scholarly journals Human Cytomegalovirus Infection of Caco-2 Cells Occurs at the Basolateral Membrane and Is Differentiation State Dependent

1999 ◽  
Vol 73 (6) ◽  
pp. 4552-4560 ◽  
Author(s):  
Michael A. Jarvis ◽  
C. Edward Wang ◽  
Heather L. Meyers ◽  
Patricia P. Smith ◽  
Christopher L. Corless ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Hcmv Infection ◽  
Cell Model ◽  
Basolateral Membrane ◽  
In Vitro Models

ABSTRACT Epithelial cells are known to be a major target for human cytomegalovirus (HCMV) infection; however, the analysis of virus-cell interactions has been difficult to approach due to the lack of in vitro models. In this study, we established a polarized epithelial cell model using a colon epithelial cell-derived cell line (Caco-2) that is susceptible to HCMV infection at early stages of cellular differentiation. Infection of polarized cells was restricted to the basolateral surface whereas virus was released apically, which was consistent with the apical and not basolateral surface localization of two essential viral glycoproteins, gB and gH. HCMV infection resulted in the development of a cytopathology characteristic of HCMV infection of colon epithelium in vivo, and infection did not spread from cell to cell. The inability of HCMV to infect Caco-2 cells at late stages of differentiation was due to a restriction at the level of viral entry and was consistent with the sequestration of a cellular receptor for HCMV. These observations provide the first evidence that restriction of HCMV replication in epithelial cells is due to a receptor-mediated phenomenon.

scholarly journals Identifying human and murine M cells in vitro

2019 ◽  
Vol 244 (7) ◽  
pp. 554-564 ◽  
Author(s):  
Ana Klisuric ◽  
Benjamin Thierry ◽  
Ludivine Delon ◽  
Clive A Prestidge ◽  
Rachel J Gibson
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
In Vitro Models ◽  
Oral Drug ◽  
M Cells ◽  
Cell Marker ◽  
M Cell ◽  
Follicle Associated Epithelium

M cells are an epithelial cell population found in the follicle-associated epithelium overlying gut-associated lymphoid tissues. They are specialized in the transcytosis of luminal antigens. Their transcytotic capacity and location in an immunocompetent environment has prompted the study of these cells as possible targets for oral drug delivery systems. Currently, the models most commonly used to study M cells are restricted to in vivo experiments conducted in mice, and in vitro studies conducted in models comprised either of primary epithelial cells or established cell lines of murine or human origin. In vitro models of the follicle-associated epithelium can be constructed in several ways. Small intestinal Lgr5+ stem cells can be cultured into a 3D organoid structure where M cells are induced with RANKL administration. Additionally, in vitro models containing an “M cell-like” population can be obtained through co-culturing intestinal epithelial cells with cells of lymphocytic origin to induce the M cell phenotype. The evaluation of the efficiency of the variations of these models and their relevance to the in vivo human system is hampered by the lack of a universal M cell marker. This issue has also hindered the advancement of M cell-specific targeting approaches aimed at improving the bioavailability of orally administered compounds. This critical review discusses the different approaches utilized in the literature to identify M cells, their efficiency, reliability and relevance, in the context of commonly used models of the follicle-associated epithelium. The outcome of this review is a clearly defined and universally recognized criteria for the assessment of the relevance of models of the follicle-associated models currently used. Impact statement The study of M cells, a specialized epithelial cell type found in the follicle-associated epithelium, is hampered by the lack of a universal M cell marker. As such, many studies lack reliable and universally recognized methods to identify M cells in their proposed models. As a result of this it is difficult to ascertain whether the effects observed are due to the presence of M cells or an unaccounted variable. The outcome of this review is the thorough evaluation of the many M cell markers that have been used in the literature thus far and a proposed criterion for the identification of M cells for future publications. This will hopefully lead to an improvement in the quality of future publications in this field.

scholarly journals Human epithelial cells in vitro – Are they an advantageous tool to help understand the nanomaterial-biological barrier interaction?

