scholarly journals Impact of Hydroxychloroquine-Loaded Polyurethane Intravaginal Rings on Lactobacilli

2015 ◽  
Vol 59 (12) ◽  
pp. 7680-7686 ◽  
Author(s):  
Yannick Leandre Traore ◽  
Yufei Chen ◽  
Anne-Marie Bernier ◽  
Emmanuel A. Ho
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Female Genital Tract ◽  
Vaginal Epithelial Cell ◽  
Content Type ◽  
Cell Monolayers ◽  
Intravaginal Rings ◽  
Vaginal Epithelial Cells ◽  
Epithelial Cell Monolayers ◽  
The Impact

ABSTRACTThe use of polymeric devices for controlled sustained delivery of drugs is a promising approach for the prevention of HIV-1 infection. Unfortunately, certain microbicides, when topically applied vaginally, may be cytotoxic to vaginal epithelial cells and the protective microflora present within the female genital tract. In this study, we evaluated the impact of hydroxychloroquine (HCQ)-loaded, reservoir-type, polyurethane intravaginal rings (IVRs) on the growth ofLactobacillus crispatusandLactobacillus jenseniiand on the viability of vaginal and ectocervical epithelial cells. The IVRs were fabricated using hot-melt injection molding and were capable of providing controlled release of HCQ for 24 days, with mean daily release rates of 17.01 ± 3.6 μg/ml in sodium acetate buffer (pH 4) and 29.45 ± 4.84 μg/ml in MRS broth (pH 6.2). Drug-free IVRs and the released HCQ had no significant effects on bacterial growth or the viability of vaginal or ectocervical epithelial cells. Furthermore, there was no significant impact on the integrity of vaginal epithelial cell monolayers, in comparison with controls, as measured by transepithelial electrical resistance. Overall, this is the first study to evaluate the effects of HCQ-loaded IVRs on the growth of vaginal flora and the integrity of vaginal epithelial cell monolayers.

scholarly journals Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2

2015 ◽  
Vol 83 (7) ◽  
pp. 2762-2770 ◽  
Author(s):  
Nitsan Maharshak ◽  
Eun Young Huh ◽  
Chorlada Paiboonrungruang ◽  
Michael Shanahan ◽  
Lance Thurlow ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Epithelial Cell ◽  
Enterococcus Faecalis ◽  
Fluorescein Isothiocyanate ◽  
Cell Permeability ◽  
Content Type ◽  
Cell Monolayers ◽  
Microbial Protease ◽  
Epithelial Cell Monolayers ◽  
Protease Activated Receptor 2

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbeEnterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) fromE. faecalisV583 andE. faecalislacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutantE. faecalisstrains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2−/−) mice were used to investigate the role of this receptor inE. faecalis-induced permeability. Gelatinase (GelE) purified fromE. faecalisV583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2−/−mice by fecal supernatants from ulcerative colitis patients was assessed. SecretedE. faecalisproteins induced permeability in epithelial cell monolayers, which was reduced in the absence ofgelEor by blocking PAR2 activity. SecretedE. faecalisproteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2−/−mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2−/−mice. Our investigations demonstrate that GelE fromE. faecaliscan regulate enteric epithelial permeability via PAR2.

The interaction of fresh and frozen-thawed ram spermatozoa with oviducal epithelial cells in vitro

2000 ◽  
Vol 12 (6) ◽  
pp. 237 ◽  
Author(s):  
L. Gillan ◽  
G. Evans ◽  
W. M. C. Maxwell
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Small Proportion ◽  
Oestrous Cycle ◽  
Cell Monolayers ◽  
Oviduct Epithelial Cell ◽  
Epithelial Cell Monolayers ◽  
Oviduct Epithelial Cells

In order to investigate the interaction of fresh and frozen–thawed spermatozoa with oviduct epithelial cells, spermatozoa were co-incubated with ovine oviduct epithelial cell monolayers (OECM) derived from either complete oviducts, at any stage of the oestrous cycle (Experiments 1 and 2), or from different regions of the oviduct at different stages of the cycle (Experiment 3). Fresh and frozen—thawed spermatozoa displayed different patterns of binding to, and release from, the OECM. Frozen—thawed spermatozoa immediately bound to the complete oviduct OECM and were released after 2 h. A small proportion of fresh spermatozoa bound immediately, increasing to a maximum after 2 h, and were gradually released thereafter. When only the cells that were released from the OECM were observed by chlortetracycline staining in Experiment 2, it was found that the presence of an OECM increased the number of capacitated fresh spermatozoa while decreasing the number of capacitated frozen–thawed spermatozoa. Overall, the OECM advanced the membrane state of both types of spermatozoa from uncapacitated to acrosome-reacted. Fresh and frozen—thawed spermatozoa bound to OECM derived from the cells of the isthmus and the ampulla in similar proportions. However, more spermatozoa were capacitated when incubated with OECM derived from isthmic rather than ampullary cells. Higher proportions of fresh spermatozoa bound to, and were acrosome-reacted following incubation with OECM derived from post- rather than pre-ovulatory tracts. Such differences were not observed for frozen—thawed spermatozoa. The findings reported in this study show that fresh and frozen—thawed spermatozoa behave differently when in contact with oviduct cells in vitro. This may be a consequence of the more advanced membrane state of the frozen spermatozoa upon thawing.

