

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Juan-Hua Quan ◽  
Byung-Hun Kang ◽  
Jung-Bo Yang ◽  
Yun-Ee Rhee ◽  
Heung-Tae Noh ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Dna Fragmentation ◽  
Trichomonas Vaginalis ◽  
Reactive Oxygen ◽  
Host Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Ros Production ◽  
Oxygen Species ◽  
Siha Cells

Trichomonas vaginalis induces apoptosis in host cells through various mechanisms; however, little is known about the relationship between apoptosis, reactive oxygen species (ROS), and NF-κB signaling pathways in the cervical mucosal epithelium. Here, we evaluated apoptotic events, ROS production, and NF-κB activity in T. vaginalis-treated cervical mucosal epithelial SiHa cells, with or without specific inhibitors, using fluorescence microscopy, DNA fragmentation assays, subcellular fractionation, western blotting, and luciferase reporter assay. SiHa cells treated with live T. vaginalis at a multiplicity of infection of 5 (MOI 5) for 4 h produced intracellular and mitochondrial ROS in a parasite-load-dependent manner. Incubation with T. vaginalis caused DNA fragmentation, cleavage of caspase 3 and PARP, and release of cytochrome c into the cytoplasm. T. vaginalis-treated SiHa cells showed transient early NF-κB p65 nuclear translocation, which dramatically dropped at 4 h after treatment. Suppression of NF-κB activity was dependent on parasite burden. However, treatment with the ROS scavenger, N-acetyl-C-cysteine (NAC), reversed the effect of T. vaginalis on apoptosis and NF-κB inactivation in SiHa cells. Taken together, T. vaginalis induces apoptosis in human cervical mucosal epithelial cells by parasite-dose-dependent ROS production through an NF-κB-regulated, mitochondria-mediated pathway.

scholarly journals Influence of icariin on inflammation, apoptosis, invasion, and tumor immunity in cervical cancer by reducing the TLR4/MyD88/NF-κB and Wnt/β-catenin pathways

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chunyang Li ◽  
Shuangqing Yang ◽  
Huaqing Ma ◽  
Mengjia Ruan ◽  
Luyan Fang ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Underlying Mechanism ◽  
Tissue Growth ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Siha Cells ◽  
Cervical Cancer Cells

Abstract Background Cervical cancer is a type of the most common gynecology tumor in women of the whole world. Accumulating data have shown that icariin (ICA), a natural compound, has anti-cancer activity in different cancers, including cervical cancer. The study aimed to reveal the antitumor effects and the possible underlying mechanism of ICA in U14 tumor-bearing mice and SiHa cells. Methods The antitumor effects of ICA were investigated in vivo and in vitro. The expression of TLR4/MyD88/NF-κB and Wnt/β-catenin signaling pathways were evaluated. Results We found that ICA significantly suppressed tumor tissue growth and SiHa cells viability in a dose-dependent manner. Also, ICA enhanced the anti-tumor humoral immunity in vivo. Moreover, ICA significantly improved the composition of the microbiota in mice models. Additionally, the results clarified that ICA significantly inhibited the migration, invasion capacity, and expression levels of TGF-β1, TNF-α, IL-6, IL-17A, IL-10 in SiHa cells. Meanwhile, ICA was revealed to promote the apoptosis of cervical cancer cells by down-regulating Ki67, survivin, Bcl-2, c-Myc, and up-regulating P16, P53, Bax levels in vivo and in vitro. For the part of mechanism exploration, we showed that ICA inhibits the inflammation, proliferation, migration, and invasion, as well as promotes apoptosis and immunity in cervical cancer through impairment of TLR4/MyD88/NF-κB and Wnt/β-catenin pathways. Conclusions Taken together, ICA could be a potential supplementary agent for cervical cancer treatment.

scholarly journals CP30, a Cysteine Proteinase Involved inTrichomonas vaginalis Cytoadherence

2000 ◽  
Vol 68 (9) ◽  
pp. 4907-4912 ◽  
Author(s):  
M. Remedios Mendoza-López ◽  
Cecilia Becerril-Garcia ◽  
Loriz V. Fattel-Facenda ◽  
Leticia Avila-Gonzalez ◽  
Martha E. Ruíz-Tachiquín ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Hela Cell ◽  
Trichomonas Vaginalis ◽  
Cysteine Proteinase ◽  
Collagen Iv ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Monolayers ◽  
Thiol Proteinase

ABSTRACT We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.

scholarly journals Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila

2010 ◽  
Vol 207 (8) ◽  
pp. 1713-1726 ◽  
Author(s):  
Christopher T.D. Price ◽  
Tasneem Al-Quadan ◽  
Marina Santic ◽  
Snake C. Jones ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Biological Function ◽  
Host Cells ◽  
Dependent Manner ◽  
Protein Farnesyltransferase ◽  
Type Iv ◽  
Eukaryotic Proteins ◽  
Bacterial Effectors

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III–VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

scholarly journals A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth ParasiteTaenia crassiceps