2012 ◽  
Vol 4 (1) ◽  
pp. 1-19 ◽  
Author(s):  
Barbara Rothen-Rutishauser ◽  
Martin J.D. Clift ◽  
Corinne Jud ◽  
Alke Fink ◽  
Peter Wick
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basic Mechanism ◽  
In Vitro Models ◽  
Advantages And Disadvantages ◽  
Culture Models ◽  
Experimental Approaches ◽  
Cell Culture Models

AbstratThe human body can be exposed to nanomaterials through a variety of different routes. As nanomaterials get in contact with the skin, the gastrointestinal tract, and the respiratory tract, these biological compartments are acting as barriers to the passage of nano-sized materials into the organism. These structural and functional barriers are provided by the epithelia serving as an interface between biological compartments. In order to initiate the reduction, refinement and replacement of time consuming, expensive and stressful (to the animals) in vivo experimental approaches, many in vitro epithelial cell culture models have been developed during the last decades. This review therefore, focuses on the functional as well as structural aspects of epithelial cells as well as the most commonly used in vitro epithelial models of the primary biological barriers with which nanomaterials might come in contact with either occupationally, or during their manufacturing and application. The advantages and disadvantages of the different in vitro models are discussed in order to provide a clear overview as to whether or not epithelial cell cultures are an advantageous model to be used for basic mechanism and nanotoxicology research.

scholarly journals Impact of Antibodies and Strain Polymorphisms on Cytomegalovirus Entry and Spread in Fibroblasts and Epithelial Cells

2017 ◽  
Vol 91 (13) ◽  
Author(s):  
Xiaohong Cui ◽  
Daniel C. Freed ◽  
Dai Wang ◽  
Ping Qiu ◽  
Fengsheng Li ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Viral Entry ◽  
Active Immunization ◽  
Cell Entry ◽  
Strain Specificity ◽  
Humoral Responses ◽  
Cmv Disease

ABSTRACT Cytomegalovirus (CMV) entry into fibroblasts differs from entry into epithelial cells. CMV also spreads cell to cell and can induce syncytia. To gain insights into these processes, 27 antibodies targeting epitopes in CMV virion glycoprotein complexes, including glycoprotein B (gB), gH/gL, and the pentamer, were evaluated for their effects on viral entry and spread. No antibodies inhibited CMV spread in fibroblasts, including those with potent neutralizing activity against fibroblast entry, while all antibodies that neutralized epithelial cell entry also inhibited spread in epithelial cells and a correlation existed between the potencies of these two activities. This suggests that exposure of virions to the cell culture medium is obligatory during spread in epithelial cells but not in fibroblasts. In fibroblasts, the formation of syncytiumlike structures was impaired not only by antibodies to gB or gH/gL but also by antibodies to the pentamer, suggesting a potential role for the pentamer in promoting fibroblast fusion. Four antibodies reacted with linear epitopes near the N terminus of gH, exhibited strain specificity, and neutralized both epithelial cell and fibroblast entry. Five other antibodies recognized conformational epitopes in gH/gL and neutralized both fibroblast and epithelial cell entry. That these antibodies were strain specific for neutralizing fibroblast but not epithelial cell entry suggests that polymorphisms external to certain gH/gL epitopes may influence antibody neutralization during fibroblast but not epithelial cell entry. These findings may have implications for elucidating the mechanisms of CMV entry, spread, and antibody evasion and may assist in determining which antibodies may be most efficacious following active immunization or passive administration. IMPORTANCE Cytomegalovirus (CMV) is a significant cause of birth defects among newborns infected in utero and morbidity and mortality in transplant and AIDS patients. Monoclonal antibodies and vaccines targeting humoral responses are under development for prophylactic or therapeutic use. The findings reported here (i) confirm that cell-to-cell spread of CMV is sensitive to antibody inhibition in epithelial cells but not fibroblasts, (ii) demonstrate that antibodies can restrict the formation in vitro of syncytiumlike structures that resemble syncytial cytomegalic cells that are associated with CMV disease in vivo, and (iii) reveal that neutralization of CMV by antibodies to certain epitopes in gH or gH/gL is both strain and cell type dependent and can be governed by polymorphisms in sequences external to the epitopes. These findings serve to elucidate the mechanisms of CMV entry, spread, and antibody evasion and may have important implications for the development of CMV vaccines and immunotherapeutics.

scholarly journals A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

2008 ◽  
Vol 22 (12) ◽  
pp. 2677-2688 ◽  
Author(s):  
Paul G. Tiffen ◽  
Nader Omidvar ◽  
Nuria Marquez-Almuina ◽  
Dawn Croston ◽  
Christine J. Watson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Oncostatin M ◽  
Specific Gene ◽  
Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

scholarly journals Human Cytomegalovirus Cell Tropism and Host Cell Receptors

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 70 ◽  
Author(s):  
Gerna ◽  
Kabanova ◽  
Lilleri
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Current Data ◽  
Immunosuppressed Patient ◽  
Cell Tropism ◽  
Virus Envelope