scholarly journals Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

2015 ◽  
Vol 308 (9) ◽  
pp. G745-G756 ◽  
Author(s):  
Susana Lechuga ◽  
Somesh Baranwal ◽  
Andrei I. Ivanov
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Actin Cytoskeleton ◽  
Critical Role ◽  
Intestinal Epithelial ◽  
Interacting Protein ◽  
Cell Monolayers ◽  
Epithelial Junctions ◽  
Epithelial Cell Monolayers ◽  
Apical Junctions

Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis.

scholarly journals Correlative light and scanning electron microscopy (CLSEM) for analysis of bacterial infection of polarized epithelial cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carina Kommnick ◽  
Andrea Lepper ◽  
Michael Hensel
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Monolayers ◽  
Epithelial Cell Monolayers ◽  
Polarized Epithelial Cells ◽  
Scanning Electron

AbstractInfection of mammalian host cells by bacterial pathogens is a highly dynamic process and microscopy is instrumental to reveal the cellular and molecular details of host-pathogen interactions. Correlative light and electron microscopy (CLEM) combines the advantages of three-dimensional live cell imaging with ultrastructural analysis. The analyses of adhesion to, and invasion of polarized epithelial cells by pathogens often deploys scanning electron microscopy (SEM), since surface structures of the apical brush border can be analyzed in detail. Most available CLEM approaches focus on relocalization of separated single cells in different imaging modalities, but are not readily applicable to polarized epithelial cell monolayers, since orientation marks on substrate are overgrown during differentiation. To address this problem, we developed a simple and convenient workflow for correlative light and scanning electron microscopy (CLSEM), using gold mesh grids as carrier for growth of epithelial cell monolayers, and for imaging infection. The approach allows fast live cell imaging of bacterial infection of polarized cells with subsequent analyses by SEM. As examples for CLSEM applications, we investigated trigger invasion by Salmonella enterica, zipper invasion by Listeria monocytogenes, and the enterocyte attachment and effacement phenotype of enteropathogenic Escherichia coli. Our study demonstrates the versatile use of gold mesh grids for CLSEM of the interaction of bacterial pathogens with the apical side of polarized epithelial cells.

The role of 17β-estrogen in Escherichia coli adhesion on human vaginal epithelial cells via FAK phosphorylation

2021 ◽  
Author(s):  
Xia Liu ◽  
Ting Luan ◽  
Wanqing Zhou ◽  
Lina Yan ◽  
Hua Qian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Specific Inhibitor ◽  
Estrogen Receptor Α ◽  
Epithelial Cell Line ◽  
Vaginal Epithelial Cell ◽  
E Coli ◽  
Vaginal Epithelial Cells

Estrogen, the predominant sex hormone, has been found to be related to the occurrence of vaginal infectious diseases. However, its role in the occurrence and development of bacterial vaginitis caused by Escherichia coli is still unclear. The objective of this study was to investigate the role of 17β-estrogen in E. coli adhesion on human vaginal epithelial cells. The vaginal epithelial cell line, VK2/E6E7, was used to study the molecular events induced by estrogen between E. coli and cells. An adhesion study was performed to evaluate the involvement of the estrogen-dependent focal adhesion kinase (FAK) activation with cell adhesion. The phosphorylation status of FAK and estrogen receptor α (ERα) upon estrogen challenge was assessed by Western blotting. Specific inhibitors for ERα were used to validate the involvement of ERα-FAK signaling cascade. The results showed that, following the stimulation with 1000 nM estrogen for 48 h, a transient activation of ERα and FAK was observed, as well as the increased average number of E. coli adhering to vaginal epithelial cell. In addition, estrogen-induced activation of ERa and FAK was inhibited by the specific inhibitor of ERα, especially when the inhibitor reached a 10 μM concentration and acted for 1 h, and a decrease in the number of adherent E. coli was observed simultaneously. However, this inhibitory effect diminished as the concentration of estrogen increased. In conclusion, FAK and ERα signaling cascades were assosiated with the increasing E. coli adherence to vaginal epithelial cells, which was promoted by a certain concentration of estrogen.