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Galileo Escobedo ◽  
Gloria Soldevila ◽  
Guadalupe Ortega-Pierres ◽  
Jesús Ramsés Chávez-Ríos ◽  
Karen Nava ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Map Kinases ◽  
Host Cells ◽  
Cross Contamination ◽  
Flow Cytometry Analysis ◽  
Cellular Processes ◽  
17Β Estradiol ◽  
Activation Motif

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasiteTaenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host andT. crassiceps, and may be considered as target for anti-helminth drugs design.

scholarly journals Ascorbic acid attenuates activation and cytokine production in sepsis-like monocytes

2021 ◽  
Author(s):  
Tobias Schmidt ◽  
Robin Kahn ◽  
Fredrik Kahn
Keyword(s):  
Flow Cytometry ◽  
Ascorbic Acid ◽  
Cytokine Production ◽  
In Vitro Study ◽  
Surface Expression ◽  
High Dose ◽  
Functional Assay ◽  
Dependent Manner ◽  
Dose Dependent

Objective To investigate the effects of high dose ascorbic acid (AA) on monocyte polarization and cytokine production in vitro Design Experimental in vitro study of cells from healthy subjects and patients with sepsis Setting University research laboratory and academic hospital Subjects Six healthy controls and three patients with sepsis Interventions Monocytes were isolated from whole blood of healthy donors (n=6) and polarized in vitro for 48hrs using LPS or LTA. Polarization was confirmed by surface marker expression using flow cytometry. As a comparison, monocytes were also isolated from septic patients (n=3) and analyzed for polarization markers. The effect of AA on monocyte polarization was evaluated. As a functional assay, AA-treated monocytes were analyzed for cytokine production of TNF and IL-8 by intracellular staining and flow cytometry following activation with LPS or LTA. Measurements and Main Results Both LPS and LTA induced polarization in healthy monocytes in vitro, with increased expression of both pro- (CD40 and PDL1, p<0.05) and anti-inflammatory (CD16 and CD163, p<0.05) polarization markers, with non-significant effects on CD86 and CD206. This pattern resembled, at least partly, that of monocytes from septic patients. Treatment with AA significantly inhibited the upregulation of surface expression of CD16 and CD163 (p<0.05) in a dose dependent manner, but not CD40 or PDL-1. Finally, AA attenuated LPS or LTA-induced cytokine production of IL-8 and TNF in a dose-dependent manner (both p<0.05). Conclusions AA inhibits upregulation of anti-, but not pro-inflammatory related markers in LPS or LTA polarized monocytes. Additionally, AA attenuates cytokine production from in vitro polarized monocytes, displaying functional involvement. This study provides important insight into the immunological effects of high dose AA on monocytes, and potential implications in sepsis.

scholarly journals Trichomonas vaginalis Lipophosphoglycan Mutants Have Reduced Adherence and Cytotoxicity to Human Ectocervical Cells

2005 ◽  
Vol 4 (11) ◽  
pp. 1951-1958 ◽  
Author(s):  
Felix D. Bastida-Corcuera ◽  
Cheryl Y. Okumura ◽  
Angie Colocoussi ◽  
Patricia J. Johnson
Keyword(s):  
Wheat Germ Agglutinin ◽  
Parent Strain ◽  
Trichomonas Vaginalis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Chemical Mutagenesis ◽  
Reduced Adherence

ABSTRACT The extracellular human pathogen Trichomonas vaginalis is covered by a dense glycocalyx thought to play a role in host-parasite interactions. The main component of the glycocalyx is lipophosphoglycan (LPG), a polysaccharide anchored in the plasma membrane by inositol phosphoceramide. To study the role of LPG in trichomonads, we produced T. vaginalis LPG mutants by chemical mutagenesis and lectin selection and characterized them using morphological, biochemical, and functional assays. Two independently selected LPG mutants, with growth rates comparable to that of the wild-type (parent) strain, lost the ability to bind the lectins Ricinnus comunis agglutinin I (RCA120) and wheat germ agglutinin, indicating alterations in surface galactose and glucosamine residues. LPG isolated from mutants migrated faster than parent strain LPG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the mutants had shorter LPG molecules. Dionex high-performance anion exchange chromatography with pulsed amperometric detection analyses revealed galactosamine, glucosamine, galactose, glucose, mannose/xylose, and rhamnose as the main monosaccharides of T. vaginalis parent strain LPG. LPG from both mutants showed a reduction of galactose and glucosamine, corresponding with the reduced size of their LPG molecules and inability to bind the lectins RCA120 and wheat germ agglutinin. Mutant parasites were defective in attachment to plastic, a characteristic associated with avirulent strains of T. vaginalis. Moreover, the mutants were less adherent and less cytotoxic to human vaginal ectocervical cells in vitro than the parental strain. Finally, while parent strain LPG could inhibit the attachment of parent strain parasites to vaginal cells, LPG from either mutant could not inhibit attachment. These combined results demonstrate that T. vaginalis adherence to host cells is LPG mediated and that an altered LPG leads to reduced adherence and cytotoxicity of this parasite.