In the 1970s–1980s, a striking increase in the number of disseminated human cytomegalovirus (HCMV) infections occurred in immunosuppressed patient populations. Autopsy findings documented the in vivo disseminated infection (besides fibroblasts) of epithelial cells, endothelial cells, and polymorphonuclear leukocytes. As a result, multiple diagnostic assays, such as quantification of HCMV antigenemia (pp65), viremia (infectious virus), and DNAemia (HCMV DNA) in patient blood, were developed. In vitro experiments showed that only low passage or endothelial cell-passaged clinical isolates, and not laboratory-adapted strains, could reproduce both HCMV leuko- and endothelial cell-tropism, which were found through genetic analysis to require the three viral genes UL128, UL130, and UL131 of the HCMV UL128 locus (UL128L). Products of this locus, together with gH/gL, were shown to form the gH/gL/pUL128L pentamer complex (PC) required for infection of epithelial cells/endothelial cells, whereas gH/gL and gO form the gH/gL/gO trimer complex (TC) required for infection of all cell types. In 2016, following previous work, a receptor for the TC that mediates entry into fibroblasts was identified as PDGFRα, while in 2018, a receptor for the PC that mediates entry into endothelial/epithelial cells was identified as neuropilin2 (Nrp2). Furthermore, the olfactory receptor family member OR14I1 was recently identified as a possible additional receptor for the PC in epithelial cells. Thus, current data support two models of viral entry: (i) in fibroblasts, following interaction of PDGFRα with TC, the latter activates gB to fuse the virus envelope with the cell membrane, whereas (ii) in epithelial cells/endothelial cells, interaction of Nrp2 (and OR14I1) with PC promotes endocytosis of virus particles, followed by gB activation by gH/gL/gO (or gH/gL) and final low-pH entry into the cell.

In vitro infection of megakaryocytes and their precursors by human cytomegalovirus

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 487-493 ◽  
Author(s):  
Kirsten Crapnell ◽  
Esmail D. Zanjani ◽  
Aniruddho Chaudhuri ◽  
Joao L. Ascensao ◽  
Stephen St. Jeor ◽  
...  
Keyword(s):  
Life Cycle ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Allogeneic Bone Marrow Transplantation ◽  
Laboratory Strain ◽  
Allogeneic Bone ◽  
Cd34 Cells ◽  
Hcmv Disease

Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34+ cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42+ MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the β-galactosidase (β-gal) gene. Using chromogenic β-gal substrates, HCMV was detected during MK differentiation of infected CD34+ cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV.

scholarly journals Microfluidic approaches for epithelial cell layer culture and characterisation

The Analyst ◽  
2014 ◽  
Vol 139 (13) ◽  
pp. 3206-3218 ◽  
Author(s):  
Roland Thuenauer ◽  
Enrique Rodriguez-Boulan ◽  
Winfried Römer
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Layer ◽  
Microfluidic Biochips ◽  
Epithelial Cell Layer

Novelin vitromodels of epithelia in which thein vivomicroenvironment of epithelial cells is precisely reconstituted can be realised with microfluidic biochips.

scholarly journals Rapid protein kinase D1 signaling promotes migration of intestinal epithelial cells

2012 ◽  
Vol 303 (3) ◽  
pp. G356-G366 ◽  
Author(s):  
Steven H. Young ◽  
Nora Rozengurt ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt
Keyword(s):  
Protein Kinase ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Transgenic Mice ◽  
Intestinal Epithelial Cell ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Protein Kinase D1

We have examined the role of protein kinase D1 (PKD1) signaling in intestinal epithelial cell migration. Wounding monolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid PKD1 activation in the cells immediately adjacent to the wound edge, as judged by immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD1 at Ser916, an autophosphorylation site. An increase in PKD1 phosphorylation at Ser916 was evident as early as 45 s after wounding, reached a maximum after 3 min, and persisted for ≥15 min. PKD1 autophosphorylation at Ser916 was prevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A kb NB 142-70-sensitive increase in PKD autophosphorylation was also elicited by wounding IEC-6 cells. Using in vitro kinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cells induced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling is required to promote migration of intestinal epithelial cells into the denuded area of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAs targeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. To test whether PKD1 promotes migration of intestinal epithelial cells in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestinal epithelium. Enterocyte migration was markedly increased in the PKD1 transgenic mice. These results demonstrate that PKD1 activation is one of the early events initiated by wounding a monolayer of intestinal epithelial cells and indicate that PKD1 signaling promotes the migration of these cells in vitro and in vivo.

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