scholarly journals Antivaccinia Activities of Acyclic Nucleoside Phosphonate Derivatives in Epithelial Cells and Organotypic Cultures

2002 ◽  
Vol 46 (11) ◽  
pp. 3356-3361 ◽  
Author(s):  
R. Snoeck ◽  
A. Holý ◽  
C. Dewolf-Peeters ◽  
J. Van Den Oord ◽  
E. De Clercq ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Active Compound ◽  
Immunocompromised Patients ◽  
Organotypic Cultures ◽  
Cell Monolayers ◽  
Epithelial Cell Monolayers ◽  
Acyclic Nucleoside ◽  
Acyclic Nucleoside Phosphonate

ABSTRACT Organotypic “raft” cultures of epithelial cells allow the reconstitution of a skin equivalent that is easily infectible with different viruses with cutaneous tropism. Among these, poxvirus and particularly vaccinia virus (VV) are good candidates for use in antiviral tests, giving histological pictures comparable to those observed in humans infected with smallpox. Therefore, we decided to evaluate a series of phosphonate derivatives for their ability to inhibit VV growth in epithelial cell monolayers, and the most powerful derivatives were tested in the organotypic cultures. The most active compound was 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA], followed by 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine, cyclic (S)-HPMPA, 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [(S)-HPMPC; cidofovir, Vistide], and cyclic (S)-HPMPC. Cidofovir, which is on the market for the treatment of human cytomegalovirus retinitis in immunocompromised patients, is potentially a good candidate for the treatment of a poxvirus outbreak, in the absence of any vaccination.

scholarly journals Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers

Membranes ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 331
Author(s):  
Yong Ho Kim ◽  
Kwang-Jin Kim ◽  
David Z. D’Argenio ◽  
Edward D. Crandall
Keyword(s):  
Epithelial Cell ◽  
Tight Junctions ◽  
Pore Radius ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Type I ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers ◽  
Hydrophilic Solutes

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on “equivalent” pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92–99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.

scholarly journals A Novel Pharmacological Approach to Enhance the Integrity and Accelerate Restitution of the Intestinal Epithelial Barrier

2020 ◽  
Vol 26 (9) ◽  
pp. 1340-1352
Author(s):  
Xuelei Cao ◽  
Lei Sun ◽  
Susana Lechuga ◽  
Nayden G Naydenov ◽  
Alex Feygin ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Cell Monolayers ◽  
Barrier Disruption ◽  
Epithelial Cell Monolayers ◽  
Immunofluorescence Labeling ◽  
E Cadherin

Abstract Background Disruption of the gut barrier is an essential mechanism of inflammatory bowel diseases (IBDs) contributing to the development of mucosal inflammation. A hallmark of barrier disruption is the disassembly of epithelial adherens junctions (AJs) driven by decreased expression of a major AJ protein, E-cadherin. A group of isoxazole compounds, such as E-cadherin-upregulator (ECU) and ML327, were previously shown to stimulate E-cadherin expression in poorly differentiated human cancer cells. This study was designed to examine whether these isoxazole compounds can enhance and protect model intestinal epithelial barriers in vitro. Methods The study was conducted using T84, SK-CO15, and HT-29 human colonic epithelial cell monolayers. Disruption of the epithelial barrier was induced by pro-inflammatory cytokines, tumor necrosis factor-α, and interferon-γ. Barrier integrity and epithelial junction assembly was examined using different permeability assays, immunofluorescence labeling, and confocal microscopy. Epithelial restitution was analyzed using a scratch wound healing assay. Results E-cadherin-upregulator and ML327 treatment of intestinal epithelial cell monolayers resulted in several barrier-protective effects, including reduced steady-state epithelial permeability, inhibition of cytokine-induced barrier disruption and junction disassembly, and acceleration of epithelial wound healing. Surprisingly, these effects were not due to upregulation of E-cadherin expression but were mediated by multiple mechanisms including inhibition of junction protein endocytosis, attenuation of cytokine-induced apoptosis, and activation of promigratory Src and AKT signaling. Conclusions Our data highlight ECU and ML327 as promising compounds for developing new therapeutic strategies to protect the integrity and accelerate the restitution of the intestinal epithelial barrier in IBD and other inflammatory disorders.

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