Glucose-restriction increasesTrichomonas vaginaliscellular damage towards HeLa cells and proteolytic activity of cysteine proteinases (CPs), such as TvCP2

Parasitology ◽  
2019 ◽  
Vol 146 (9) ◽  
pp. 1156-1166 ◽  
Author(s):  
Jesús F. T. Miranda-Ozuna ◽  
Luis Alberto Rivera-Rivas ◽  
Rosa Elena Cárdenas-Guerra ◽  
Mar Sarai Hernández-García ◽  
Sarahí Rodríguez-Cruz ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Hela Cells ◽  
Trichomonas Vaginalis ◽  
Host Cells ◽  
Cellular Damage ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Monolayers ◽  
Glucose Restriction ◽  
Effect Of Glucose

AbstractTrichomonas vaginalisinduces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose onT. vaginaliscytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface ofT. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.

AMN107 Appears Equipotent and May Synergise with Imatinib at the CML Stem Cell Level through Interaction with ABCG2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1080-1080 ◽  
Author(s):  
H. Jorgensen ◽  
E. Allan ◽  
N. Jordanides ◽  
A. Hamilton ◽  
J. Mountford ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Single Agent ◽  
Cell Number ◽  
Total Cell ◽  
Dependent Manner ◽  
Cell Level ◽  
Clinical Resistance

Abstract AMN107 (Novartis) is a novel Abl tyrosine kinase inhibitor specifically developed to be more selective for BcrAbl. AMN107 also maintains activity against the most common mutations associated with clinical resistance to imatinib mesylate (IM). In preclinical studies in cell lines and animal models, AMN107 was found to have greater potency than IM. By 3H-thymidine proliferation assays, the IC50 for AMN107 in K562 cells was 30 +/− 10nM compared with 600 +/− 60nM for IM. AMN107 and IM reduced K562 output cell number to 25% of input at 50 and 1000nM respectively, at 72h. These data are in keeping with the reported 20-fold increase in potency of AMN107 over IM. In addition, we have tested AMN107 for in vitro activity against primary CD34+Ph+ CML cells during 72h of culture in 5 growth factors. In CML cells (n=5), AMN107 and IM failed to reduce input cell number although the total cell output was restricted to 50% of PBS treated control at 2 +/− 1μM for AMN107 and to 31 +/− 7% of PBS treated control for 5μM IM suggesting the drugs were equipotent. The ability of the drugs to inhibit BcrAbl activity was then measured indirectly via the phosphorylation status of CrkL using a specific antiphospho-CrkL antibody and flow cytometry. Once again AMN107 and IM appeared equipotent in CML cells with 5μM of each compound leading to equal de-phosphorylation of CrkL. We next tested the efficacy of AMN107 as a single agent and in combination with IM against quiescent CML cells using in vitro dye (CFSE) tracking experiments. We evaluated by flow cytometry the proportion of input cells remaining alive, CD34+ and undivided (CFSEmax) or in first division. Compared to PBS treated control, 1.7, 2.5, 3.8 and 4.7-fold increases were found in the proportion of input CD34+ cells recovered in divisions 0 and 1 after 3 days exposure to 0.005, 0.05, 0.5 and 5μM AMN107, respectively. This was less accumulation than observed in the IM (5μM)-treated cells (11.0-fold). The combination of IM and AMN107, each at 5μM, was more effective in terms of total cell kill (54 and 74% fewer total cells remaining than with IM and AMN107 alone, respectively) and resulted in fewer viable cells recovered in divisions 0 and 1 than with either agent alone (for the combination, 1.9-fold on PBS treated recovery). We finally assessed the role of ABCG2 in modulating AMN107’s access to its intracellular BcrAbl target. We have previously shown ABCG2 to be over-expressed on CML stem cells and to interact with IM (Blood (2004); 104: 205a). We hypothesised that AMN107 and IM may co-operate as ABCG2 substrates or inhibitors to increase the intracellular levels of either or both drugs thus amplifying their efficacy against target protein specifically in CML stem cells. In competition assays with a known fluorescent substrate of ABCG2 (ie BODIPY-prazosin, BP), a specific inhibitor of the ABCG2 pump (fumitremorgin C, FTC) and an ABCG2 stably transfected AML cell line (AML6.2), the sample treated with BP plus FTC is taken to have greatest retention (100%). AMN107 inhibited efflux in a dose dependent manner to a maximum of 88% at 5μM, similarly to IM. Thus, AMN107 was equipotent with IM in primary CML stem cells in terms of restricting cell growth, inhibiting BcrAbl activity and interacting with ABCG2. However, AMN107 alone lead to less accumulation of quiescent CML cells in vitro as compared to IM, with the combination even more effective in this regard. The apparent co-operative effect of AMN107 and IM at the stem cell level would be predicted to improve clinical responses if tolerated in patients.

scholarly journals Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein

2001 ◽  
Vol 75 (11) ◽  
pp. 5335-5342 ◽  
Author(s):  
Kartik Chandran ◽  
Xing Zhang ◽  
Norman H. Olson ◽  
Stephen B. Walker ◽  
James D. Chappell ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Cell Attachment ◽  
Molecular Genetic ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
Dependent Manner ◽  
Viral Attachment ◽  
Structure Assembly

ABSTRACT Mammalian reoviruses, prototype members of theReoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.

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